8^2^ PATHOGENIC 
MICROORGANISMS 
A PRACTICAL MANUAL FOR STUDENTS, PHYSICIANS 
AND HEALTH OFFICERS 
BY 
WILLIAM HALLOCK jPARK, M.D. 
PROFESSOR OF BACTERIOLOGY AND HYGIENE, UNIVERSITY AND BELLEVUE HOSPITAL MEDICAL 
COLLEGE, AND DIRECTOR OF THE BUREAU OF LABORATORIES OF THE DEPARTMENT 
OF HEALTH, NEW YORK CITY 
AND 
ANNA W. WILLIAMS, M.D. 
ASSISTANT DIRECTOR OF THE BUREAU OF LABORATORIES; CONSULTING PATHOLOGIST TO THE NEW YORK 
INFIRMARY FOR WOMEN AND CHILDREN 
FIFTH EDITION, ENLARGED AND THOROUGHLY REVISED 
WITH 210 ENGRAVINGS AND 9 FULL-PAGE PLATES 
LEA & F EBI GER 
NEW YORK AND PHILADELPHIA 
1914 Entered according to the Act of Congress, in the year 1914, by 
LEA & FEBIGER 
in the office of the Librarian of Congress. All rights reserved. PREFACE TO FIFTH EDITION. 
The first edition of this book was called Bacteriology in Medicine 
and Surgery. It was written to make available for others the practical 
knowledge which had been acquired in the work of the bacteriologic 
laboratories of the city of New York, and was intended more for medical 
practitioners than for medical students or laboratory workers. When 
the second edition had been exhausted the improvement in methods of 
cultivating and studying the protozoa had reached a point rendering 
it advantageous to include the animal as well as the vegetable germs. 
This was done, and the title of the third edition was altered to conform 
with the text, which had been broadened to give in outline practically 
the whole field of pathogenic microorganisms. 
The book in its later editions has come to be used in an ever-increasing 
degree by medical students, so that while its point of view has remained 
the same, namely, to dwell especially on the relations of microorganisms 
to disease in man, it has been thought wise to touch on other aspects, 
such as the bacteria concerned in agriculture and in some of the 
important fermentations. 
References are frequently given to some of the related investiga- 
tions, but no effort has been made to afford a complete or uniform 
bibliography. The question as to the practical limits of bibliographic 
references in such a hand-book as this is an open one. Hence the lack 
of uniformity in this respect. 
In this fifth edition we have rearranged the material in order to bring 
more closely together all of the pathogenic organisms. Under this 
arrangement Part I deals with the general characteristics and methods 
of study of all the microorganisms considered (molds, yeasts, bacteria, 
and protozoa). Part II includes the study of the individual pathogenic 
microorganisms and their near relatives. Part III presents certain prac- 
tical aspects of the subject under the title Applied Microbiology. The 
nine plates included in this edition have been arranged and drawn 
especially for this book. Owing to recent advances in the knowledge 
of microbiology, we have added much new material and rewritten 
several parts of the book. For example, the subject of complement IV 
PREFACE TO FIFTH EDITION 
fixation has increased so much in importance that a full description 
of its nature and its various applications has been given in a separate 
chapter. The whole subject of immunity has been extensively revised. 
A new chapter has been written on filtrable viruses. The chapter on 
the preparation and uses of media has been rewritten, and the recent 
methods for the use of anilin dyes, as well as other new procedures, have 
been incorporated. Other chapters which have been extensively changed 
are those dealing with the streptococcus, the pneumococcus, the diph- 
theria bacillus, the tetanus bacillus, the hemoglobinophilic bacilli, the 
bacillus of pertussis, the molds, the amebse, and the chapter on rabies. 
The chapter on complement fixation has been written by M. P. 
Olmstead, who for several years past has been in charge of much of 
the work on this subject, which is carried on in the New York City 
Health Department. The chapter on media has been rewritten by 
C. Krumwiede, one of the assistant directors of the laboratories of 
the Health Department. He also revised the chapters on the coli- 
typhoid group, on the group of acid-fast bacilli, and on the cholera 
spirillum and relatives. The chapter on the bacillus malleus and that 
on the meningococcus were revised respectively by B. Van H. Anthony, 
M.D.,and P. L. DuBois, M.D., both bacteriologists in our laboratories. 
We are further indebted to our associates in the laboratory for aid 
in many different ways. 
W. H. P. 
A. W. W. 
New York, 1914. CONTENTS. 
PART 1. 
PRINCIPLES OF MICROBIOLOGY. 
CHAPTER I. 
Introductory. Historical Sketch 17 
CHAPTER II. 
Classification and General Characteristics of Microorganisms . . .24 
CHAPTER III. 
The Microscope and the Microscopic Examination of Microorganisms 66 
CHAPTER IV. 
General Methods Used in the Cultivation of Microorganisms . . 86 
CHAPTER V. 
The Use of Animals for Diagnostic and Test Purposes .... 122 
CHAPTER VI. 
The Procuring and Handling of Material for Microbiologic Exami- 
nation from Those Suffering from Disease 125 
CHAPTER VII. 
The Relation of Microorganisms to Disease 132 
CHAPTER VIII. 
Immunity, Active and Passive. The Antagonism Existing between 
the Living Body and Microorganisms 151 
CHAPTER IX. 
Nature of the Protective Defences of the Body and their Manner 
of Action. Ehrlich's "Side Chain" and Other Theories . . . 158 
CHAPTER X. 
Complement Fixation: The Technique of the Test and its Practical 
Applications 177 VI 
CONTENTS 
CHAPTER XI. 
The Nature of the Substances Concerned in Agglutination . . . 193 
CHAPTER XII. 
Opsonins. Extract of Leukocytes 208 
CHAPTER XIII. 
Active Immunization. Vaccines 219 
CHAPTER XIV. 
Protein Hypersensitiveness or Anaphylaxis 224 
PART II. 
PATHOGENIC MICROORGANISMS INDIVIDUALLY 
CONSIDERED. 
CHAPTER XV. 
The Pathogenic Molds (Hyphomycetes, Eumycetes) and Yeasts 
(Blastomycetes) 229 
CHAPTER XVI. 
The Pyogenic Cocci 241 
CHAPTER XVII. 
The Diplococcus of Pneumonia (Pneumococcus, Streptococcus Pneu- 
moniae. Micrococcus Lanceolatus) ' 262 
CHAPTER XVIII. 
Meningococcus or Micrococcus (Intracellularis) Meningitidis, and 
the Relation of it and of Other Bacteria to Meningitis . . . 273 
CHAPTER XIX. 
The Gonococcus or Micrococcus Gonorrhceae. Micrococcus Melitensis 283 
CHAPTER XX. 
The Bacillus and the Bacteriology of Diphtheria 292 
CHAPTER XXL 
The Bacillus and the Bacteriology of Tetanus 320 
CHAPTER XXII. 
Intestinal Bacteria 334 CONTENTS 
VII 
CHAPTER XXIII. 
The Colon-typhoid Group of Bacilli 344 
CHAPTER XXIV. 
The Typhoid Bacillus 355 
CHAPTER XXV. 
Paratyphoid Group 374 
CHAPTER XXVI. 
Dysentery Group 378 
CHAPTER XXVII. 
Bacillus Pyocyaneus (Bacillus of Green and of Blue Pus). Bacillus 
Proteus (Vulgaris) 383 
CHAPTER XXVIII. 
The Bacillus and the Bacteriology of Tuberculosis 388 
CHAPTER XXIX. 
Other Acid-fast Bacilli: Bacillus of Leprosy, Bacillus of Rat 
Leprosy, Bacillus of Johne's Disease in Cattle and the Group 
of Non-pathogenic Acid-fast Bacilli 420 
CHAPTER XXX. 
Glanders Bacillus (Bacillus Mallei) 425 
CHAPTER XXXI. 
The Group of Hemoglobinophilic Bacilli. Bordet-Gengou Bacillus 432 
CHAPTER XXXII. 
Microorganisms Belonging to the Hemorrhagic Septicemia Group . 445 
CHAPTER XXXIII. 
The Anthrax Bacillus and the Pathogenic Anaerobes 450 
CHAPTER XXXIV. 
The Cholera Spirillum (Cholera Vibrio) and Similar Varieties . . 464 
CHAPTER XXXV. 
Pathogenic Microorganisms Belonging to the Higher Bacteria 
(Trichomycetes) 476 
CHAPTER XXXVI. 
Filtrable Viruses. Diseases of Unknown Etiology 488 
CHAPTER XXXVII. 
Flagellata 496 VIII 
CONTENTS 
CHAPTER XXXVIII. 
Trypanosoma 503 
CHAPTER XXXIX. 
Spirocheta and Allies 515 
CHAPTER XL. 
Bodo. Polymastigida 528 
CHAPTER XLI. 
Ameba 532 
* 
CHAPTER XLII. 
Sporozoa. Ciliata 543 
CHAPTER XLIII. 
The Malarial Organisms. Babesia 548 
CHAPTER XLIV. 
Smallpox (Variola) and Allied Diseases 565 
CHAPTER XLV. 
Rabies. Yellow Fever 572 
PART III. 
APPLIED MICROBIOLOGY. 
CHAPTER XLVI. 
The Bacteriologic Examination of Water, Air, and Soil. The Con- 
tamination and Purification of Water. The Disposal of Sewage 599 
CHAPTER XLVII. 
The Bacteriology of Milk in its Relation to Disease 611 
CHAPTER XLVIII. 
The Soil Bacteria and their Functions. Sewage Bacteria. Bacteria 
in Industries 629 
CHAPTER XLIX. 
The Destruction of Bacteria by Chemicals. Practical Use of Dis- 
infectants 637 
CHAPTER L. 
Practical Disinfection and Sterilization (House, Person, Instru- 
ments, and Food). Sterilization of Milk for Feeding Infants . 650 PATHOGENIC MICROORGANISMS. 
PART I. 
PRINCIPLES OF MICROBIOLOGY. 
CHAPTER I. 
INTRODUCTORY. HISTORICAL SKETCH 
Although most of the more important discoveries in microbiology 
which place it on the footing of a science are of comparatively recent 
date, the foundations of its study were laid over two centuries ago. 
From that time the history of microorganisms has been intimately 
associated with that of medicine. Indeed, it is only through the inves- 
tigations into the life history of these minute forms that our present 
knowledge of the etiology, course, and prevention of the infectious 
diseases has been acquired. The prominent position which the study 
of microorganisms already holds toward medicine is, moreover, daily 
increasing in importance. Original discoveries are constantly adding 
to our knowledge of germ diseases, and the outlook is favorable for 
eventually obtaining, through serums, through attenuated cultures, or 
through the toxic substances produced by microorganisms themselves, 
means for immunizing against, if not of curing, an increased number 
of the specific infections. Even at present, bacterial products and pro- 
tective serums are used successfully as preventive or curative agents 
in several of the most prevalent infectious diseases. Our knowledge 
concerning other microorganisms has enabled us largely to limit their 
dissemination and so to prevent disease. An acquaintance, therefore, 
with the main facts concerning these microorganisms is most necessary 
to the education of the modern physician. 
The vast majority of the known microorganisms which cause disease 
belong to the closely related groups of lowest plants and animals, 
i. e., the bacteria, the molds, the yeasts, and the protozoa. A few of 
the pathogenic metazoa in some of their stages are also microscopic 
(some of the parasitic worms); therefore, microscopic methods of study 
are also applicable to them; but since they are fully presented in works 
on clinical microscopy they are not given here. 
2 18 
PRINCIPLES OF MICROBIOLOGY 
Before entering into a detailed consideration of the subject it 
may be interesting and instructive to review very briefly a few of the 
important steps which led to the development of the science, and upon 
which its foundation rests, in which we shall see that the results obtained 
were gained only through long and laborious research and after many 
obstacles were met and overcome by accurate observation and experi- 
ment. 
Probably the first authentic observations of living microscopic 
organisms of which there is any record are those of Kircher, in 1659. 
This original investigator demonstrated the presence in putrid meat, 
milk, vinegar, cheese, etc., of "minute living worms," but did not 
describe their form or character. 
Not long after this, in 1675, Leeuwenhoeck observed in rain-water 
putrid infusions, and in his own and other saliva and diarrheal evacu- 
ations living, motile "animalcula" of most minute dimensions, which 
he described and illustrated by drawings. Leeuwenhoeck practised 
the art of lens-grinding, in which he eventually became so proficient 
that he perfected a lens superior to any magnifying glass obtainable 
at that day, and with which he was enabled to see objects very much 
smaller than had ever been seen before. "With the greatest astonish- 
ment," he writes, "I observed distributed everywhere through the 
material which I was examining animalcules of the most microscopic 
size, which moved themselves about very energetically." The work 
of this observer is conspicuous for its purely objective character and 
absence of speculation; and his descriptions and illustrations are done 
with remarkable clearness and accuracy, considering the imperfect 
optical instruments at his command. It was not until many years 
later, however, that any attempt was made to define the characters 
of these minute organisms and to classify them systematically. 
At that time all of the microscopic organisms seen were classed together 
as little animals. Indeed, all of the microorganisms first described at 
any length were probably protozoa, and only after further improvement 
of lenses and a more minute study of the organisms were bacterial forms 
gradually recognized as a separate class. 
The same skepticism that is seen in the acceptance of most new dis- 
coveries was displayed by doubters of the truth of these early reports 
of microscopic findings. Chief among the skeptics must be placed 
Linnaeus, who in the first edition of his Systema Naturae (1735) absolutely 
denies the existence of Leeuwenhoeck's animalcula, though in the later 
editions he grudgingly admits them under the significant generic name 
of Chaos {Chaos proteus {Ameba), etc.). 
The first ideas of the structure of the protozoa were drawn from 
analogy. The early observers thought that each tiny organism possessed 
an internal structure made up of organs and tissues similar to those 
in metazoa. They could not conceive of motion without articulation, 
tendons, and muscles; nor of food absorption without an alimentary 
tract, and they were so impressed with the ideas of what they thought 
they ought to see that they were convinced that they really saw many INTRODUCTORY 
19 
of the complicated structures possessed by metazoa. For example 
the contractile vacuole, a characteristic pulsating vesicle of the protozoa, 
discovered by Joblot in 1754, was thought by many to be lungs, other 
vacuoles were said to be stomachs, the mouths were often seen and the 
rest of the alimentary tract was supplied from the imagination; the red 
pigment spots of many forms were interpreted as true eyes, etc. There 
were many opponents to these view's, however, and the idea of the cell 
being the unit of structure, which was advanced by Schleiden in 1838, 
helped determine the fact that protozoa were single cells with no definite 
organic structure. 
With the publications of Dujardin (1835-41) a correct idea of the struc- 
tural simplicity of the microorganisms gained ground. But for some 
time after, the controversy regarding the simple nature of protozoa was 
strenuously carried on. It is a most instructive bit of history in research 
work, showing how the lack of minute observation, the exercise of a too 
vivid imagination, and the close reasoning from analogy may lead one 
astray, while the proper use of these functions may bring out the truth. 
Kolliker, Biitschli, Engelmann, and Hertwig, with many others, 
(1870-80) finally demonstrated fully the unicellular nature of the 
protozoa. 
The most important characteristic of a protozoon, its life history, 
was first partially made out by Trembley in 1744-47. Biitschli helped 
determine the sexual activities of the members of this group, while 
Maupas (1889) was the first to demonstrate the conditions leading to 
their conjugation. 
From the earliest investigations into the life history and properties 
of germs, microorganisms have been thought to play an important 
part in the causation of infectious diseases. Shortly after the first 
investigations into this subject the opinion was advanced that puer- 
peral fever, measles, smallpox, typhus, pleurisy, epilepsy, gout, and 
many other diseases were due to contagion. In fact, so widespread 
became the belief in a causal relation of these minute organisms to 
disease that it soon amounted to a veritable craze, and all forms and 
kinds of diseases were said to be produced in this way upon no other 
foundation than that these organisms had been found in the mouth 
and intestinal contents of men and animals and in water. 
Among those who were specially conspicuous at this time for their 
advanced views on the germ-theory of infectious diseases was Marcus 
Antonius Plenciz, a physician of Vienna. This acute observer, who 
published his views in 1762, maintained that not only were all infec- 
tious diseases caused by microorganisms, but that the infective mate- 
rial could be nothing else than a living organism. On these grounds 
he endeavored to explain the variations in the period of incubation of 
the different infectious diseases. He also insisted that there were 
special germs for each infectious disease by which the specific disease 
was produced. Plenciz believed, moreover, that these organisms were 
capable of multiplication in the body, and suggested the possibility 
of their being conveyed from place to place through the air. 20 
PRINCIPLES OF MICROBIOLOGY 
These views, it is true, were largely speculative, and rested upon 
insufficient experiment, but they were so plausible, and the arguments 
put forward in their support were so logical and convincing, that they 
continued to gain ground, in spite of considerable opposition and 
ridicule, and in many instances the conclusions reached have since 
been proved to be correct. The mode of infection, its unlimited devel- 
opment among large numbers of individuals, and gradual spread over 
wide areas-the incubation, course of, and resulting immunity in 
recovery from infectious diseases-all pointed to a living organism 
as the probable cause. 
Among other distinguished men of the day whose observations 
exerted a most powerful influence upon the doctrine of infection, may 
be mentioned Henle. His writings (Pathological Investigations, 1840 
and Text-book of Rational Pathology, 1853), in which he described 
the relation of microorganisms to infectious diseases, and defined the 
character and action of bacteria upon certain phases and symptoms 
of these affections, are remarkable for their clearness and precision. 
Origin of Microorganisms.-But, meanwhile, the question which most 
interested these investigators into the cause of infectious diseases was: 
Whence are these microorganisms derived which were supposed to 
produce them? Were they the result of spontaneous generation due 
to vegetative changes in the substances in which the organisms were 
found, or were they reproduced from similar preexisting organisms- 
the so-called vitalistic theory? This question is intimately connected 
with the investigations into the origin and nature of fermentation and 
putrefaction. 
Spallanzani in 1769 demonstrated that if putrescible infusions of 
organic matter were placed in hermetically sealed flasks and then 
boiled the liquids were sterilized; neither were living organisms found 
in the solutions, nor did the infusions decompose; they remained 
unchanged for an indefinite period. 
The objection was raised to these experiments that the high tem- 
perature to which the liquids had been subjected so altered them that 
spontaneous generation could no longer take place. Spallanzani met 
the objection by cracking one of the flasks and allowing air to enter, 
when living organisms and decomposition again appeared in the boiled 
infusions. 
Another objection raised by the believers in spontaneous generation 
was that, in excluding the oxygen of the air by hermetically sealing 
the flasks, the essential condition for the development of fermentation, 
which required free admission of this gas, was interfered with. This 
objection was then met by Schulze, in 1836, by causing the air admitted 
to the boiled decomposable liquids to pass through strong sulphuric 
acid. Air thus robbed of its living organisms did not produce 
decomposition. 
Schwann in 1839 obtained similar results in another way: he deprived 
of microorganisms the air admitted to his boiled liquids by passing 
it through a tube which was heated to a temperature high enough ORIGIN OF MICROORGANISMS 
21 
to destroy germs. To this investigator is also due the credit of having 
discovered the specific cause-the yeast plant, or Saccharomyces cerevisioe 
-of alcoholic fermentation, the process by which sugar is decomposed 
into alcohol and carbonic acid. 
Again it was objected to these experiments that the heating of the 
air had perhaps brought about some chemical change which hindered 
the production of fermentation. Schroeder and von Dusch in 1854 
then showed that by a simple process of filtration, which has since 
proved of inestimable value in bacteriologic work, the air can be 
mechanically freed from germs. By placing in the mouth of the flask 
containing the boiled solutions a loose plug of cotton, through which 
the air could freely circulate, it was found that all suspended micro- 
organisms could be excluded, and that air passed through such a filter, 
whether hot or cold, did not cause fermentation of boiled infusions. 
Similar results were obtained by Hoffmann in 1860, and by Chevreul 
and Pasteur in 1861, without a cotton filter, by drawing out the neck 
of the flask to a fine tube and turning it downward, leaving the mouth 
open. In this case the force of gravity prevents the suspended bacteria 
from ascending, as there is no current of air to carry them upward 
through the tube into the flask containing the boiled infusion. 
These facts have since been practically confirmed on a large scale 
in the preservation of food by the process of sterilization. Indeed, 
there is scarcely any biologic problem which has been so satisfactorily 
solved or in which such uniform results have been obtained; but all 
through the experiments of the earlier investigators irregularities were 
constantly appearing. Although in the large majority of cases it was 
found possible to keep boiled organic liquids sterile in flasks to which 
the oxygen of the air had free access, the question of spontaneous 
generation still remained unsettled, inasmuch as occasionally, even 
under the most careful precautions, decomposition did occur in such 
boiled liquids. 
This fact was explained by Pasteur in 1860 by experiments showing 
that the temperature of boiling water was not sufficient to destroy 
all living organisms, and that, especially in alkaline liquids, a higher 
temperature was required to insure sterilization. He showed, how- 
ever, that at a temperature of 110° to 112° C., which he obtained by 
boiling under a pressure of one and one-half atmospheres, all living 
organisms were invariably killed. 
Pasteur at a later date (1865) demonstrated the fact that the organ- 
isms which resist boiling temperature are, in fact, reproductive bodies, 
which are now known as spores. 
In 1876 the development of spores was carefully investigated and 
explained by Ferdinand Cohn. He, and a little later Koch, showed 
that certain rod-shaped organisms possess the power of passing into a 
resting or spore stage, and when in this stage they are much less sus- 
ceptible to the injurious action of higher temperatures than in their 
normal vegetative condition. 
When and how life began no one is yet able to say. That spontaneous 22 
PRINCIPLES OF MICROBIOLOGY 
generation may even be taking place now under unknown conditions 
is conceivable, but all such ideas are purely hypothetical and there is 
no evidence that under present conditions any of the knpwn micro- 
organisms have originated in any way except from a previous similar cell. 
Stimulated by the establishment of the fact, thrpugh Pasteur's 
investigations, that fermentation and putrefaction were due to the 
action of living organisms reproduced from similar preexisting forms, 
and that each form of fermentation was due to a special microorganism, 
the study of the causal relation of microorganisms to disease was taken 
up with renewed vigor. Reference has already been made to the opinions 
and hypotheses of the earlier observers as to the microbic origin of 
infectious diseases. The first positive grounds, however, for this doc- 
trine, founded upon actual experiment, were the investigations into the 
cause of certain infectious diseases in insects and plants. Thus, Bassi 
in 1837 demonstrated that a fatal infectious malady of the silkworm 
-pebrine-was due to a parasitic protozodn. Pasteur later devoted 
several years' study to an exhaustive investigation into the same sub- 
ject; and in like manner Tulasse and Kiihne showed that certain specific 
affections in grains, in the potato, etc., were due to the invasion of 
parasites. 
Very soon after this it was demonstrated that microorganisms were 
probably the cause of certain infectious diseases in man and the higher 
animals. Davaine, a famous French physician, has the honor of having 
first demonstrated the causal relation of a microorganism to a specific 
infectious disease in man and animals. The anthrax bacillus was dis- 
covered in the blood of animals dying from this disease by Pollender 
in 1849 and by Davaine in 1850; but it was not until 1863 that the last- 
named observer demonstrated by inoculation experiments that the 
bacillus was the cause of anthrax. 
The next discoveries made were those relating to wounds and the 
infections to which they are liable. Rindfleisch in 1866 and Waldeyer 
and von Recklinghausen in 1871 were the first to draw attention to the 
minute organisms occurring in the pyemic processes resulting from in- 
fected wounds, and occasionally following typhoid fever. Further 
investigations were made by Billroth, Fehleisen, and others, in erysipe- 
latous inflammations secondary to injury, who agreed that in these 
conditions microorganisms could almost always be detected in the lymph 
channels of the subcutaneous tissues. 
The brilliant results obtained by Lister in 1863-1870, in the antiseptic 
treatment of wounds to prevent or inhibit the action of infective 
organisms, exerted a powerful influence on the doctrine of bacterial 
infection, causing it to be recognized far and wide and gradually lessen- 
ing the number of its opponents. Lister's methods were suggested 
to him by Pasteur's investigations on putrefaction. 
In 1877 Weigert and Ehrlich recommended the use of the aniline 
dyes as staining agents and thus made possible a more exact micro- 
scopic examination of microorganisms in cover-glass preparations. 
In the year 1880 Pasteur published his discovery of the bacillus of ORIGIN OF MICROORGANISMS 
23 
fowl cholera and his investigations upon the attentuation of the virus 
of anthrax and of fowl cholera, and upon protective inoculation against 
these diseases. In the meantime he showed that pure cultures might 
be obtained by the dilution method. 
Laveran in the same year announced the discovery of parasitic bodies, 
the first pathogenic protozoa described, in the blood of persons sick 
with malarial fever, and thus stimulated investigations upon the 
immensely important unicellular animal parasites. 
In 1881 Koch published his fundamental researches upon pathogenic 
bacteria. He introduced solid culture media (agar had already been 
used by Frau Hess) and the "plate method" for obtaining pure cultures, 
and showed how different organisms could be isolated, cultivated 
independently, and, by inoculation of pure cultures into susceptible 
animals, could be made, in many cases, to reproduce the specific disease 
of which they were the cause. To him more than to any other are due 
the methods which have enabled us to prove absolutely, in a broad 
sense, the permanence of bacterial varieties. It was in the course of this 
work that the Abbe system of substage condensing apparatus was first 
used in bacteriology. 
In 1882 Pasteur published his first communication upon rabies. 
The method of treatment devised by him is still in general use. A 
little later came the investigations of Loftier and Roux upon the diph- 
theria bacillus and its toxins, and that of Kitasato upon tetanus. These 
researches paved the way for Behring's work on diphtheria antitoxin, 
which in its turn stimulated investigation upon the whole subject of 
immunity. The number of investigators rapidly- increased as the 
importance of the earlier fundamental discoveries became apparent. 
Additions to the science of microbiology have been made from many 
sides, and the practical application of the facts learned from these 
investigations are steadily increasing. The most important of these 
are given in the following pages. CHAPTER II. 
CLASSIFICATION AND GENERAL CHARACTERISTICS OF 
MICROORGANISMS. 
In general the lowest forms of life are microscopic. Among these 
microscopic organisms are many which have in common the ability 
to produce disease in the higher animals and plants. Some of these 
disease-producing ' or pathogenic microorganisms are plants, others 
are animals, while some are difficult to classify. Those only that are 
of direct importance to man are considered in this work. 
CLASSIFICATION OF MICROORGANISMS. 
The classification of microorganisms is still in the transition stage. 
This is due chiefly to the difficulties encountered in studying the indi- 
vidual morphologic characteristics of such minute bodies and in deter- 
mining their limits of variation. 
There is no one distinctive characteristic known which separates 
the lowest plants from the lowest animals. While the lowest micro- 
organisms of all-the bacteria-are usually classed as plants, their struc- 
ture is so simple and their biologic characteristics are so varied that 
their relationship to the vegetable kingdom is not clear-cut. In their 
possession of more or less rigid plasmolysible bodies, in the tendency 
of many to grow in filaments, and in the ability of some to use simple 
elements as food, they resemble plants; while in the motility of many, 
the non-possession by all of chlorophyl, and in the necessity of many 
for complex food, they resemble animals. 
There is a similar difficulty in definitely classifying many members of the 
other groups of closely related microorganisms, namely, the protozoa, the 
yeasts, and the molds, and it has been suggested that under the name Protista 
a third kingdom be formed consisting of all of these lowest microorganisms. 
As a rule, in the more minute organisms genera are based upon mor- 
phologic characteristics, and species upon biochemic, physiologic, or 
pathogenic properties. This is due to the facts that the morphology 
of varieties may vary extremely under different conditions and that 
morphology may give no indication whatever of the relation of micro- 
organisms to disease and fermentation-the chief characteristics which 
give them their importance to human beings. 
The properties of bacteria, for example, which are fairly constant 
under uniform conditions and which have been more or less used in 
systems of classification, are those of spore and capsule formation, 
motility (flagella formation), reaction to staining reagents; relation to 
temperature, to oxygen, and to other food material, and, finally, their CLASSIFICATION OF MICROORGANISMS 
25 
relation to fermentation and disease. But any one of these properties 
under certain conditions may so vary that, taking it as a basis for classi- 
fication, an organism could be dropped from the group with which it 
had been classified and be placed in an entirely different group. Thus, 
the power to produce spores or flagella may be held in abeyance for a 
time or, in the case of the former, be totally lost; the relations to oxygen 
may be gradually altered, so that an anaerobic bacterium grows in the 
presence of oxygen; parasitic organisms may be so cultivated as to 
become saprophytic varieties, and those which have no power to grow 
in the living body may acquire pathogenic properties. 
The possibility of making any thoroughly satisfactory classification 
is rendered still more difficult by the fact that many necessarily imper- 
fect attempts have already been made, so that there is a great deal 
of confusion, which is steadily increased as new varieties are found 
or old ones reinvestigated and classified differently in the different 
systems. 
Until a more universally accepted classification can be determined 
it seems wiser to note only such a broad, simple grouping as the one 
given below. 
Nomenclature.-It is well to call attention to the fact that in naming 
bacterial species, especially among the bacteria, the binomial law of 
nomenclature has been frequently violated. Such names as Bacillus 
coli communis should not be accepted; the name Bacillus coli is sufficient 
as well as correct. 
Working Classification of Pathogenic Microorganisms in Outline. 
Classes in which patho- 
genic species occur. 
Genera in which chief patho- 
genic species occur. 
Kingdom. 
Subkingdom. 
Molds 
(Hyphomy cetes) 
Mycomycetes 
Phycomycetes 
Unclassified (Fungi 
Imperfecti) 
Aspergillus, Penicillium. 
Mucor. 
Microspora, Trichophyta, Sporo- 
tricha, Achoria. 
Plants 
(fungi) 
Yeasts 
(Blastomycetes) 
Oidea 
Saccharomycetes 
Cocci (Coccacese) 
Oidium. 
Saccharomyces. 
Micrococcus, Diplococcus, Strep- 
tococcus, Tetracoccus. 
Bacilli (Bacteriacese) 
Spirilla (Spirillace®) 
Higher bacteria (Tricho- 
bacteria) 
Bacillus (Bacterium). 
Spirillum [Spirocheta,Treponema]. 
Cladothrix, Nocardia (Strepto- 
/ thrix). 
Bacteria 
(Schizomycetes) 
Flagellates 
(Mastigophora) 
Flagellata 
Cercomonas,Leptomonas, Herpet- 
omonas, Trypanosoma, Leish- 
mania, Trichomonas, Lamblia 
[Spirocheta, Treponema]. 
Entameba. 
Amebae 
(Sarcodina) 
Sporozoa 
Rhizopoda 
Animals 
(Proto- 
zoa) 
Telosporidia, 
Neosporidia 
Eimeria, Hemogregarinida, Pro- 
teosoma, Hemameba, Babesia, 
Rhinosporidium, Myxobolus, 
Nosema, Sarcocystis, etc. 
Balantidium. 
Ciliates 
Ciliata 
Ultramicroscopic organisms. 26 
PRINCIPLES OF MICROBIOLOGY 
Permanence of Species.-When we come to study special varieties 
or groups of microbes, such as the bacilli which produce typhoid fever, 
diphtheria, or tuberculosis, it is of great importance for us to deter- 
mine, if possible, to what extent the peculiar characteristics which each 
of these groups of microbes possess are permanent in the generations 
which develop from them. 
We cannot believe that the multitude of varieties which now exist 
have always existed. The probability is very strong that with succeeding 
generations and changing conditions new varieties have developed 
with new characteristics. 
From time to time the changing conditions under which life pro- 
gresses probably expose certain animals to the invasion of varieties 
which never before have gained access to them. If the microorganisms 
find some means of transmission to other animals equally susceptible, a 
parasitic species becomes established which at first, perhaps, finds 
conditions only occasionally favorable to it. Thus in some such way 
a multitude of groups have arisen, some of which accustom themselves 
to the conditions present in living plants, others to those in fishes, 
others to those in birds, and others still to those in man and the higher 
animals. 
These are, however, theories. What has been actually observed in 
the few years during which bacteria have been studied? In this short 
time the pathogenic species as observed in disease have remained 
practically unaltered. The diphtheria bacilli are the same today as 
when Loffler discovered them in 1884, and the disease itself is evidently 
the same as history shows it to have been before the time of Christ. 
The same permanence of disease type is true for tuberculosis, smallpox, 
hydrophobia, leprosy, etc. Under practically unchanged conditions, 
therefore, such as exist in the bodies of men, germs which have once 
become established as parasites continue to reproduce new generations 
which retain their peculiar (specific) characteristics. It is true that 
among the countless organisms developed some fail to hold the parasitic 
characteristics. These either continue as saprophytes or cease to exist. 
Whether new disease varieties are coming into existence from time to 
time is, of course, a possibility, but not a certainty. 
The fact that the chief pathogenic varieties of microorganisms which 
excite disease in man seem to have retained for centuries their charac- 
teristics, in no way proves that when placed under different conditions 
they would remain stable. As will be shown, certain characteristics 
of some bacteria can be radically altered by changed conditions, such 
as being grown outside the natural host, either in the test-tube or in an 
unaccustomed host. When these new surroundings are unfavorable, 
the organisms, while retaining their morphology, may lose their power 
of developing and producing specific poisons in the original host. Such 
attenuation may also occur in certain organisms when retained for a 
long time in an apparently immune host, as is seen in the streptococci 
and pneumococci of the throat or in the colon bacilli of the intestines. 
The recovery of poison production is often brought about by developing MOLDS 
27 
the microorganism for a considerable length of time under the condi- 
tions best suited for it. The recovery of the ability to grow in the body 
of any animal species is brought about by causing the germ to develop 
in a series of animals of the same species whose resistance has been 
overcome by reducing their vitality through poisons, heat, cold, etc., or 
by giving enormous doses of microbes to produce the first infection. 
Another method is to accustom the microorganism to the animal's body 
by letting it remain surrounded by the animal fluids, but protected 
from phagocytes in a pervious capsule in the peritoneal cavity or by 
growing it in unheated fresh serum or blood media. 
Certain groups of microorganisms seem to undergo variations more 
readily than others. The coli group and the streptococcus group are 
notable examples. 
The above examples of variations may be classed under those known 
as fluctuating variations. True mutations or discontinuous variations 
among bacteria have been from tune to time recorded, but whether or 
not the changed characteristics may be considered species characteristics 
cannot at present be decided. 
GENERAL CHARACTERISTICS OF EACH GROUP OF MICRO- 
ORGANISMS. 
Introduction.-The knowledge gained from the fact that each variety 
of microbe possible of cultivation may grow in distinctive ways upon 
so-called artificial culture media has been an immense aid in studying 
the characteristics of the more minute forms, for the individual cell of 
most varieties is so minute that even the highest magnification we have 
may show little if any morphologic difference between organisms which 
produce distinctly different diseases, or between a pathogenic and a 
non-pathogenic form. There are, however, certain morphologic and 
biologic characteristics of the single cell which are pronounced, and we 
therefore study these before going on to the study of cultures-that is, 
of microorganisms in masses. 
Morphologic descriptions should always be accompanied by a definite 
statement of the habitat-if from a culture, age of the growth, the 
medium from which it was obtained, and the temperature at which 
it was developed. 
MOLDS (HYPHOMYCETES, EUMYCETES). 
The molds (see Plate I) are closely related to the higher bacteria. 
Like them they grow in branching filaments or threads forming a 
mycelium, but these filaments or hyphae, as they are called, are more 
definite than those of the trichobacteria, and they are multicellular; 
that is, the filaments are septate, in some varieties always (myco- 
mycetes) and in others when forming spores (phycornycetes). These 
two groups of molds have other differential characteristics, particularly 
in their fruiting organs. 28 
PRINCIPLES OF MICROBIOLOGY 
The mycomycetes are divided into two groups: (1) Ascomyeetes, 
which form a spore sac, containing a definite number of spores, a mul- 
tiple of two, usually eight (ascospores); (2) Basidiomycetes, which form 
a definite spore-bearing cell called the basidium or conidiphore, from 
which four (usually) conidia or sterigmata protrude (mushrooms, etc.). 
The sexual fruiting organs are varied. 
Yeasts produce a definite sporangium containing a definite number 
of spores (two or a multiple of two), therefore they are closely related 
to the ascomyeetes. 
The phycomycetes are characterized by the formation of no definite 
basidium or ascus. The asexual fruiting organs are formed in three 
different ways: 
1. Ends of hyphse swell and are shut off by septum, forming sporangium 
in which (a) swarm spores develop. These become free by rupture of 
sporangium wall, swim about, and then form a new plant; (6) spores 
form a cell wall while in sporangium. 
2. Spores produced directly by constriction at end of certain hyphse. 
Such spores are called conidia and the hyphse conidophores; each 
conidium is able to develop into a new plant. 
3. Spores produced by separation of hyphse into short-walled seg- 
ments or oidia. When the spores are thick walled they are called 
chlamydophores. 
Sexual reproduction occurs in one of two ways: 
1. The ends of two similar hyphse become attached, then they seg- 
ment from the rest of the hyphse and become surrounded by a thick 
wall. The partition in the centre dissolves and the protoplasm mixes. 
The resulting body is called a zygospore. It produces a new plant. 
The members of this group are called zygomycetes. 
2. The swollen ends of two dissimilar hyphse, one large (female) 
and one smaller (male), become attached, fuse, segment, and become 
surrounded by a thick wall. The body is called an oospore. The plants 
are called oomycetes. 
Fungi Imperjecti.-The molds that are not definitely classified are 
divided into two groups: 
1. Known forms which do not fit in the above group. 
2. Not fully known forms. To this group belong most of the molds 
causing human disease. 
EXPLANATION OF PLATE I. 
Partly schematic. Rearranged and drawn by Williams from the indicated authors. 
Fig. 1.-Aspergillus glaucus. Fruiting hyphse growing from mycelium: A, conidiophore; 
B, sterygma; C, conidia; D, beginning perithecium; E, conidiophore bearing spores; 
F, perithecium containing rudiments in section; G, ascus containing eight spores (De Bary). 
Fig. 2.-Penicillium, showing formation of conidia, A. 
Fig. 3.-Mucor mucedo: A, sporangium containing spores; B, spores liberated; C, 
lamydospores; D, E, F, stages in the formation of a zygospore. 
Fig. 4.--Oidium lactis. 
Fig. 5.-Sporotrichum schenki, showing formation of whorled spores on branched 
mycelium. 
Fig. 6.-Yeast from human infection in culture showing mycelium-like growth. 
Fig. 7.-Saccharomyces cerevisise (Hansen). A, budding; B, spore formation. PLATE I 
A. W. WILLIAMS, DEL. YEASTS 
29 
YEASTS (BLASTOMYCETES). 
These microorganisms have been for many centuries of the greatest 
importance in brewing and baking. They are not uncommonly present 
in the air and in cultures made from the throat. .Certain recent 
experiments have shown that some varieties when injected are capable 
of producing tumor-like growths. Certain varieties are pathogenic for 
mice, and in recent years (since 1894) there have been reported a 
number of cases of human infection from yeasts. 
The position which the yeasts occupy in systematic biology has not, 
thus far, been accurately determined. In fact, it is still a question as to 
whether they constitute distinct fungi or whether they should be classed 
under the molds. 
The chief characteristic of the yeasts is their peculiar method of 
reproduction, which in most cases is by means of budding. For this 
reason these organisms go by the name of blastolnycetes in contrast 
to the fission fungi, or schizomycetes, and the thread fungi, or hypho- 
mycetes. The fact was mentioned above that some yeasts form asco- 
spores and that they are therefore related to the ascomycetes. The 
soar fungus, which at one time grows to long threads, at another time 
(under certain conditions almost exclusively) multiplies by budding, 
is also a transition form. It is thus seen that no hard-and-fast line exists 
between yeasts and molds. - 
The most important property of yeasts, though one not possessed 
by all to the same degree, is that of producing alcoholic fermentation. 
In brewing we distinguish between the yeasts that can be employed 
practically, "culture yeasts," and those which often act as disturbing 
factors, so-called "wild" yeasts. 
The shape of most of the culture yeasts is oval or elliptic (Plate I, 
Fig. 7). Round or globular forms are more often met with among the 
wild species which usually excite only a slight degree of fermentation. 
They are known as "torula" forms. But sausage-shaped and thread 
forms are also met with. Pathogenic forms may also be round. 
The individual yeast cells are strongly refractive, so that under 
the microscope at times they have almost the lustre of fat droplets. 
This is important because in examining fresh tissues the yeast cells 
may be hard to distinguish from fat droplets, often requiring the aid 
of certain reagents for their identification. 
The size of the individual yeast cells varies enormously, even in 
those of the same species or the same culture. In old colonies indi- 
viduals may be found no larger than cocci, 0.5/z to 2^ in diameter, 
while in other colonies, especially on the surface of a liquefied medium, 
giant yeast cells are found often attaining a diameter of 40^ or more. 
In spite of these wide fluctuations, however, the various species are 
characterized by a fairly definite average in size and form. Each cell 
contains a more or less definite nucleus, which is demonstrated by the 
usual chromogenic stains. 
During the process of budding the nucleus of the cell moves toward 30 
PRINCIPLES OF MICROBIOLOGY 
the margin, where it divides. At this point the limiting membrane of 
the cell ruptures or usually a hernia-like protrusion develops which has 
the appearance of a button attached to the cell. The daughter-cell so 
formed rapidly increases in size and gradually assumes the shape and 
size of the mother-cell. 
A fact of the utmost importance for the propagation of the blasto- 
mycetes and continuation of the species is the formation of spores 
(Plate I, Fig. 7). In this also the cell nucleus takes part, dividing into 
several fragments, each of which becomes the centre of a new cell lying 
within the original cell. These new cells possess a firm membrane, 
a cell nucleus, and a little dense protoplasm. The number of spores 
developed in the yeast cells varies, but is constant for a given species. 
As a rule, one cell does not produce more than four endogenous spores, 
but species have been observed-e. g., Schizosaccharomyces octosporus 
(Beijerinck)-in which eight spores are found. 
Guilliermond has described conjugation in yeasts before the formation 
of spores. 
The vitality of yeasts is truly enormous. Hansen as well as Lindner 
were able to obtain a growth from cultures twelve years old. Busse 
succeeded in getting a luxuriant growth from a dry potato culture seven 
and a half years old, which was almost as hard as bone. 
BACTERIA. 
Definition.-Bacteria may be defined as extremely minute, simple, 
unicellular microorganisms, which reproduce themselves with exceeding 
rapidity, usually by transverse division, and grow without the aid of 
chlorophyl. They have no morphologic nucleus, but contain nuclear 
material which is generally diffused throughout the cell body in the form 
of larger or smaller granules. 
Natural Habitat.-There are such wonderful differences in the con- 
ditions of life and nutrition which suit the different varieties, that bac- 
teria are found all over the known world. Wherever there is sufficient 
moisture, one form or another will find other conditions adequate for 
multiplication. Thus, we meet with bacterial life between 0° and 
75° C. Some live only in the tissues of men, others in lower animals, 
a larger number may grow in both man and lower animals, others still 
grow only in plants, but by far the greater number live in dead organic 
matter. For some free oxygen is necessary to life; for others it is a poison. 
Morphology (see Plates II and III).-The form and dimensions of 
bacterial cells at their stage of complete development must be distin- 
guished from those which they possess just after or just before they have 
divided. As a spherical cell develops preparatory to its division into two 
cells it becomes elongated and appears as a short oval rod; at the moment 
of its division, on the contrary, the transverse diameter of each of its 
two halves is greater than their long diameter. A short rod becomes 
in the same way, at the moment of its division, two cells, the long BACTERIA 
31 
diameter of each of which may be even a trifle less than its short 
diameter, and thus they appear on superficial examination as spheres. 
Size.-The dimensions of the adult individual vary greatly in the dif- 
ferent species as well as in members of the same species. The largest 
bacillus recorded is 50m to 60m1 long and 4g to 5g wide (B. butschlii, 
see Fig. 6). One of the smallest forms known (B. influenzae) is 
0.5g x 0.2g. 
Some pathogenic organisms (supposed to be bacteria) are so small 
(ultramicroscopic, see p. 70) as to be invisible with any magnification 
which we now possess. We know of their existence either by the fact 
that they may be cultivated on artificial media, producing appearances 
of mass growth, and that such cultures when inoculated into susceptible 
animals cause the characteristic disease, or by the fact that the filtrates 
alone are infectious. Some filtrates are infectious after passing through 
the pores of the finest filter (see Filtrable Viruses). 
Shape.-The basic forms of the single bacterial cells are threefold- 
the sphere, the rod, and the segment of a spiral. Although under 
different conditions the type form of any one species may vary con- 
siderably, yet these three main divisions under similar conditions are 
constant; and, so far as we know, it is never possible, by any means, to 
bring about changes in the organisms that will result in the permanent 
conversion of the morphology of the members of one group into that of 
another-that is, micrococci- always, under suitable conditions, pro- 
duce micrococci, bacilli produce bacilli, and spirilla produce spirilla. 
As bacteria multiply the cells produced from the parent cell have 
a greater or less tendency to remain attached. This is on account of 
the slimy envelope which is more or less developed in all bacteria. 
In some varieties this tendency is extremely slight, in others it is marked. 
This union may appear simply as an aggregation of separate bacteria 
or so close that the group appears as a. single cell. According to the 
method of the cell division and the tenacity with which the cells hold 
together, there are different groupings of bacteria, which aid us in 
their differentiation and identification. Thus, in cocci we get the 
bacterial cell dividing into one, two, or three planes (Plate II, Figs. 1-5), 
while in bacilli and spirilla the division is generally in only one plane 
(Plate II, Figs. 6-9). 
1. Spherical Form, or Coccus.-The size varies from about 0.3m 
as minimum diameter to 3 m as maximum; the average size of the patho- 
genic cocci is 0.8m- The single elements are, at the moment of their 
complete development, so far as we can determine, practically spherical; 
but when seen in the process of multiplication through division the 
form is seldom that of a true sphere. Here we have elongated or lancet- 
shaped forms, as frequently seen in the diplococcus of pneumonia, or 
the opposite, as in the diplococcus of gonorrhea, where the cocci appear 
to be flattened against one another. Those cells which divide in one 
direction only and remain attached are found in pairs (diplococci) 
1 A p, or micromillimeter, is 2'^0 of an inch. 32 
PRINCIPLES OF MICROBIOLOGY 
or in shorter or longer chains (streptococci). Those which divide in 
two directions, one at right angles to the other, form groups of four 
(tetrads). Those which divide in three directions and cling together 
form packets in cubes (sarcinse). Those which divide in any axis form 
irregularly shaped, grape-like bunches (staphylococci). 
2. Rod Form, or Bacillus.-The type of this group is the cylinder. 
The length of the fully developed cell is always greater than its breadth. 
The size of the cells of different varieties varies enormously: from a 
length of 30/x and a breadth of 4 id to a length of 0.2^ and a breadth of 
0.1 id. The largest bacilli met with in disease do not, however, usually 
develop over 3m x 1/d while the average is 2/x x 0.5/z. Bacilli are roughly 
classed, according to their form, as slender when the ratio of the long 
to the transverse diameter is from 1:4 to 1: 10, and as thick when the 
proportions of the long to the short diameter is approximately 1:2. 
The characteristic form of the bacillus has a straight axis, with 
uniform thickness throughout, and flat ends; but there are many excep- 
tions to this typical form. Thus frequently the motile bacteria have 
rounded ends, many of the more slender forms have the long axis, slightly 
bent; some few species, as for example the diphtheria bacilli, invariably 
produce many cells whose thickness is very unequal at different 
portions. Spore formation also causes an irregularity of the cell outline. 
The bacilli, except when they develop from spores or granules, divide 
only in the plane perpendicular to their long axis. A classification, 
therefore, of bacilli according to their manner of grouping is much 
simpler than in the case of the cocci. We may thus have bacilli as 
isolated cells, as pairs (diplobacilli), or as longer or shorter chains 
(streptobacilli). 
3. Spiral Form, or Spirillum.-The members of the third mor- 
phologic group are spiral in shape, or only segments of a spiral. Here, 
EXPLANATION OF PLATE II. 
Fig. 1.-Illustrating cocci single or in irregular groups (micrococcus, staphylococcus), 
micrococcus from air. X 1000. 
Fig. 2.-Illustrating cocci in twos-Diplococcus pneumonia from peritoneal exudate 
of rabbit. X 1000. 
Fig. 3.-Illustrating cocci in chains--Streptococcus pyogenes. X 1000. 
Fig. 4.-Illustrating cocci in fours-Micrococcus tetragenus from spleen of mouse. 
X 1000. 
Fig. 5.-Illustrating cocci in packets-Sarcina lutea from air. X 1000. 
Fig. 6.-Illustrating large single bacilli: B. subtilis (hay bacillus). X 1000. 
Fig. 7.-Illustrating small bacilli; mostly in twos-B. hoffmanni from human throat 
X 800. (Park.) 
Fig. 8.-Illustrating bacilli in chains-Anthrax bacillus from spleen of mouse. X 500. 
Fig. 9.-Illustrating bacilli in bunches-Typhoid bacillus from human spleen. X 500. 
Fig. 10.-Illustrating bacilli in threads-Anthrax bacilli from blood of frog. 
Fig. 11.-Spirillum undula, single coccus, showing flagellse. X 1000. 
Fig. 12.-Cholera spirilla:, gelatin culture. X 1000. 
Fig. 13.-Large spirillae in chains from water. X 1000. 
Fig. 14.-Smaller spirillse in chains-Spirillum rubrum. X 1000. 
Fig. 15.-Streptothrix candida-Broth culture (Zettnow, from Kolle and Wassermann). 
Fig. 16.-Streptothrix hominis from sputum (Zettnow, from Kolle and Wassermann). 
Unless otherwise indicated the photographs are from Frankel and Pfeiffer. PLATE II 
TYPES OF BACTERIA 
I SPHERE (COCCI OR COCCACEAE ) 
H CYLINDER ( BACILLI OR BACTERIACE/E ) 
UISPIRAL (SPIRILLA OR SPIRI LLACE/E ) 
rVHIGHER BACTERIA ( TRICHOBACTERIA ) 
A. W. WILLIAMS, DEL. BACTERIA 
33 
too, we have large and small, slender and thick spirals. The twisting 
of the long axis, which here lies in two planes, is the chief characteristic 
of this group of bacteria. Under normal conditions the twisting is 
uniform throughout the entire length of the cell. The spirilla, like the 
bacilli, divide only in one direction. A single cell, a pair, or the union 
of two or more elements may thus present the appearance of a short 
segment of a spiral or a comma-shaped form, an S-shaped form, or a 
complete spiral or corkscrew-like form. 
Structure of Bacterial Cells.-When examined living in a hanging 
drop (see p. 72) under the microscope bacteria appear usually as color- 
less refractive bodies with or without spores or other more highly refrac- 
tive areas. It is only by the use of stains that we are able to see more 
of their structure. 
Capsule.-Special staining methods (see p. 79) show that many 
bacteria (some investigators say all) under certain conditions possess a 
capsule (Plate III, Figs. 14-16), a gelatinous envelope which is supposed 
to be formed from the outer layer of the cell membrane. Some bacteria 
easily develop a much thicker capsule than others. Such forms are 
known as capsule bacteria. These generally produce a slimy growth 
on cultivation (e. g., B. mucosus). 
Capsules develop best in animal tissues. In cultures, with a few 
exceptions, they require for their development special albuminous 
culture media, such as milk, blood serum, bronchial mucus, etc. In 
ordinary nutrient media or on potatoes the capsule may be visible 
in the first culture generations when grown from the body, but usually 
it shows very indistinctly if at all. The capsule is distinguished by a 
diminished power of staining with ordinary aniline dyes, therefore, 
unlss special staining methods are used, the bacteria may appear to be 
lying in a clear unstained area. With certain dyes the inner portion 
of the capsule stains, giving the bacteria an apparent greater diameter. 
The demonstration of the capsule is often of help in differentiating 
between different but closely related bacteria; e. g., some forms of strep- 
tococcus and pneumococcus. 
Cell Membrane.-That all bacteria possess a cell membrane is shown 
(1) by special staining methods (e. g., flagella stains, see p. 80) and (2) 
by plasmolysis, demonstrated by placing the bacteria in a 1 per cent, 
solution of sodium chloride when the central portion (entoplasm?) con- 
tracts and separates in places from the membrane. In some bacteria 
the membrane is slightly developed, while in others (e. g., B. tuberculosis) 
it is well developed. It is different in composition from the membrane 
of higher plants in not possessing cellulose. In some forms, however, 
a similar carbohydrate, hemicellulose, has been demonstrated. In 
certain forms a substance related to chitin, found in the cyst walls 
of protozoa, has been found. Some observers consider the cell mem- 
brane merely a concentrated part of the cytoplasm, similar to the ecto- 
plasm of higher cells. That it is closely related to the living part of the 
cell is shown by the connection of the organs of locomotion (flagella) 
with it (Plate III, Figs. 17 and 21). 
3 34 
PRINCIPLES OF MICROBIOLOGY 
The Cell Substance.-The chief views in regard to the nature and the 
structure of the cell substance contained within the membrane may be 
summarized as follows: 
1. Bacteria have a definite morphologic, more or less centrally situated 
nucleus (Feinberg, Nakanischi, Schottelius, Swellengrebel, and others). 
2. Bacteria have no nucleus or differentiated nuclear material (Fischer, 
Migula, Massart, and others). 
3. The whole organism, except the membrane which is a delicate layer of 
cytoplasm, is a nucleus (Biitschli, Lbwit, Boni, and others). 
4. The nuclear material is in the form of distributed chromatin granules 
throughout the cytoplasm (Hertwig, Schaudinn, Guilliermond, Zettnow, and 
others). 
5. A variety of the fourth view is that bacteria possess both chief elements 
of a cell, namely, cytoplasm and karyoplasm, but that these are so finely 
mixed that they cannot be morphologically differentiated (Weigert, Mitro- 
phanow, Gotschlich). 
6. The latest view advanced, which is a variation of the views 3, 4, and 5, is 
that the bacterial cell is a relatively simple body-a cytode in Haeckel's sense, 
or the plasson of Van Beneden-which possesses both chromatin and plastin, 
the relative amounts of these chief substances of a cell corresponding more 
to the amounts found in the nuclei of higher cells than in their cytoplasm 
(Ruzicka, Ambroz). 
These last authors call attention to the fact that both nucleus and cytoplasm 
in the higher cells are composed of a mixture of chromatin and plastin and 
that the chief difference between the two mixtures is one of amount and not 
of kind. 
EXPLANATION OF PLATE III. 
Partly schematic (Williams). Stained by Giemsa except where otherwise indicated. 
Figs. 1 to 4.-Anthrax bacilli at different stages of development. 
Fig. 1.-Shows chromatic staining masses (red), in plastin-staining cells (light blue). 
Fig. 2.-Shows same organism after one hour on fresh medium, chromatic substance 
distributed throughout medium and whole organism taking deeper, more homogeneous 
stain. 
Fig. 3.-Organism showing two kinds of granules according to staining powers. 
Fig. 4.-Same organism placed on fresh medium extrudes some granules (waste-prod- 
ucts) and redissolves others (chromatin granules). 
Figs. 5 to 9.--Similar granules in diphtheria bacilli at various staes of development. 
Fig. 10..-Metachromatic granules in B. pyocyaneus, Neisser stain. 
Fig. 11.-Metachromatic granules in Sarcinse, Neisser stain. 
Fig. 12.-Metachromatic granules in B. influenzae. 
Fig. 13.-Metachromatic granules in gonococcae. 
Fig. 14.-Pneumococcus, capsule stained by Hiss method. 
Fig. 15.-Pneumococcus mucosus (Streptococcus mucosus) by Welch stain. 
Fig. 16.-Rhinoscleroma bacillus by Hiss stain for capsules. 
Fig. 17.-Plasmolysis in cholera bacillus showing capsule. 
Figs. 18 to 21.-Types of flagella by Loffler stain. 
Fig. 18.-Monotricha, cholera spirillum. 
Fig. 19-Amphitricha, water bacillus. 
Fig. 20.-Lophotricha, spirillum undula. 
Fig. 21.-Peritricha, typhoid bacillus. 
Fig. 22.-Formation and end germination of spores in anthrax. 
Fig. 23.-Lateral germination of spore in B. subtilis. 
Fig. 24.-Central germination of spores in B. alvei (Wilson). 
Fig. 25.-Type of spores: A, central; B, eccentric; C, end. 
Fig. 26.-Diphtheria bacilli in old cultures. 
Fig. 27.-Plague bacilli in old cultures. 
Fig. 28.-Influenza bacilli in old cultures. PLATE III 
STRUCTURE OF BACTERIA 
NUCLEAR MATERIAL AND CELL GRANULES 
CAPSULES AND MEMBRANES 
FLAGELLA 
SPORES 
IRREGULAR FORMS IN OLD CULTURES (INVOLUTION FORMS I 
A. W. WILLIAMS, DEL. BACTERIA 
35 
Our own studies of the structure of bacteria lead us to agree with 
the views expressed in Nos. 4 and 6 of the above summary-that is, 
bacteria possess both chief elements of a cell, namely, chromatin and 
plastin, and according to the stage of growth and division (varying 
with species) the chromatin may be in the form of morphologic granules 
or may be so finely divided and mixed with the plastin as to be indis- 
tinguishable from it. (See Plate III, Figs. 1, 2,5, 6, 7.) 
Metachromatic Granules (Plate III, Figs. 1-13).-These granules appear 
in unstained bacteria as light-refracting, in stained preparations as deeply 
stained areas. They have a great affinity for dyes, and so stain readily 
and give up the stain with some difficulty. With complex stains they 
show a greater affinity than the rest of the bacillus for certain con- 
stituents of the stain-e. g., with poly chromic methylene blue they 
take up more of the azur, thus appearing red and indicating at the 
same time their nuclear nature. In certain bacteria, such as the diph- 
theria bacilli, they are especially well marked in young, vigorous 
cultures. Here they have diagnostic value. At least some of these 
granules are nuclear in character. 
Besides the metachromatic granules there are certain other granules 
which take up stains readily and others still which absorb stains with 
difficulty; some of these granules are of the nature of starch and some 
of fat or other food products. Certain saprophytic forms have sulphur, 
others iron granules. 
Organs of Motility.-The outer surface of spherical bacteria is almost 
always smooth and devoid of appendages; but that of the rods and 
spirals is frequently provided with fine, hair-like appendages, or flagella, 
which are organs of motility (Plate III, Figs. 18-21). These flagella, 
either singly or in tufts, are sometimes distributed over the entire body 
of the cell, or they may only appear at one or both ends of the rod. 
The polar flagella appear On the bacteria shortly before division. The 
flagella are believed to be formed from the outer cell layer (ectoplasm) 
or possibly from the capsule, though they have been described by 
certain authors as arising in endoplasmic granules. So far as we know, 
the flagella are the only means of locomotion possessed by the bacteria. 
They are not readily stained, special staining agents being required for 
this purpose (see p. 80). The envelope of the bacteria, which usually 
remains unstained with the ordinary dyes, then becomes colored and 
more distinctly visible than is commonly the case. Occasionally, how- 
ever, some portion of the envelope remains unstained, when the flagella 
present the appearance of being detached from the body of the bacteria 
by a narrow zone. In stained cultures of richly flagellated bacteria 
peculiar pleated masses sometimes are observed, consisting of flagella 
which have been detached and then matted together. Bacteria may 
lose their power of producing flagella for a series of generations. Whether 
this power be permanently lost or not we do not know. 
Bacteria are named according to the number and position of the 
flagella they possess as follows: Monotricha (a single flagellum at 
one pole; e. g., cholera spirillum); Amphitricha (a flagellum at each 36 
PRINCIPLES OF MICROBIOLOGY 
pole; e. g., many spirilla); lophotricha (a tuft of flagella at one pole;1 
e. g., Spirillum undulans'); peritricha (flagella projecting from all parts of 
surface; e. g., B. alvei, B. typhosus, and others) (Plate III, Figs. 18-21). 
Fig. 1 
Fig. 2 
Bacilli showing one polar flagellum. 
Bacilli showing multiple flagella. 
So far, in only a few bacteria (the largest spirilla) have flagella been demon- 
strated during life, and then only under special conditions (see K. Reichert 
for bibliography). We have, however, an organism belonging to the B. alvei 
group, which shows its flagella very distinctly during life when a small portion 
of the viscid growth in a liquefying Lbffler blood-serum tube is transferred 
to a hanging mass of agar (p. 73) and examined under high magnification. 
The flagella on this organism may also be seen with dark-field illumination. 
Reichert claims that all motile bacteria show their flagella by this method.1 
Physiologic Characteristics of Bacteria.-The essential physiologic 
activities of bacteria are: motility, growth, reproduction, and spore 
formation. 
Motility.-Many bacteria when examined under the microscope 
are seen to exhibit active movements in fluids. The movements are 
of a varying character, being described as rotary, undulatory, sinuous, 
etc. At one time they may be slow and sluggish, at another so rapid 
that any detailed observation is impossible. Some bacteria are very 
active in their movements, different individuals progressing rapidly 
in different directions, while with many it is difficult to say positively 
whether there is any actual motility or whether the organism shows 
only molecular movements-so-called "Brownian" movements, or 
pedesis-a dancing, trembling motion possessed by all finely divided 
organic particles. In order to decide definitely with regard to the 
motility of any bacterial preparation, it is well to make two hanging 
drops. To one, 5 per cent, of formalin is added, which of course kills 
the organism. If, now, the live culture shows motility, which is not 
shown by the killed culture, it is an indication that one is dealing 
with a motile culture. Very young cultures, of but three to four hours' 
1 Some investigators consider that every flagellum is essentially a tuft, composed of 
many small fibrils. BACTERIA 
37 
development, in neutral nutrient bouillon should be examined at a 
temperature suitable for their best growth. Not all species of bacteria 
which have flagella exhibit at all times spontaneous movements; such 
movements may be absent in certain culture media and at too low 
or too high temperatures, or with an insufficient or excessive supply 
of oxygen; hence one should examine cultures under various conditions 
before deciding as to the non-motility of any organism. 
The highest speed of which an organism is capable has been approxi- 
mately estimated with some forms, and the actual figures show an 
actual slow rate of movement, though, comparatively, when the size 
of the organism is considered, the movement is rapid. Thus, the 
cholera spirillum may travel for a short time at the rate of 18 cen- 
timeters per hour. 
Movement is influenced by many factors, such as chemicals (the 
oxygen in the air especially), heat, light, and electricity. The tactile 
property which enables microorganisms to take cognizance of various 
forces is known as taxis; when forces attract, the phenomenon is known 
as positive taxis and when they repel it is called negative taxis. Chemo- 
taxis, or the effect of chemicals, is taken up in detail on page 58. 
Growth and Reproduction.-Under favorable conditions bacteria 
grow rapidly to a certain size, more or less constant for each species, 
and then divide by fission into approximately equal halves. The average 
time required for this cycle is twenty to thirty minutes. Probably 
in all species the nuclear material divides first. This is certainly the 
case in the group to which the B. diphtheria belongs, where division 
of the nuclear granules may be observed in the living organism before 
the characteristic snapping of the cell body and where division into 
equal halves seldom occurs. 
Fig. 3 
Successive stages in division of B, diphtheria showing relation of line of division to 
metachromatic granule. Continuous observation of living bacillus drawn without camera 
lucida. (Williams.) 
According to our observations on the living cell of members of this group, 
division takes place at a point occupied by a metachromatic granule (Fig. 3). 
Before division of the cell body the metachromatic granule, which appears 
to contain nuclear substance, elongates and shows a darker line at or near 
its centre. This seems to divide and form two lines, each of which has at a 
point near the surface a very tiny, refractive granule, staining deeply with 
chromatin stains. Between these two lines the cell body suddenly divides 
with a snap, like the opening of a jackknife, division beginning at the point 
between the two tiny granules, and the two new cells remain for a variable 
time attached at opposite points, thus giving the V-shaped forms. Kurth 
and Hill also called attention to division by snapping in members of the 
diphtheria-bacillus group, though neither recognized the relation between the 38 
PRINCIPLES OF MICROBIOLOGY 
position of the metachromatic granules and the point of division. The tiny 
granules are probably similar to the cell-partition granules described by 
various observers. 
It is not often that the favorable conditions mentioned above for 
the production of equal and rapid division obtain for any time, since 
even in pure cultures bacteria in their growth soon produce an environ- 
ment unfavorable for further multiplication. Several factors help 
to make this environment: First, the using up of suitable food and 
moisture; second, the disintegration of food substances into various 
injurious products, such as acids, alkalies, ferments; third, in mixed 
cultures the overgrowth of one or more varieties. As these unfavorable 
conditions are more or less constantly present, we seldom see such 
absolute symmetry in the growth and division of bacteria as is usually 
described. In fact, except under ideally favorable conditions (e. g., 
rapid successive transfers from young cultures on the most favorable 
food medium), we can never see absolutely equal fission among bacteria; 
and in some species, notably the diphtheria group, division is extremely 
irregular even in our usual twenty-four cultures on favorable media. 
Involution and Degeneration Forms.-It follows, from the conditions 
considered above, that, as cultures grow older or when media unfavor- 
able to equal division are used, the bacteria may show extremely irregular 
forms, absolutely different from the young forms, such as long threads 
or filaments with irregular thickenings, coccus forms from bacilli and 
spirilla which have divided without increasing in length, bacillar forms 
from cocci which have grown without dividing, and apparently branched 
forms from many varieties of bacilli and spirilla. These have been 
called involution or degeneration forms. 
In our study of the so-called branched forms of the diphtheria bacillus we 
have observed the following interesting fact: Under certain conditions, 
marked apparent branching appears at a definite time in the age of the culture. 
The conditions are: slightly disturbed growth in pellicle on nutrient broth. 
When ' such pellicles are examined every day they are found to contain, on 
the sixth to the twelfth day, varying chiefly with the amount of disturbance, 
many large intensely staining forms with one to several apparent branches 
and many large metachromatic granules (Figs. 4 and 5). The facts that 
these forms were the only ones to show active growth and division when 
examined on a hanging mass of agar and that in such growth the metachro- 
matic granules seem to fuse (Fig. 5) before fission led us to suppose that these 
forms represent a primitive sexual process, a sort of autogamy. Schaudinn 
(Fig. 6) has shown a primitive conjugation (autogamy) and a relationship 
between the chromatin granules, or nuclear substance, and the spores in certain 
bacteria. 
Although elongation in the greater diameter and complete division 
at right angles to this is the rule for the majority of bacteria, there are 
certain groups which, instead of becoming separated from each other 
as single cells, tend to produce an incomplete segmentation, the cells 
remaining together in masses, as the sarcinse, for example, which divide 
more or less regularly in three directions. The indentations upon these 
masses or cubes, which indicate the point of incomplete fission, give to BACTERIA 
39 
these bundles of cells the appearance commonly ascribed to them- 
that of a bale of rags. The rod-shaped bacteria never divide 
longitudinally. 
Fig. 4 
Fig. 5 
B. diphtheriae "No. 8" from 9 days' 
broth pellicle, showing many "branched" 
forms. Stained with carbol-fuchsin. X 
1500 diameters. 
B. diphtheria} "No. 8" from 10 days' 
broth pellicle, showing longitudinal fusion 
and position of metachromatic granules. 
Stained with Loffler's methylene blue. 
X 2000 diameters. 
Fig. 6 
Bacillus butschlii: a to c, incomplete division of the cell; d to f, gradual collection of 
chromatin granules at ends of cells; g to i, formation of end spores from these chromatin 
end masses. (After Schaudinn.) 
Spore formation must be distinguished from vegetative reproduction. 
This is the process by which the organisms are enabled to enter a stage 
in which they resist deleterious influences to a much higher degree 
than is possible for them to do in the growing or vegetative condition. 
It is true that in all non-spore-bearing cultures a certain proportion of 
the bacteria are more resistant than the average. No marked differ- 
ence in protoplasm, however, has been noted in them other than the 
ability to stain more intensely and sometimes to show strong meta- 40 
PRINCIPLES OF MICROBIOLOGY 
chromatic areas. The difference between these and the less resistant 
forms is not great. Some have believed that this resistance is due to 
certain bodies called arthrospores, which are abnormally large cells with, 
usually, a thickened cell wall and increased staining properties, formed 
as a rule in old cultures. Fullerton and others have described similar 
forms in some of the higher bacteria and consider them spores. (See 
Nocardia.) 
The true spores (endospores) of the lower bacteria are definite 
bodies. These are strongly refractile and glistening in appearance, 
oval or round in shape, and composed of concentrated protoplasm 
developed within the cell and surrounded by a very dense envelope 
(Plate III, Figs. 22-25). They are characterized by their power of 
resisting the injurious influences of heat, desiccation, and chemical 
disinfectants up to a certain limit. (See page 79 for details.) Spores 
also stain with great difficulty (see chapter on Disinfection). 
The production of endospores in the different species of bacteria, 
though not identical in every instance, is very similar. The conditions 
under which they are produced in nature are supposed to be similar to 
those observed in artificial cultures, but they may not always be similar, 
hence we must not consider a bacterium a non-spore bearer because 
it has not been seen to form spores in the laboratory. Usually the for- 
mation of spores in any species is best observed in a streak culture 
on nutrient agar or potato, which should be kept at the temperature 
nearest the optimum for the growth of the organism to be examined. 
At the end of twelve, eighteen, twenty-four, thirty, thirty-six hours, 
etc., specimens of the culture are observed, first unstained in a hang- 
ing drop or on an agar mass, and then, if round or oval, highly refractile 
bodies are seen, stained for spores. Each bacillus, as a rule, produces 
but one spore, and more than two have never been observed. 
Motile bacteria usually come to a state of rest or immobility previous 
to spore formation. Several species first become elongated. The anthrax 
bacillus does this, and a descriptuon of the method of its production of 
spores may serve as an illustration of the process in other bacteria (Plate 
III, Fig. 22, A-E). In the beginning, the protoplasm of the elongated 
filaments is homogeneous, but after a time it becomes turbid and finely 
granular. These fine granules are then replaced by a smaller number 
of coarser granules, the so-called sporogenous granules supposed to 
be chiefly nuclear in nature, which by coalescence finally amalgamate 
into a spherical or oval refractive body. This is the spore. As soon as 
the process is completed there may appear between each two spores a 
delicate partition wall. For a time the spores are retained in a linear 
position by the cell membranes of the bacilli, but these are later dissolved 
or broken up and the spores are set free. Not all the cells that make 
the effort to form spores, as shown by the spherical bodies contained 
in them, bring these to maturity; indeed, many varieties, under certain 
cultural conditions, lose altogether their property of forming spores. 
The following are the most important spore types: (a) the spore lying 
in the centre of the cell; which may be much distended in its central BACTERIA 
41 
portion, giving it a spindle shape or clostridium, e. g., Bacillus butyricus; 
(6) the spore lying at the extremity of a cell much enlarged at that 
end-the so-called "head spore" or plectridium, e. g., the tetanus 
bacillus; (c) the spore lying eccentrically (Plate III, Fig. 25, A-C)- 
According to Schaudinn and others, in certain spore-bearing bacteria the 
spore formation is part of a sexual-like process (see under Reproduction). 
The germination of spores takes place as follows (Plate III, Figs. 22-24): 
By the absorption of water they become swollen and pale in color, 
losing their shining, refractive appearance. Later, a little protuberance 
is seen upon one side (equatorial germination) or at one extremity of the 
spore (polar germination), and this rapidly grows out to form a rod 
which consists of soft-growing protoplasm enveloped in a membrane, 
which is formed of the endosporium or inner layer of the cellular envelope 
of the spore. The outer envelope, or exosporium, is cast off and may 
be seen in the vicinity of the newly formed rod; or it may be absorbed. 
The chief spore formers among the pathogenic bacteria are the 
anaerobes (tetanus, malignant edema, intestinal bacteria). Only one 
distinctly pathogenic aerobe produces endospores-the anthrax bacillus. 
The Higher Forms of Bacteria (see end of Section II).-Some forms 
grow out into true or false branching threads and thus make a group 
of organisms intermediate between bacteria and the molds. These have 
been called higher bacteria or trichobacteria (see table, p. 25). They 
show increased complexity of structure and function: (1) in forming 
irregularly segmented filaments composed of elements similar to those 
found in the lower forms and showing either true or false branching; 
(2) in developing certain portions of their substance into reproductive 
bodies from which the new individuals grow (Plate II, Figs. 15 and 16). 
The filaments seen sometimes among the lower forms have inde- 
pendent segments, which may easily separate and grow as tiny unicel- 
lular forms, while in the higher forms the filaments in their growth 
show a certain interdependence of their parts. For example, growth 
often occurs from only one end of the filament while the other becomes 
attached to some fixed object. 
The members of the higher bacteria which are pathogenic for man 
have as yet been incompletely studied and classified. The following 
divisions serve as an attempt at differentiation: 
1. Leptothrix grows in stiff, almost straight threads, in which division 
processes are seldom or never observed, and no branching has been seen. 
2. Cladothrix grows in threads which rapidly fragment and produce 
false branching, that is, the terminal cell remains partly attached, but 
is pushed to one side by further growth from the parent thread; thus 
a Y-shaped growth is produced, and then bacillary characteristics in old 
cultures. 
3. Actinomyces grows in threads with true branching. Spores have 
been observed. It is characterized by the radiating wreath-like forms 
which it alone produces in the living body. 42 
PRINCIPLES OF MICROBIOLOGY 
4. Nocardla (Streptothrix) grows in threads which produce abundant 
true branching; later there is fragmentation and formation of conidia. 
Reproduction among the Higher Bacteria.-These forms increase in 
length for a time'and then, at the free ends, or at intervals along the 
filaments, they produce small rounded cells, called conidia or spores 
from which new individuals are formed. The terminal spores may be 
flagellated after their separation from the parent filament. 
The flagellated forms frequently resemble certain flagellata among 
the protozoa. 
PROTOZOA. 
Definition.-A protozoon (the lowest form of animal life) is a mor- 
phologically single-celled organism composed of protoplasm which is 
differentiated into cytoplasm and nucleus (or nuclear substance) both 
of which show many variations throughout the more or less complicated 
life cycle that each individual undergoes. 
They are of higher grade than the bacteria because of their greater 
complexity in structure and life cycle (Plate IV). 
Their shape and size vary so widely that no general description will 
fit all types. Some forms are small enough to pass through a Berkefeld 
filter, while the largest varieties described are about two-thirds of an 
inch long. 
The cytoplasm consists of a mixture of substances, the most important 
of which belong to the proteids. It is more or less fluid, but, because 
of differences in the density and solubility of the several parts, it often 
presents an alveolar, linear, or granular appearance, which may come 
out clearly in fixed and stained specimens, but is usually not well seen 
in the living cells. 
Ectoplasm and Entoplasm.-Frequently the protozoon cytoplasm is 
differentiated into a concentrated, viscid, more homogeneous, or hyaline 
outer layer called the ectoplasm and a more fluid granular central portion 
called the entoplasm. These two portions have different functions. 
The ectoplasm helps form the various organs (organelles) of motion, 
contraction, and prehension such as pseudopods (false feet), flagella 
(whip-like threads), cilia (hair filaments), suctorial tubules (through 
which food passes), and myonemes (contractile fibrils found in ciliates, 
gregarines, and a few flagellates). The entoplasm digests the food 
and contains the nucleus, as well as various granules and vacuoles. 
Some vacuoles serve as food digesters, and hence contain digestive 
ferments The so-called contractile vacuoles which periodically fill 
and empty themselves may be considered as excretory organelles. 
Other substances are seen from time to time in the entoplasm, such 
as bacteria, red-blood cells, fatty granular pigments, bubbles of gas, 
crystals, etc. 
The Nucelus.-The simplest morphologic nucleus is a vesicular body 
which is differentiated from the cytoplasm by its essential constituent 
chromatin, so-called because it has a strong affinity for certain basic PROTOZOA 
43 
staining materials. Chromatin consists mostly of nuclein and appears in 
the form of smaller or larger granules, masses, or rods. 
Generally, the chromatin particles are mixed with a second less 
intensely staining substance with more of an affinity for acid stains, 
called plastin or paranuclein, similar to the substance from which 
the true nucleolus of the metazobn cell seems to be formed. This 
substance may appear in one or more distinct rounded bodies. Most 
of the chromatic substances of the nucleus in many protozoa are often 
massed together in an intensely staining ball-like body called the 
karyosome which undergoes various cyclic changes during the growth 
and development of the organism. The centrosome is generally 
imbedded in the karyosome. The chromatin and plastin lie imbedded 
in a third substance in the form of an achromatic network called linin, 
which is closely related to the cytoplasmic network. There may or 
may not be a definite nuclear membrane. Sometimes there is no defi- 
nitely structured nucleus, but the nuclear substance in the form of small 
chromatin masses or granules is distributed throughout the cytoplasm 
(the so-called "distributed nucleus") similar to that seen in bacteria. 
Somatic and Generative Chromatin.-Some chromatin substances of the cell 
have physiologic properties different from others. At times substances which 
have only vegetative properties are active, forming the so-called somatic or 
trophic chromatin; at other times, substances appear during sexual activities 
called generative or sexual or idiochromatin, and from these the vegetative 
(somatic) chromatin for the new cells is again formed. In the ciliata both 
these chromatin elements are present as distinct morphologic bodies during 
the entire life of the organism, the somatic form in the macronucleus and the 
generative form in the micronucleus. 
Chromidia.-The chromatin elements, in the form of granules, small irregu- 
lar masses, threads, network, etc., which at certain stages pass from the 
nucleus into the cytoplasm, or which at times are, possibly, formed in the 
cytoplasm, were named "Chromidien" by R. Hertwig, who in 1899 first de- 
scribed their appearance. Their function in generative processes was demon- 
strated in 1903 by Schaudinn. During their formation the nucleus may entirely 
disappear, so that morphologically the cell may be considered non-nuclear. 
At a definite time thereafter new typical nuclei may be formed from these 
chromidial substances. 
Locomotor Nucleus {Kinetic Nucleus').-In flagellates still another definite 
physiologic chromatin is seen in the small body called the kinetic nucleus, 
which is either apart from or merged into a smaller body, the blepharoplast, 
forming the root of the flagellum. The kinetic nucleus is so called because it 
produces the locomotor apparatus. Both the kinetic and trophic nuclei may 
contain somatic and generative chromatin at the same time. 
The Centrosome.-This is a small body which is always present in meta- 
zoan cells, playing an important part in cell division, but it has not been demon- 
strated as a morphologic entity in many varieties of protozoa; part of the 
karyosome, however, may take its place, or there may always be a true centro- 
some within the karyosome. Whenever a centrosome appears in protozoa, 
it has its origin in the nucleus, resembling in this the kinetic nucleus and 
blepharoplast. All these four bodies, therefore, centrosome, blepharoplast, 
kinetic nucleus, and karyosome, may be considered as having a similar 
morphologic origin. 
Physiologic Characteristics of Protozoa.-In common with all other 
living organisms protozoa possess the characteristics of motility, nutri- 
tion, respiration and reproduction. 44 
PRINCIPLES OF MICROBIOLOGY 
Motility.-All protozoa react in certain characteristic ways toward 
chemic, mechanic, and electric stimuli. Many are affected by light, 
while probably none react to sound. They manifest the reaction usually 
by motion of some sort. Most animal parasites, especially the higher 
forms, exert a positive taxis for leukocytes, principally for the large 
mononuclears and the eosinophiles. This fact is made use of in clinical 
diagnosis. 
Nutrition.-Many protozoa, especially the pathogenic forms, absorb 
fluid food directly through the body wall; but the majority take in 
solid food, such as small animal or vegetable organisms and organic 
waste, some through more or less definite regions of the body, others 
through any part of the surface by extending pseudopodia and entirely 
surrounding the food object, forming a so-called gastric vacuole. 
After the food is digested the waste products are excreted, sometimes 
by osmosis, generally through special structures as the contractile 
vacuoles which regularly eject fluid substances to the outside of the 
organism. 
Respiration.-It is supposed that the contractile vacuole has a respi- 
ratory as well as an excretory function. The interchange of gases is 
always going on, if not through a contractile vacuole, then by osmosis 
through any part of the wall. 
Growth and Reproduction.-Under favorable conditions, new protoplasm 
is constructed rapidly, and the mass increases faster than the surface 
which, according to Spencer, initiates cell division. The changes gen- 
erally appear first in the nucleus. The simplest variety of reproduction 
is a two-celled fission which may be either longitudinal or transverse, 
either of which may be direct (amitotic) or indirect (mitotic). A modifi- 
cation of equal fission is the so-called budding which may be single or 
multiple. When growth occurs so that fission is for a time incomplete, 
one cytoplasm containing several nuclei which finally separate into 
as many daughter organisms, the process is called multiplicative repro- 
duction, or brood formation or internal budding. In the most extreme 
cases of multiplicative reproduction as it occurs among sporozoa the 
mother-cell with its nucleus separates simultaneously into large numbers 
of tiny daughter-cells. Such a process, when it occurs without conjuga- 
tion and encystment, is called schizogony and the new cells are called 
merozoites. When such a multiplicative division occurs (generally 
after fertilization) within a cyst, it is spoken of as sporogeny and the 
new cells are called sporozoites. 
Sexual Phenomena.-Sexual phenomena (syngamy) fundamentally 
similar to those seen in metazoa have been observed in all groups of 
protozoa studied. The reproduction by the usual division or budding 
is interrupted at certain times in the life history of each organism and 
individuals come together in such a way that their nuclei fuse, usually 
after having undergone characteristic reduction divisions. 
When the union is permanent, we speak of it as copulation and liken the 
process to that of the fecundation of the ovum by a spermatozoon. When the 
union is transient we call it conjugation. Here the two cells fuse for a time PROTOZOA 
45 
when the nuclei interchange protoplasm and then the cells separate and each 
one continues to grow and divide independently. When in a partly divided 
cell or in an apparently single cell, two nuclei, after undergoing reduction 
division, or its like, fuse, the process is called autogamy. 
The Developmental Cycle of a protozoon consists of all the changes 
which occur in its growth from one act of fertilization to another (Plate 
IV, Fig. 3). Many protozoa carry on the sexual part of their life 
cycle in one host and the asexual part in another (e. g., malarial 
organisms). 
Cyst Formation.-If protozoa do not get the required amount 
of water or air or suitable food they cease their special movements, 
round out into more or less of a sphere and form a resisting membrane 
of chitin within which they may live for a long time, withstanding 
periods of desiccation, extreme heat and cold, and they may be blown 
about as dust until they find conditions again favorable for renewed 
growth; when water is absorbed the cyst is ruptured and active life 
begins anew. 
In parasitic forms encystment plays an important part in the passage from 
the old host to the new. The majority of forms would not be able to exist 
outside of the body of the host without having some protective membrane. 
The cyst may be formed simply for protection from drought, etc., when it is 
called a hypnocyst, from which the organism may emerge in about the same 
form as when it encysted; or the cyst may precede reproduction by spore 
formation or simple division, when it is called a sporocyst. In either case it 
may consist of a simple wall or it may be formed of several walls to enable it 
to resist prolonged desiccation, when it is called a resting cyst. 
Characteristics of Each of the Four Groups of Protozoa.-Flagellata. 
-Flagellata are protozoa which move in the adult forms by one to 
several flagella or whip-like processes. If pseudopodia develop, they 
are transitory. 
Generally the flagella arise from the anterior part of the organism, 
and in motion the larger ones (primary flagella) are directed forward, 
while smaller ones (secondary flagella) are directed backward, acting 
as rudders. Certain flagellata possess a modification of their bodies 
in what is called the undulating membrane, which consists of a fluted 
protoplasmic process attached along one side of the organism, the free 
edge of which is prolonged as the flagellum. It has been shown that 
flagella are not simple protoplasmic processes, but that they have more 
or less of a framework of elastic fibres as well, hence their power in 
locomotion can be better understood. Except with special stains, which 
bring out these fibres, they appear homogeneous. 
The flagella arise from some definite place in the cytoplasm, some- 
times from a distinctly differentiated chromatic body which has been 
given various names, such as blepharoplast, kinetic nucleus or centro- 
some, sometimes near this from a basal granule, microsome, diplosome, 
or flagellum root, sometimes directly from the nucleus. The basal 
granules seem to be derived primarily from the kinetonucleus, and 
may be considered from a physiologic stand-point, as a part of the 
motor nuclei. 46 
PRINCIPLES OF MICROBIOLOGY 
The body of the flagellates is generally more or less elongated and, 
except in most primitive ones, is fixed in its outline. The latter charac- 
teristic is chiefly due to the fact that the organisms usually possess 
definite though delicate membranes containing elastic fibrils. The 
cytoplasm is usually not differentiated into an ento- and ectoplasm. 
It often contains one to several contractile vacuoles, as well as food 
EXPLANATION OF PLATE IV. 
Partly schematic. Rearranged and drawn by Williams. All stained by Giemsa. 
I. Flagellates. 
Fig. 1.-Illustrating one flagellum. Leishmania: A, intracellular form ; B, cultural forms. 
Fig. 2.-Illustrating undulating membranes: A, Trypanosoma lewisi; B, Trypanosoma 
brucei; C, Trypanosoma gambiense. 
Fig. 3.-Illustrating two flagella. Bode lacertse (after Prowazek). 
Fig. 4.-Illustrating four flagella. Trichomonas. 
II. Amebte. 
.Fig. 1.-Illustrating points considered differential in the two chief types of amebic 
(entamebse) described as parasitic in human beings. 
Fig. 1.-Entameba coli, vegetative stage. 
Fig. 2.-Dividing nucleus. 
Fig. 3.-Entameba coli cyst containing eight nuclei. 
Fig. 4.-Entameba histolytica, vegetative stage. 
Fig. 5.-Four-nucleate cyst. 
III. Sporozoa. 
A, description of Figs. 1 to 17. (After Schaudinn.) The life cycle of Eimeria schubergi. 
In 1, the sporozoites, becoming free by bursting the sporocysts, pass out through 
an aperture in the wall of the oocyst, and are ready to enter the epithelial cells 
of the host. 2 to 6 represent the asexual reproduction or schizogony, commencing 
with infection of an epithelial cell by a merozoite or a sporozoite; the merozoite 
after stage 6 may start again (5) at stage 2, as indicated by the arrows, or it may 
go on to the formation of gametocytes (9 to 11). 9 to 11 represent the sexual 
generation, the line of development becoming split into two lines-male (d) and 
female (?)-culminating in the highly differentiated gametes, which conjugate 
and become again a single line, shown in 12-14. The zygote thus formed goes on 
the production of spores, 15 and 16. 2 and 3 represent epithelial cells showing 
penetration of a merozoite or a sporozoite and its change into a schizont; 
4, the nucleus of the schizont divided into numerous daughter-nuclei; 6, seg- 
mentation of the schizont into numerous merozoites, about a central mass of 
residual protoplasm, which in this figure is hidden by the merozoites; 5, mero- 
zoites passing to reinfect host cell and repeat the process of schizogony; 7, 8, 
merozoites to be differentiated into male and female gametocytes; 9, the two 
gametocytes within a host cell; the microgametocyte (d) has fine granulations; 
the macrogametocyte (?) has coarse granulations. 11, a female gametocyte 
undergoing maturation; 13, mature macrogamete, freed from the host cell, and 
sending a cone of reception toward an approaching microgamete. In 12 the nuclei 
of the last stage have become microgametes, each with two flagella. The free 
microgametes are swimming to find a macrogamete. 14, the zygote (fertilized 
macrogamete), surrounded by a tough membrahe or oocyst, which allows no more 
microgametes to enter, and containing the female chromatin, which is taking the 
form of a spindle, and the male chromatin in a compact lump. 15, the nucleus 
of the zygote divided-the nuclei of the sporoblasts. In 16 the four sporoblasts 
become distinct, leaving a small quantity of residual protoplasm; each sporoblast 
has formed a membrane, the sporocyst. Within each sporocyst two sporozoites 
form about a sporal residuum. 
B, Babesia infecting red-blood cells: 1, pear-shaped bodies; 2, dividing forms; 3, 
eight pear-shaped bodies in a cell; 4, irregular ring-like bodies; 5, large, irregular 
body; 6, body with a flagellum-like projection. 
IV. Ciliates. 
Fig. 1.-Balantidium coli: A, adult form (after Hartmann); B, C, dividing forms; 
D, conjugating forms. PLATE IV 
TYPES OF PROTOZOA 
I.FLAGELLATES-M0VIN6 BY FLAGELLA 
H. AMEB/E - MOVING BY PSEUDOPODS 
IISPOROZOA-INTRACELLULAR. PRODUCING MANY SPORES 
IV. CILIATES-MOVING BY CILIA 
A. W. WILLIAMS, DEL. PROTOZOA 
47 
vacuoles, and there is frequently a definite opening or cytostom for 
the entrance of food. There are usually many granules and inclusions 
of various kinds scattered throughout the cytoplasm,, and myoneme 
striations are seen in some forms. The nucleus, as a rule situated ante- 
riorly, varies much according to different species and to different stages 
of development. 
The flagellata multiply either in the free motile condition or after 
encystment. In the first case, as a general thing, they divide longi- 
tudinally. The basal granules divide with the nuclei and the flagella 
of the daughter organisms are usually formed anew. Multiple division 
is also observed. In the second case, they may or may not conjugate 
before they encyst. Then they divide within the cyst. 
The sexual cycle varies much in different species. Isogamy has been 
noticed between fully grown individuals as well as between smaller 
forms. The union of different-sized forms, or anisogamy, has also been 
observed. Also autogamy is said not to be infrequent. It is claimed 
that certain of the flagellates pathogenic in man require a second host 
for the development of their sexual cycle. 
The flagellates are subdivided into several orders, in only two of which 
are forms found which are pathogenic for man. (See Part II.) The 
different types of pathogenic flagellates are shown in Plate IV, I, Figs. 1-4. 
Among the most important pathogenic forms are the trypanosomes. 
Trypanosoma.-The name trypanosoma (boring animal) was given 
by Gruby, in 1843, to certain free-swimming hemoflagellates found by 
him in the blood of frogs. Much later similar flagellates were found 
in the blood-plasma of many different species of vertebrates and in 
the intestinal tract of several blood-sucking invertebrates. Some of 
the forms, including those found in man, are pathogenic. A number 
of the blood-sucking insects are carriers of the diseased species to 
healthy animals. 
Typical trypanosomes are characterized by a comparatively long, 
spirally twisted body, along one side of which is attached an undulating 
membrane having a cord-like edge that is continued forward as a free 
whip (flagellum). The flagellum arises near the posterior end of the 
organism in a small granule called the basal granule, which may be 
connected with the blepharoplast, a larger chromatin mass, called 
also the kinetonucleus because of its control over the motor apparatus. 
The nuclear apparatus consists of a macro- or trophonucleus, and of 
the above-mentioned kinetonucleus or blepharoplast, which last func- 
tions as a centrosome. The trophonucleus is usually situated near 
the middle of the organism; it is granular, thick, and egg-shaped, but 
varies somewhat in size and shape. The cytoplasm is faintly alveolar 
or granular, varying with age, environment, and possibly species 
Toward the straighter border of the cell the cytoplasm is more or less 
striated and in a few species definite myonemes are seen. 
Reproduction occurs usually by longitudinal, occasionally by multiple 
division. The life cycle is not well known. Though transmission 
occurs through the bites of various invertebrates, notably flies, the few 
sexual changes described as taking place in the intestines of some of 48 
PRINCIPLES OF MICROBIOLOGY 
these intermediate hosts have not been fully corroborated. That an 
intermediate host is not necessary for the continued life of at least one 
species of trypanosome seems to be proved by the fact of direct trans- 
mission of T. equiperdum from horse to horse through coitus. 
Leishmania.-Another important pathogenic flagellate is Leishmania. 
In humans this grows chiefly within large mononuclear cells. It shows 
its flagellated forms in cultures. (Plate IV, Fig. 1. See also Part II.) 
Amebida.-Under amebida (syn., amebee) we include forms composed 
of naked, simply constructed protoplasm having the power of producing 
lobose pseudopodia which are used as organs of motion and of nutrition. 
The pseudopodia are protoplasmic processes which are projected 
in irregular succession from different parts of the surface of the cell, 
producing in this way an irregular motion. The form of the pseudopodia 
varies considerably in the different species. For instance, there are 
broad, blunt processes or narrow, less blunted ones, and each may 
be short or long, single or slightly branched. The entoplasm may or 
may not take a share in their formation. The forms of course vary 
within limits according to the condition of the medium in which the 
amebse are living. Movements are always called forth by some physic 
or chemic excitant. When such an excitant is desirable for food the 
pseudopods flow around it, and it is subsequently absorbed in the cyto- 
plasm of the organism. 
The members of this group may possess one nucleus or several. 
Ameba binucleata has two nuclei in the young adult stage, and Pelomyxa 
palustris, living in the bottom ooze of ponds, has an enormous number 
of nuclei. A marked feature of the nuclear apparatus is the formation 
of chromidia which, as has already been noted, may play such an impor- 
tant part in sexual reproduction. Generally each ameba has one contrac- 
tile vacuole, but occasionally some are seen with several or with none. 
Saprophytic forms belonging to this order are common. They may 
be found wherever there are moisture and decaying vegetable matter. 
The pathogenic forms are not so frequent. Because of the possibility of 
the still unknown causes of certain diseases (see Rabies and Smallpox) 
being organisms related to this order, it is especially important to study 
both saprophytic and pathogenic varieties, since a knowledge of the 
former which are more easily studied may help us understand obscure 
points in the life history of the latter. 
Notwithstanding the common occurrence of saprophytic forms, the 
full life history of few of them has been worked out, and until the full 
cycle of development of any so-called ameba is known it is impossible 
to say whether that particular form belongs among rhizopoda or whether 
it is a developmental form of another group, as ameboid forms may 
occur at some time in the life history of all groups. It is quite possible 
that some of the organisms described as belonging to this order are 
really members of entirely different orders. For instance, it is known 
that the flagellate Trichomonas loses its flagella before copulation 
and crawls about by means of short blunt pseudopods as a typical 
ameba. PROTOZOA 
49 
Ameboe reproduce by simple fission, by budding, and by brood for- 
mation. In the last case the reproduction is usually preceded by encyst- 
ment. Two forms have been described most frequently as parasitic 
in man. The differences between them are shown in Plate IV, n, 
Figs. 1-5. 
The Sporozoa.-The sporozoa are a group of exclusively parasitic 
protozoa of very widespread occurrence, living in the cells, tissues, and 
cavities of animals of every class. Generally they are harmless, but 
some varieties may produce pathologic changes and even fatal diseases 
severely epidemic. 
As their name indicates, they are all characterized by reproduction 
through spore formation, but they exhibit the utmost diversity of 
structural and developmental characteristics. As a rule, each species 
is parasitic on one kind of tissue of a particular species of host. They 
are generally taken into the system in the spore stage either (1) with 
the food of the host, (2) by the bites of insects, or (3) by inhalation. 
The spore membranes are dissolved by the fluids of the host, and thus 
one or more germs or sporozoites are set free to bore into the special 
cells of the host. Here they grow, some remaining permanently intra- 
cellular, others only in the young stages. The latter either pass different 
phases of their more or less complicated life history in different parts 
of the body of one and the same host or they pass some phases of their 
life cycle in the cells of an intermediate host. 
The sporozoa vary widely in size as well as in other characteristics. 
From the smallest, several of which can be contained in a single blood- 
cell, there are all gradations in size up to those that may be seen by 
the naked eye (Porospora gigantea, 16 mm.). 
Besides being characterized by the power to produce more or less 
resisting spores, the sporozoa are also characterized by the fact that 
as a class they possess none of the special organs found in other pro- 
tozoa for ingesting or digesting solids. Many develop flagella during 
sexual phases or show ameboid movement during certain stages of 
their life cycle, but the flagella and pseudopodia are organs of loco- 
motion and not of nutrition. Food vacuoles or contractile vacuoles 
have not been found. 
The life cycle of a typical sporozoon is rearranged and condensed 
from Schaudinn in Plate IV, m, Figs. 1-16. 
Ciliata.-The ciliata (Plate IV) belong to the most complex of the 
protozoa. They possess a definite entoplasm containing nuclei and food 
vacuoles, and a definite ectoplasin containing basal granules from which 
arise the cilia which give the group its name. They have organoid 
structures which receive the food, some have definite mouth openings, 
indeed, and definite places for excreting waste products. The food 
vacuoles may contain acid or alkaline digestive products. The nuclear 
material is differentiated into two forms, a large macronucleus and a 
much smaller micronucleus. The function of the macronucleus is sup- 
posed to be vegetative, and that of the micronucleus reproductive. 
The macronucleus varies in size and shape and is completely filled with 
4 50 
PRINCIPLES OF MICROBIOLOGY 
an alveolar chromatin. The micronucleus also varies in size and shape, 
but unless in reproductive phases is generally vesicular in structure, 
with the chromatin heaped in one mass. Division of the nuclei takes 
place by mitosis in the case of the micronuclei, and by amitosis, as a rule, 
in the case of the macronuclei. Under conditions unfavorable for growth 
the ciliata may encyst. 
Conjugation is necessary to the life activity of these organisms. 
The phenomena of conjugation in the ciliata has been well worked out. 
The micronuclei play the most important part, whereas the macro- 
nuclei simply break up and disappear in the protoplasm. 
According to the arrangement of the cilia, the ciliata are divided 
into the four orders given in the general classification. Among these, 
the second, the order of the Heterotricha, interests us. In the Hetero- 
tricha the cilia are uniform over most of the body, while a specialized 
set fused into a series of firm vibratory plates is found about the 
mouth. Only one genus, Balantidium, has been observed in man 
(Plate IV; also Part II). 
CHEMICAL COMPOSITION OF MICROORGANISMS. 
Qualitatively considered, the bodies of microorganisms consist largely 
of water, salts (chiefly phosphorus, potassium, chlorine, calcium and 
sulphur), fats, and albuminous substances. There are also present, in 
smaller quantities, extractive substances soluble in alcohol and in 
ether. Special varieties contain unusual substances, as wax and hemi- 
cellulose in tubercle bacilli. Microorganisms possess the capacity in a 
high degree of accommodating their chemical composition to the variety 
of soil in which they are growing. Each variety, furthermore, yields 
proteid substances peculiar to itself, as shown in the effects produced 
by animal inoculation. At present we know but little concerning the 
differentiation of these specific substances. This subject will be taken 
up in detail under Toxins, etc. According to Cramer, many bacteria 
contain amyloid substances which give a blue reaction with iodine. 
True cellulose has not been found in bacteria, but large quantities of a 
gelatinous carbohydrate similar to hemicellulose have been obtained. 
Nuclein is found in all microorganisms. The nuclein bases-zanthin, 
guanin, and adenin-have been obtained in considerable amounts. 
There is a group of bacteria which contain large amounts of sulphur- 
viz., the Beggiatoa-and another group, the Cladothrix, is capable of 
separating ferric oxide from water containing iron. 
The percentage of water contained in microbes grown on solid culture 
media, as well as the amount of residue and ash, depends largely on the 
composition of the media. Thus, Bacillus prodigiosus when grown 
on potato contains 21.5 per cent, of dry residue and 2.7 per cent, of 
ash; when cultivated on turnips it contains 12.6 per cent, of dry residue 
and 1.3 per cent, of ash. Besides the concentration of the culture, its 
temperature and age also influence the amount of residue and ash 
produced. The residue varies, moreover, qualitatively in the same EFFECTS OF SURROUNDING FORCES UPON MICROORGANISMS 
51 
species under the influence of the culture media employed. Thus 
it appears that an additional quantity of peptone in the culture media 
tends to increase the percentage of nitrogenous matter in the bacillus, 
while the addition of glucose decreases it. 
The chemical composition of the bodies of animal parasites is an 
almost unexplored field. The ectoplasm and the cyst sacs in general 
are made up principally of a substance called chitin. Glycogen has 
been isolated from many forms. Proteolytic enzymes and acid secre- 
tion in digestive vacuoles have been demonstrated. 
Microchemical Reactions.-To a certain degree the chemical com- 
position of the individual organism may be studied both in the living 
and in the dead individual by the addition of the testing substances 
to a hanging drop or to a spread of such organism and the examination 
of it under the microscope. 
Of special importance in this regard is the resistance which bacteria 
possess to diluted alkalies. Inasmuch as the majority of animal tissues 
are dissolved when treated with alkalies, this method has been adopted 
for rendering visible unstained bacteria in tissues. (See also Principles 
of Staining, p. 76). 
EFFECTS OF SURROUNDING FORCES UPON MICROORGANISMS. 
1. Food.-Naturally, the effect of food upon organisms is marked. 
Though the majority of pathogenic microorganisms grow easily on 
certain artificial foods (culture media), some of them, like most of the 
protozoa we have not yet been able to cultivate outside of the body 
of their host. Those microorganisms which seem to depend entirely 
upon a living host for their existence are known as strict parasites; those 
which live only upon dead organic (a few on inorganic) substances are 
called strict saprophytes; those which can lead a saprophytic existence, 
but which usually thrive only within the body of a living animal, are 
called facultative parasites. The strict saprophytes, which represent the 
large majority of all microorganisms are not only harmless to living 
organisms, but perform many exceedingly important functions in nature, 
such as the destruction of dead organic matter and its preparation for 
plant food through decomposition, putrefaction, and fermentation, 
while one group (see below, the Nitrifying Bacteria) are constructive 
in their activities. The parasites, on the contrary, may be harmful 
invaders of the body tissues, exciting by their growth and products 
many forms of disease. The substances essential for the majority of 
those forms which can be grown artificially are organic material, as a 
source of carbon and nitrogen, an abundance of water and certain salts. 
Either calcium or magnesium and sodium or potassium salts are usually 
required, also sulphur and phosphorus salts. Iron is demanded by a 
few varieties. The demands of microorganisms for food of a certain 
composition vary considerably. (See chapter on Cultivation of Micro- 
organisms.) 52 
PRINCIPLES OF MICROBIOLOGY 
While it is true that very wide differences in relative composition 
and total concentration of food media riiay have slight effect upon the 
general development of a given organism, slight changes in composition 
and reaction of the media often have a great effect upon morphology, 
rate of growth, motility, and specific products of growth. 
Reaction of Media.-The reaction of the media is of very great impor- 
tance. Most bacteria, particularly the pathogenic forms, grow best 
on those media that are slightly alkaline or neutral to litmus. Yeasts 
and molds grow best on a slightly acid medium. An amount of acid 
or alkali insufficient to prevent the development of bacteria may still 
suffice to rob them of some of their most important functions, such as 
the production of poison. The different effect upon closely allied varieties 
of bacteria of a slight excess of acid or alkali is sometimes made use of 
in separating those which may be closely allied in many other respects. 
Influence of One Species upon the Growth of Another.-When one 
species of organism is grown in a food medium, that medium usually 
becomes less suitable for the growth of its kind and of other organisms. 
This is due partly to the impoverishment of the foodstuffs, but more 
to the production of chemical substances or enzymes. When different 
species are grown together, the antagonistic action of one upon the 
other may be shown from the beginning. Some species, however, 
have a cooperative or symbiotic action with other species. 
In nature, microorganisms usually occur in mixed cultures (e. g., in 
water, milk, intestinal contents of all animals), and here we may see 
antagonistic action in the prevalence of one species over others (e. g., 
the lactic acid formers in the intestines), or cooperative action in the 
equal and luxuriant growth of two or more species (e. g., pneumococcus 
and influenza bacillus in the lungs). 
Experimentally, the existence of antagonisms can be demonstrated 
by inoculating alternate streak cultures of various bacteria on gelatin 
or agar media. It is found that many species will grow not at all or only 
sparingly when in close proximity to some other species. This antag- 
onism, however, is often only one-sided in character. Again, when 
gelatin or agar plates are planted with a mixture of two species of bac- 
teria it may be observed that only one of the two grows. A third method 
of making this experiment is simultaneously to inoculate the same liquid 
medium with two species, and then to examine them later, both micro- 
scopically and by making plate cultures; not infrequently one species 
may take precedence of the other which after a time it may entirely 
overcome. 
Finally, it may be shown experimentally that microorganisms may 
oppose one another as antagonists in the animal body. For instance, 
Emmerich has shown that animals infected with anthrax may often 
be cured by a secondary infection with the streptococcus. 
The symbiotic or cooperative action of microorganisms may be 
demonstrated experimentally in the following examples: 
(a) Pneumococci, when grown together with a bacillus obtained from 
the throat, produce very large, succulent colonies. The influenza EFFECTS OF SURROUNDING FORCES UPON MICROORGANISMS 
53 
bacillus, which will not grow alone upon ordinary nutrient agar, will 
grow well there in the presence of certain other bacteria. Some anae- 
robic species grow even with the admission of air if only some aerobic 
species are present (tetanus bacilli with diphtheria bacilli). 
(6) Certain chemical effects, as, for instance, the decomposition of 
nitrates, cannot be produced by many species of bacteria alone, but only 
when two are associated. 
2. Behavior toward Oxygen and other Gases.-The majority of micro- 
organisms absolutely require free oxygen for their growth, but a con- 
siderable minority fail to grow unless it is excluded. This latter fact, 
noted first by Pasteur, led him to divide germs into aerobic and anae- 
robic forms. Between these two groups we have those that can grow 
either with or without the presence of oxygen, called respectively 
facultative anaerobic and facultative aerobic organisms. 
(a) Aerobic Organisms.-Growth only in the presence of free oxygen. 
The slightest restriction of air inhibits development. Spore formation, 
especially, requires the free admission of air. 
(6) Anaerobic Organisms.-Growth and spore formation only on the 
total exclusion of free oxygen. Among this class of organisms are the 
bacillus of malignant edema, the tetanus bacillus, the bacillus of symp- 
tomatic anthrax, and many soil bacteria. Exposed to the action of 
oxygen, the vegetative forms of these bacteria are readily destroyed; 
the spores, on the contrary, are very resistant. Anaerobic germs being 
unable to use, deprived the air of oxygen-the chief source of energy 
used by the aerobic species to oxidize the nutritive substances in the 
culture media-are dependent for their oxygen upon decomposable 
substances, such as grape-sugar. 
(c) Facultative Aerobic and Facultative Anaerobic Organisms.-The 
greater number of aerobic germs, including most of the pathogenic 
species, are capable of withstanding, without being seriously affected, 
some restriction in the amount of oxygen admitted, and many, indeed, 
grow equally luxuriantly with the partial exclusion of oxygen. Life in 
the animal body, for example, as in the intestines, necessitates existence 
with diminished supply of oxygen. If in any given variety of bacteria, 
the amount of oxygen present is unfavorable, there will be more or 
less restriction in some of the life processes of this variety, such as 
pigment and toxin production, spore formation, etc. Pigment forma- 
tion almost always ceases with the exclusion of oxygen, but poisonous 
products of decomposition may be more abundantly produced. 
It has been observed not infrequently that certain species which 
on their isolation at first show more or less anaerobic development- 
that is, a preference to grow in the depth of an agar-stick culture, for 
instance-after a while seem to become strict aerobes, growing only 
on the surface of the medium. 
Those organisms that grow best or grow exclusively when the oxygen 
is only partly removed are called micro-aerophilic organisms. 
Other Gases.-While all facultative organisms as well as strict anae- 
robes grow well in nitrogen and hydrogen, they behave very differently 54 
PRINCIPLES OF MICROBIOLOGY 
toward carbonic acid gas. A large number of these species do not grow 
at all, being completely inhibited in their development until oxygen is 
again admitted-for example, B. anthracis and B. subtilis and other 
allied species. It has been found in some species, as glanders and cholera, 
that the majority of the organisms are quickly killed by CO2, while 
few, such as staphylococci, offer a great resistance, rendering impossible 
complete sterilization by means of this gas. The streptococcus as well 
as the staphylococcus exhibits a scanty growth. A mixture of one- 
fourth air to three-fourths carbonic acid gas seems to have no injurious 
effect on bacteria which cannot grow in an atmosphere of pure CO2. 
Under pressure CO2 is more effective (page 57). 
Sulphureted hydrogen in large quantity is a strong bacterial poison. 
Even in small amounts it kills some bacteria. 
3. Effect of Temperature.-Some form of microbic life is possible 
within the limits of 0° and 70° C. The maximum and minimum tem- 
perature for each individual species ordinarily lies from 10° to 30° C. 
apart, and the optimum covers about 5°. Usually the temperature 
of the soil in which the germs are deposited is the controlling factor 
in deciding whether growth will or will not take place. Thus, nearly 
all parasitic microorganisms require a temperature near that of the 
body of their host for their development, while many, saprophytic 
forms can grow only at much lower temperatures. Microbes when 
exposed to lower temperature than suffices for their growth, while 
having their activities decreased, may not be otherwise injured unless 
actually frozen for a certain time; while exposure to higher temperatures 
than allows of growth more or less quickly destroys the life of the organism. 
Microorganisms have been classified according to the temperatures 
at which they develop, as follows: 
Psychrophilic Microbes.-Minimum at 0° C., optimum at 15° to 20° 
C., maximum at about 30° C. To this class belong many of the water 
microorganisms, such as the phosphorescent bacteria in sea-water; and 
many molds and yeasts. 
Mesophilic Microbes.-Minimum at 5° to 25° C., optimum about 37° 
C., maximum at about 43° C. To this class belong all pathogenic 
bacteria, most parasitic and many saprophytic forms. 
Thermophilic Microbes.-Minimum at 25° to 45° C., optimum at 50° 
to 55° C., maximum at 60° to 70° C. This class includes a number 
of soil bacteria which are almost exclusively spore-bearing bacilli. 
They are also found widely distributed in feces. 
By carefully elevating or reducing the temperature the limits within 
which a species will grow can be altered. Thus, the anthrax bacillus 
may be gradually made to accommodate itself to a temperature of 42° C., 
and pigeons, which are comparatively immune to anthrax, partly on 
account of their high body temperature (42° C.), when inoculated with 
this anthrax succumb to the infection. Another culture accustomed 
to a temperature of 12° C. kills frogs kept at 12° C. We have culti- 
vated a very virulent diphtheria bacillus so that it will grow at 43° C. 
and produce strong toxin. EFFECTS OF SURROUNDING FORCES UPON MICROORGANISMS 
55 
Effect of Low Temperature.-Temperatures even far under 0° C. are 
only slowly injurious to microorganisms, different species being affected 
with varying rapidity. This has been demonstrated by numerous 
experiments in which they have been exposed for weeks in a refriger- 
ating mixture at -18° C. If a culture of typhoid bacilli is frozen, about 
50 to 70 per cent, of the organisms are killed at the time. At the end 
of one week not more than 10 per cent, survive, and at four weeks not 
over 1 per cent. After six months none survive. More resistant bac- 
teria live longer and spores may survive in ice for years. Bacteria 
have even been subjected to a temperature of -175° C. by immersing 
them in liquid air kept in an open tube for two hours, and 15 to 80 per 
cent, were found still to grow when placed in favorable conditions. 
We found about 10 per cent, of typhoid bacilli alive after thirty minutes' 
exposure to this low temperature. Staphylococci were more resistant. 
Spores were scarcely affected at all. 
Effect of High Temperatures.-Prolonged temperatures from 5° to 10° 
C. over the optimum affect microorganisms injuriously in several 
respects. For instance, varieties may be produced of diminished activity 
of growth, the virulence and the property of causing fermentation may 
be decreased, and the power of spore formation may be gradually lost. 
If the maximum temperature is exceeded, the organism dies. The 
thermal death point for the psychrophilic species is about 37° C., for 
the mesophilic species about 45° to 55° C., and for the thermophilic 
species about 75° C. There are no non-spore-bearing bacteria which 
when moist are able to withstand a temperature of 100° C. even for a 
few minutes. A long exposure to temperatures between 60° and 80° C. 
has the same result as a shorter one at the higher temperatures. Ten 
to thirty minutes' exposure to moist heat will at 60° C. kill the cholera 
spirillum, the streptococcus, the typhoid bacillus, and the gonococcus, 
and at 70° C. the staphylococcus, the latter being among the most 
resistant of the pathogenic organisms which show no spores. A much 
shorter exposure will kill a large percentage of any mass of these bacteria. 
Effect of Dry Heat.-When microorganisms in a desiccated condi- 
tion are exposed to the action of heated dry air the temperature required 
for their destruction is much above that required when they are in a 
moist condition or when they are exposed to the action of hot water 
or steam. A large number of pathogenic and non-pathogenic species 
are able occasionally to resist a temperature of over 100° C. dry heat 
for from ten minutes to one hour. In any large number of bacteria 
a few are always more resistant then the majority. A temperature of 
120° to 130° C. dry heat maintained for one and a half hours will destroy 
all bacteria, in the absence of spores. 
Resistance of Spores to Heat.-Spores possess a great power of resist- 
ance to both moist and dry heat. Dry heat is comparatively well- 
borne, many bacterial spores resisting a temperature of over 130° C. 
for as long as three hours. Exposed to 150° C. for one hour, practically 
all spores are killed. Moist heat at a temperature of 100° C., either 
boiling water or free-flowing steam, destroys the spores of most varieties 56 
PRINCIPLES OF MICROBIOLOGY 
of bacteria within fifteen minutes; certain pathogenic and non-patho- 
genic species, however, resist this temperature for hours. The spores 
of a bacillus from the soil required five and a half to six hours' exposure 
to streaming steam for their destruction. They were destroyed, however, 
by exposure for twenty-five minutes in steam at 113° to 116° C. and in 
two minutes at 127° C. The spores from tetanus bacilli may require 
longer than fifteen minutes' exposure to kill them. 
The resistance of spores to moist heat is tested by suspending threads, 
upon which the spores have been dried, in boiling water or steam. 
The threads are removed from minute to minute and laid upon agar 
or in broth and kept at a suitable temperature for the germination of 
any living spores. 
4. Influence of Light.-A large number of microorganisms are inhibited 
in growth by the action of bright daylight, more are affected by direct 
sunlight, and when the action of the sun's rays is prolonged they lose 
their power of developing when later placed in the dark. 
Some motile organisms move toward the point of greatest luminosity, 
others away from it. Light-seeking protozoa have green or yellow 
chromatophores, and usually at the anterior end a red pigment spot. 
The violet and blue rays are more active than other parts of the spectrum 
in determining motion. 
The susceptibility of bacteria to light may be tested, according to 
H. Buchner, by suspending a large number of bacteria in nutrient 
gelatin or agar and pouring the media, while still fluid in a Petri dish, 
upon which has been pasted a strip of black paper on the side exposed to 
the light. The action of heat may be excluded by allowing the ray of 
light to pass through a layer of water or alum of several centimeters' 
thickness. After the plate has been exposed to the light for one-half, 
one, one and a half, two hours, etc., it is taken into a dark room and 
allowed to stand at 20° or 35° C. a sufficient length of time to allow of 
growth, and then examined to see whether there are colonies anywhere 
except on the spot covered by the paper; when the colonies exposed 
to the light have been completely destroyed there is lying in a clear 
sterile field a sharply defined region of the shape of the paper strip 
crowded with colonies. 
Protected by ordinary non-colored glass the sun's rays act very 
slowly. 
Only the ultraviolet, violet, and blue rays of the spectrum seem to 
possess bactericidal action; green light has very much less; red and 
yellow light none at all. The action of light is apparently assisted by 
the admission of air; aneorbic species, like the tetanus bacillus, and 
facultative anaerobic species, such as the colon bacillus, are able to 
withstand quite well the action of intense, direct sunlight in the absence 
of oxygen, for four hours. 
According to Richardson and Dieudonne, the mechanism of the 
action of light may be at least partially explained by the fact that in 
agar plates exposed to light for a short time (even after ten minutes' 
exposure to direct sunlight) hydrogen peroxide (H2O2) is formed. EFFECTS OF SURROUNDING FORCES UPON MICROORGANISMS 
57 
This is demonstrated by exposing an agar plate half covered with 
black paper, upon which a weak solution of iodide of starch is poured, 
and over this again a dilute solution of sulphate of iron; the side exposed 
to the light turns blue-black. In gases containing no oxygen, hydrogen 
peroxide is not produced, and the light has no injurious effect. Access 
of oxygen also explains the effect which light produces on culture media 
which have been exposed to the action of sunlight, as standing in the 
sun for a time, when afterward used for inoculation. The bacteria 
subsequently introduced into such media grow badly-far worse than 
in fresh culture media which are kept in the shade. 
Influence of Radium.-Radio-active fluids have a slight inhibiting 
effect on microbal growth, but nothing decided enough to be used for 
therapeutic purposes has been evolved up to the present time. 
Influence of X-rays.-These rays have a slight inhibiting effect on 
microorganisms when they are directly exposed to them. 
5. Influence of Electricity.-The majority of the observations hereto- 
fore made on this subject would seem to indicate that there is no direct 
action of the galvanic current on bacteria; but the effect of heat and the 
electrolytic changes in the culture liquid resulting from the electrolysis 
may destroy them. 
Protozoa may be contracted by moderate induction shocks and killed 
by strong ones. 
When a current of electricity is passed through a liquid medium 
most active protozoa swim with their long diameters in the direction 
of the lines of force to assemble behind the cathode. Most flagellates 
and a few ciliates, however, move toward the anode. The direction 
of motion has been shown by Dale to vary with the nature and concen- 
tration of the medium. This whole question has been little studied. 
6. Influence of Agitation.-Meltzer has shown that the vitality of 
bacteria is destroyed by protracted and violent shaking, which causes 
a disintegration of the cells. Many species are more quickly autolyzed 
after violent shaking. 
7. Influence of Pressure.-Microorganisms in fluids which are sub- 
jected to great pressure are for a time inhibited in their growth. When 
oxygen or nitrogen are used the same moderate inhibition occurs. 
Influence of Carbonic Acid Under Pressure.-D'Arsonval and Charrin 
submitted a culture of Bacillus pyocyaneus to a pressure of fifty atmos- 
pheres under carbonic acid. At the end of four hours cultures could 
still be obtained, but the bacillus had lost its power of pigment produc- 
tion. A few colonies were developed after six hours' exposure to this 
pressure, but after twenty-four hours no development occurred. Other 
bacteria subjected to pressure have exhibited more resistance. We have 
subjected broth and milk containing typhoid, dysentery, diphtheria, and 
colon bacilli to the gas under a pressure of seventy-five and one hundred 
and fifty pounds. Within twenty-four hours 99 per cent, of those in 
the broth and 98 per cent, of those in the milk were destroyed. Within 
one week the broth was sterile and within four weeks the milk was sterile. 
Tubercle bacilli and staphylococci were much more resistant, but little 58 
PRINCIPLES OF MICRORIOLOGY 
effect being noticed in twenty-four hours. The results were the same 
whether the cultures were kept at 10° or 25°. Bottled waters charged 
with carbonic acid are usually sterile. 
8. Effect of Drying.-For growth, microorganisms require much 
moisture. Want of water affects them in different ways. Upon dried 
culture media development soon ceases; but on media dried gradually 
at the room temperature (nutrient agar, gelatin, potato) they live often 
for a long time, even when there are no spores to account for their lon- 
gevity. A shrunken residue of such cultures placed in bouillon has often 
been found, after a year or more, to yield living bacteria. The yeasts 
and molds are still more resistant. The question as to how long the 
non-spore-bearing forms are capable of retaining their vitality when 
dried on a cover-glass or silk threads has been variously answered. We 
know now that there are many factors which influence the retention 
of vitality; spores, of course, being more resistant than vegetative forms. 
The varying results sometimes reported by different observers may 
be explained by the fact that the conditions under which they were 
made were different, depending upon the desiccator used, the medium 
upon which the cultures were grown, and the use of silk threads or cover- 
glasses. In all these experiments, of course, it should be previously 
determined that in spore-bearing species there are no spores present. 
Even when a dried culture lives for a long time the majority of the 
organisms die in a few hours after drying. We have found 1,500,000 
colon bacilli to be reduced to 100,000 after three hours' drying. In 
pathogenic tissues or exudates they resist drying much longer than 
when unprotected. 
Encysted protozoa withstand long periods of desiccation. Most 
forms when dried quickly remain viable much longer than when dried 
slowly. 
Duration of Life in Pure Water.-When microorganisms which require 
much organic food for their development (and these include most of 
the pathogenic species) are placed in distilled water they soon die- 
that is, within a few days; even in sterilized well-water or surface water 
their life duration does not usually exceed eight to fourteen days, and 
they rarely multiply. Instances, however, of much more extended life 
under certain conditions are recorded. 
9. Tactic Effect of Chemicals.-Chemotaxis.-The deleterious effect 
of chemicals, especially those used as germicides, will be considered under 
Disinfection. 
Some chemical substances exert a peculiar attraction for micro- 
organisms, known as positive chemotaxis, while others repel them- 
negative chemotaxis. Moreover, all varieties are not affected alike. 
Oxygen, for example, attracts aerobic and repels anaerobic bacteria, 
and for each variety there is a definite proportion of oxygen, which most 
strongly attracts. The chemotactic properties of substances are tested 
by pushing the open end of a fine capillary tube, filled with the substance 
to be tested, into the edge of a drop of fluid containing the organisms 
and examining under the microscope. We are able thus to watch the PRODUCTS OF MICROBAL GROWTH 
59 
action of the microbes and note whether they crowd about the tube 
opening or are repelled from it. Among substances showing positive 
chemotaxis for nearly all microorganisms are peptone and urea, while 
among those showing negative chemotaxis are alcohol and many of the 
metallic salts. Such experiments are of course rough. The diffusion 
of the substances from the tube into the surrounding medium must 
play an extremely active role in the final result. 
Microorganisms not only are acted upon by their surroundings, as 
has already been shown, but they themselves act, often markedly, upon 
these surroundings. We have spoken, under the effect of food (p. 51) 
of the great changes which may be produced in growths by slight changes 
in the food medium. So, many of the products, as noted below, are 
influenced to a greater or a less extent by environment. 
Production of Light.-Microorganisms which have the property of 
emitting light are quite widely distributed in nature, particularly in 
media rich in salt, as in sea-water. Many of these have been accurately 
studied. The emission of light is a property of the living protoplasm 
of the organism, and is not usually due to the oxidation of any photo- 
genic substance given off by them; at least only in two instances has such 
substance been claimed to have been isolated. While these organisms 
cannot emit light except during life, they can live without emitting 
light. They are best grown under free access of oxygen in a culture 
medium prepared by boiling fish in sea-water (or water containing 3 
per cent, sea-salt), to which 1 per cent, peptone, 1 per cent, glycerin, 
and 0.5 per cent, asparagin are added. The power of emitting light is 
soon lost unless the organism is constantly transplanted to fresh media. 
Thermic Effects.-The production of heat by microorganisms does 
not attract attention in our usual cultures because of its slight amount, 
and even fermenting culture liquids with abundance of bacteria cause 
no sensation of warmth when touched by the hand. Careful tests, 
however, show that heat is produced. The increase of temperature in 
organic substances when stored in a moist condition, as tobacco, hay, 
manure, etc., is due, partly at least, to the action of bacteria. 
Chemical Effects.-The chemical changes which take place in sub- 
stances as they are split up by microorganisms depend on the nature 
of the substances involved and the conditions under which they exist, and 
on the varieties of germs present. Chemists can as yet only enumerate 
some of the substances evolved, and describe, in a few cases, the manner 
in which they were produced. The chemical activity may be divided 
into the following four types: (1) Production of substances which help 
in some way the life of the cell. These substances may be secreted 
and retained within the cell, or liberated from it; e. g., ferments or 
enzymes; true toxins (?). (2) Production of substances liberated by 
the bacteria as waste products. (3) Production of substances by the 
breaking down of the food media; e. g., putrefactive products, due. 
PRODUCTS OF MICROBAL GROWTH. 60 
PRINCIPLES OF MICROBIOLOGY 
largely to enzyme action. (4) The production of substances which 
help form the protoplasm of the bacterial cell itself. 
Fermentation.-Fermentation may be defined broadly as a chemical 
decomposition of an organic compound, induced by the life processes 
of living organisms (organized ferments), or by chemical substances 
thrown off from the organisms (unorganized or chemical ferments or 
enzymes). It has been shown by Buchner and others that, in those 
cases of fermentation in which formerly it was believed the organized 
cell itself was necessarily concerned, ferments causing the same changes 
as the organized cells may be separated by various methods, such as 
crushing, filtering and so on. This brings fermentation by unorganized 
and organized ferments very closely together, the one being a substance 
thrown off from the cell, the other a substance ordinarily retained within 
the cell. The elaboration of both ceases with the death of the bacteria 
producing them. Fermentation, therefore, requires the living agent or 
its enzyme. It furthermore demands the proper nutriment, tempera- 
ture, moisture, and the absence of deleterious substances. 
Fermentation yields products that are poisonous to the ferment; 
hence fermentation ceases when the nutriment is exhausted or the 
fermentation is in excess. That the process will often begin again 
after diluting the fermented medium, shows that the concentration of 
the harmful products plays an important part in the inhibitory action. 
Characteristics of Ferments or Enzymes.-Ferments are non-dialyz- 
able. They withstand moderate dry heat, but are usually destroyed 
in watery solutions on exposure of ten to thirty minutes to a tempera- 
ture of 60° to 70° C. They are injured by acids, but are resistant to all 
alkalies. They, even when present in the most minute quantities, 
are able partly to split up or decompose complex organic compounds 
and thus to render the foodstuff suitable for bacterial growth. A simple 
example of bacterial fermentation of carbohydrates produced by an 
enzyme is that of grape-sugar. 
CbHi2O6 
Grape-sugar 
= 2C2H6O 
2 Alcohol. 
+ 2CO2 
2 Carbon dioxide. 
Or, 
CoHnOo 
Grape-sugar. 
Or, 
CbH^Og 
Grape-sugar. 
= 2C3H6O3 
2 Lactic acid. 
= 3C2H4O2 
3 Acetic acid. 
Far less common is oxidizing fermentation, such as occurs, for example, 
in the production of acetic acid from alcohol. Here the energy is 
acquired not from the decomposition, but by the oxidation of the 
alcohol. 
The Proteolytic Ferments.-The proteolytic ferments which are 
somewhat analogous to trypsin-being capable of changing albumin- 
ous bodies into soluble and diffusible substances-are very widely 
distributed. The liquefaction of gelatin, which is chemically allied PRODUCTS OF MICROBAL GROWTH 
61 
to albumin, is due to the presence of a proteolytic ferment or trypsin. 
The production of proteolytic ferments by different cultures of the 
same variety of bacteria varies considerably-far more than is generally 
supposed. 
Bitter-tasting products of decomposition may be formed by cer- 
tain liquefying bacteria in media containing proteid, as, for example, 
in milk. 
Diastatic Ferments.--Diastatic ferments convert starch into sugar. 
This action is demonstrated by mixing starch paste with suitable 
cultures to the resulting mixture of which thymol has been added, 
and keeping the digestion for six to eight hours in the incubating oven; 
then, on the addition of Fehling's solution and heating, the reaction for 
sugar appears-the reddish-yellow precipitate due to the reduction 
of the copper. 
Inverting Ferments.-Inverting ferments (that is, those which convert 
polysaccharides into monosaccharides) are of very frequent occurrence. 
Bacterial invertin withstands a temperature of 100° C. for more than an 
hour, and is produced in culture media free from proteid. 
Rennin-like Ferments.-Rennin-like ferments (substances having the 
power of coagulating milk with neutral reaction, independent of acids) 
are found not infrequently among bacteria. 
Alkaline Products and the Fermentation of Urea.-Aerobic bacteria 
always produce alkaline products from albuminous substances. Many 
species also produce acids from sugars, which explain the fact that 
neutral or slightly alkaline broth often becomes acid at first from the 
fermentation of the sugar contained in the meat used for making the 
media. When the sugar is used up the reaction often becomes alkaline, 
as the production of alkalies continues. The substances producing 
the alkalinity in cultures are chiefly ammonia, amine, and the ammonium 
bases. 
The conversion of urea into carbonate of ammonia by Micrococcus 
urece affords an example of the production of alkaline substances by 
bacteria: 
CO(NH2)2 
Urea. 
+ 2H2O 
2 Water. 
= CO3(NH4)2 
Ammonium carbonate. 
The poisonous products of microorganisms are considered in Chap- 
ter VII, pages 145-150. 
Pigment Production.-Pigments have no known importance in con- 
nection with disease, but are of interest and have value in identifying 
bacteria. Their chemical composition is not generally known. 
Red and Yellow Pigments.-Of the twenty-seven red and yellow 
chromogenic bacteria studied by Schneider, almost all produce pig- 
ments soluble in alcohol and insoluble in water. The large majority 
of these pigments possess in common the property of being colored 
blue green by sulphuric acid and red or orange by a solution of potash. 
Though varying considerably in their chemical composition and in 
their spectra, they may be classified, for the most part, among that large 62 
PRINCIPLES OF MICROBIOLOGY 
group of pigments common to both the animal and vegetable kingdoms 
known as lipochromes, and to which belong the pigments of fat, yolk of 
eggs, the carotin of carrots, turnips, etc. 
Violet Pigments.-Certain bacteria produce violet pigments, also 
insoluble in water and soluble in alcohol, but insoluble in ether, benzol, 
and chloroform. These are colored yellow when treated in a dry state 
with sulphuric acid, and emerald green with potash solution. 
Blue Pigments.-Blue pigments, such as the blue pyocyanin produced 
by B. pyocyaneus; and the fluorescent pigment common to many so-called 
fluorescent bacteria (bacteriofluorescence) are examples. In cultures the 
pigment is at first blue; later, as the cultures become alkaline, it is 
green. 
Numerous investigations have been made to determine the cause 
of the variation in the chromogenic function of bacteria. All condi- 
tions which are unfavorable to the growth of the bacteria decrease the 
production of pigment, as cultivation in unsuitable media or at too 
low or too high a temperature, etc. The B. prodigiosus seldom makes 
pigment at 37° C., and when transplanted at this temperature, even 
into favorable media, the power of pigment production is gradually lost. 
B. pyocyaneus does not produce pigment under anaerobic conditions. 
Occasionally colored and uncolored colonies of the same species of bac- 
teria may be seen to occur side by side in one plate culture, as, for 
example, in the case of Staphylococcus pyogenes. 
Reduction Processes.-The following processes depend wholly or 
in part upon the reducing action of nascent hydrogen: 
1. Sulphuretted Hydrogen (H2S). All bacteria, according to Petri 
and Maassen, possess the power of forming sulphuretted hydrogen, 
particularly in liquid culture media containing much peptone (5 to 10 
per cent.); only a few bacteria form H2S in bouillon in the absence 
of peptone, while about 50 per cent, in media containing 1 per cent, 
peptone possess the property of converting sulphur into sulphuretted 
hydrogen, for which purpose is required the presence of nascent 
hydrogen. (For demonstration see page 120.) 
2. The reduction of blue litmus pigments, methylene blue, and 
indigo to colorless substances. The superficial layer of cultures in 
contact with the air shows often no reduction, only the deeper layers 
being affected. By agitation with access of air the colors may be 
again restored, but at the same time, if acid has been formed, the 
litmus pigment is turned red. 
3. The reduction of nitrates to nitrites, ammonia, and free nitrogen. 
The first of these properties seems to pertain to a great many bacteria. 
Aromatic Products of Decomposition.-Many microbes produce 
aromatic substances as the result of their growth. The best known of 
these are indol, skatol, phenol and tyrosin. Systematic investigations 
have only been made with regard to the occurrence of indol and phenol. 
Decomposition of Fats.-Pure melted butter is not a suitable culture 
medium for microbes. The rancidity of butter is brought about (1) PRODUCTS OF MICROBAL GROWTH 
63 
as the result of a purely chemical decomposition of the butter by the 
oxygen of the air under the influence of sunlight, and (2) through the 
formation of lactic acid from the milk-sugar left in the butter. Fats 
are, however, attacked by bacteria with the consequent production of 
acid when mixed with gelatin and used as culture media. 
Putrefaction.-By putrefaction is understood in common parlance 
every kind of decomposition due to microbes which results in the pro- 
duction of malodorous substances. Scientifically considered, putre- 
faction depends upon the decomposition of albuminous substances, 
which are frequently first peptonized and then further decomposed. 
Typical putrefaction occurs only when oxygen is absent or scanty; 
the free passage of air through a culture of putrefactive bacteria-an 
event which does not take place in natural putrefaction-very much 
modifies the process: first, biologically, as the anaerobic bacteria are 
inhibited, and then by the action of the oxygen on the products or 
by-products of the aerobic and facultative anaerobic bacteria. 
As putrefactive products we have peptone, ammonia, and amines, 
leucin, tyrosin, and other amino substances; oxyfatty acids, indol, 
skatol, phenol, ptomains, toxins, and, finally, sulphuretted hydrogen, 
mercaptans, carbonic acid, hydrogen, and possibly marsh-gas. 
Nitrifying Bacteria.-According to recent observations, nitrification 
is produced by a special group of bacteria, cultivated in the laboratory 
with difficulty, which do not grow on our usual culture media. From 
the investigations of Winogradsky it would appear that there are 
two common microorganisms present in the soil, one of which con- 
verts ammonia into nitrites and the other converts nitrites into 
nitrates. 
Conversion of Nitrous and Nitric Acids into Free Nitrogen.-This 
process is performed by a number of bacteria. 
The practical importance of these organisms is that by their action 
large quantities of nitrates in the soil, and especially in manure, may 
become lost as plant food by being converted into nitrogen. 
By the aid of certain root bacteria, which gain entrance to the roots of 
legumes and there produce nodular formations, the leguminous plants 
are enabled to assimilate nitrogen from the atmosphere. It is not 
known exactly how this assimilation of nitrogen occurs, but it is assumed 
that the zooglea-like bacteria, called bacteroids, constantly observed in 
the nodules, either alone or in a special degree, possess the property of 
assimilating and combining nitrogen. It seems, moreover, to have 
been recently established that, independently of the assistance of the 
legumes, certain bacteria exist free in the soil, which accumulate nitrogen 
by absorbing it from the air. These various nitrifying, denitrifying, and 
nitrogen-fixing bacteria are described in detail in the special chapter 
upon bacteria in nature. 
Formation of Acids from Carbohydrates.-Free acids are formed 
by many microbes in culture media containing some form of sugar or 
other fermentable carbohydrates, such as the alcohol mannite; the 
production of acid in ordinary bouillon takes place on account of the 64 
PRINCIPLES OF MICROBIOLOGY 
presence of sugar, which is usually found in small quantities in the 
meat. 
If after the sugar is consumed, not enough acid has been formed 
to kill the bacteria, the acid is neutralized gradually and in the end the 
reaction becomes less acid or even alkaline. 
Among the acids produced the most important is lactic acid; also 
traces of formic acid, acetic acid, propionic acid, and butyric acid, 
and not infrequently some ethyl alcohol and aldehyde or acetone are 
formed. Occasionally no lactic acid is present. 
Formation of Gas from Carbohydrates and Other Fermentable 
Substances of the Fatty Series.-The only gas produced in visible 
quantity in sugar-free culture media is nitrogen. If sugar is vigorously 
decomposed by bacteria, as long as pure lactic acid or acetic acid is 
produced there may be no development of gas, as, for instance, with 
the B. typhosus on grape-sugar, but frequently there is much gas devel- 
oped, especially in the absence of air. About one-third of the acid- 
producing species also develop gas abundantly, this consisting chiefly 
of CO2, which is always mixed with H. Marsh-gas is seldom formed 
by bacteria, with the exception of those decomposing cellulose. (For 
demonstration see page 119.) 
Formation of Acids from Alcohol and Other Organic Acids.-It has 
long been known that Bacillus aceti and allied bacteria convert dilute 
solutions of ethyl alcohol into acetic acid by oxidization: 
ch3 
I 
ch2oh 
+ Oa 
ch3 
I 
COOH. 
+ h2o. 
The higher alcohols-glycerin, dulcit, mannite, etc.-are also con- 
verted into acids. 
Finally, numerous results have been obtained from the conversion 
of the fatty acids and their salts into other fatty acids by bacteria. 
References. 
■ Ambroz. Entwickelungszyklus des B. nitri n. sp., etc., CentralbL f. Bakt., etc., I. 
Abt., orig., 1909, 51, 193 (with bibliography on structure and development of bacteria). 
Buchner. Berichte d. Deutsch, chem. Gesellsch., xxx, 117-124 and 1110-1113. 
Buschke. Die Sprosspilze in Kolle und Wassermann's Die Mikroorganismen, Jena, 
2d edition, 1913. 
Calkins. The Protozoa, 1st edition, New York, 1901. Also article entitled, The 
Protozoa, in Osler's Modern Medicine, Philadelphia, 1907, vol. i; also Protozoology, 
New York and Philadelphia, 1909. 
Dojlein. Lehrbuch der Protozoenkunde, 2d edition, Jena, 1909; Handbuch der 
pathogenen Mikroorganismen, Kolle und Wassermann, 2d edition, Jena, 1913. 
Jennings. Behavior in Lower Organisms, New York. Macmillan & Co., 1906. 
Lang. Protozoa in Vergleichende Anatomie der wierbellosen Thiere, new edition, 1909 
Lankester. Treatise on Zoology, 1st edition, London, Part I, first and second fascicles, 
1909. 
Meyer. Flora, 1908, p. 95. 
Migula. System der Bakterien, Jena, 1897. 
Moore. The Pathology of Infectious Diseases of Animals, 1st edition, Ithaca, 1902. PRODUCTS OF MICROBAL GROWTH 
65 
Petruschky. Die pathogenen Mychomyceten, in Kolle und Wassermann's Die Mikro- 
organismen, Jena, 2d edition, 1913. 
Plaut. Die Hyphenpilze in Kolle und Wassermann's Die Mikro-organismen, Jena, 2d 
edition, 1913. 
Ruzicka. Cytologie der sporenbildenden Bakterien, etc., Centralbl. f. Bakt., 1909, 
II, Abt., vol. xxvii. 
Schaudinn. Beitrage zur Kenntnis der Bakterien, etc., Arch. f. Protistenk, 1902, i, 306, 
and 1903, ii, 416. 
Zettnow. Romanowski's Farbung bei Bakterien, Zeitschr. f. Hyg., etc., 1899, xxx, 1; 
and Centralbl. f. Bakt., 1900, Abt. I, xxvii, 803. 
5 CHAPTER III. 
THE MICROSCOPE AND THE MICROSCOPIC EXAMINATION 
OF MICROORGANISMS. 
THE MICROSCOPE. 
If lenses were capable of refracting all light equally, and bringing 
to a focus in one plane all rays proceeding from one plane in the object 
the microscope would be a comparatively simple instrument. But 
simple lenses have several serious optical defects. 
1. Spherical Aberration.-Points in the plane of the object are imaged 
on the curved surface of the spherical lens. This defect may be some- 
what diminished by combining convex and concave lenses, and by 
restricting the size of the field. Objectives corrected in this way are 
called aplanatic. 
2. Chromatic Aberration.-This defect is due to the fact that the rays 
of light vary in their refraction according to their wave lengths (colors), 
e. g., the red rays have the longest focus and the violet the shortest. 
This is considerably corrected by combining planoconcave lenses of flint 
glass with bi-convex lenses of crown glass-achromatic objectives. 
Still more of a correction is made by combining several different 
kinds of lenses with a lens of fluorite-apochromatic objectives. 
Monochromatic light may be employed and thus chromatic aberra- 
tion may be entirely avoided. 
3. Diffraction.-Less luminous secondary images about the primary 
image, due to scratches or foreign particles or other defects may occa- 
sionally occur in the very best lenses. 
In order to understand fully the principle of the microscope, works 
on optics should be consulted. 
Different Parts of the Microscope (Figs. 7 and 8).-A complete 
instrument usually has four oculars, or eye-pieces, A, which are num- 
bered from 1 to 4, according to the amount of magnification which 
they yield. Nos. 2 and 4 are most useful for bacteriologic work. The 
objective-the lens, B, at the distal end of the barrel-serves to give 
the main magnification of the object. For stained bacteria, the 
achromatic oil-immersion lens is regularly employed; for photographic 
purposes the apochromatic lenses are needed, although even here they 
are not indispensable. A y-g lens may at times be useful, but hardly 
necessary; a No. 4 ocular and a lens give a magnification of about 
1000 diameters (Fig. 9). For unstained bacteria we employ either 
the immersion or 7 dry lens, according to the purpose for which we 
study the bacteria; for the examination of colonies where, as a rule, THE MICROSCOPE 
67 
we do not wish to see individual bacteria, but only the general appear- 
ance of whole groups, we use lenses of much lower magnification (Fig. 10). 
The stage C-the platform upon which the object rests-should be 
large enough to support the Petri plates if culture work is to be done. 
The distance between the optical axis of the instrument and the pillar 
must be great enough to permit one to examine rather more than half 
the surface of the Petri dish without revolving it. The iris diaphragm 
Fig. 7 
Fig. 8 
-Eye-lens 
-Diaphragm 
> of the Ocular 
Field-lens 
-Draw Tube 
-Tube Length 160 mm- 
Axis 
Optical 
-Body Tube 
Draw Tube Diaphragm 
with Society Screw 
•Society Screw 
-Mount 
Back-lens 
Middle-lens 
Front-lens 
Working Distance 
of the 
Objective 
Coverglass 
Slide 
Object 
Microscope. 
Internal structure of the microscope. 
7) opens and closes, and, as the iris of the eye controls, the amount of 
light. Its opening is diminished or increased by moving a small arm 
underneath the stage. The reflector or mirror E placed beneath the 
stage serves to direct the light to the object to be examined. It has 
two surfaces-one concave and one plane. The concave surface must 
not be employed when the substage condenser is being used, otherwise 
the rays of light reaching the stage from the condenser will not be 68 
PRINCIPLES OF MICROBIOLOGY 
correctly focused. The concave surface may be used when unstained 
objects, such as colonies, or hanging drops are examined. At the same 
time the Abbe condenser should be lowered and the iris blender regulated. 
The coarse adjustment F is the rack-and-pinion arrangement by which 
the barrel of the microscope can be quickly raised or lowered. It is 
used to bring the bacteria roughly into focus. If the bearings become 
loose tighten the little screws at the back of the pinion box. Keep the 
teeth clean. If the bearings need oiling use an acid-free lubricant, such 
as paraffin oil. The fine adjustment G serves to raise and lower the 
barrel very slowly and evenly, and is used for the exact study of the 
bacteria when high-power lenses are used. It is necessarily of limited 
range and delicate in its mechanism. If, when looking into the eye-piece, 
no change of focus is noticed by turning the micrometer head, or if the 
micrometer head ceases to turn, the adjustment has reached its limit. 
Raise the barrel of the microscope by means of the coarse adjustment, 
Fig. 9 
Fig. 10 
Anthrax bacilli and blood cells. 
X 1000 diameters. 
Colonies of diphtheria bacilli. 
X 200 diameters. 
then turn the micrometer back to bring the fine adjustment midway 
within its range. When the fine adjustment head stops do not force 
it. For the microscopic study of microbes it is essential that we magnify 
the organisms as much as possible and still have their definition clear 
and sharp. For this purpose the microscope should be provided with 
an oil-immersion system and a substage condensing apparatus. In 
using the oil-immersion lens a drop of oil (oil of cedar) of the same 
index of refraction as the glass is placed upon the face of the lens, so 
as to connect it with the cover-glass when the bacteria are in focus. 
There is thus no loss of light through deflection, as is the case in the dry 
system. If the lenses become dirty they should be wiped gently with 
Japanese lens paper or a clean, soft, old-linen handkerchief. If necessary 
breathe on the lens before wiping, and if this does not succeed use a 
little xylol or chloroform. These substances are not to be used unless 
necessary. An immersion objective should always be cleaned imme- THE MICROSCOPE 
69 
diately after using. The objective should always be kept covered so as 
to prevent dust dropping in. 
Light. -The best light is obtained from white clouds or a blue sky 
with a northern exposure. Avoid direct sunlight. If necessary use 
white shades to modify the sunlight. Artificial light has one advantage 
over daylight in that it is constant in quality and quantity. The 
Welsbach burner and a whitened incandescent bulb give a good light. 
A blue glass between the artificial light and the lens is often of value. 
An eye-shade may be helpful. 
Substage condensing apparatus H is a system of lenses situated 
beneath the central opening of the stage. It serves to condense the 
light passing through the reflector to the object in such a way that 
it is focused upon the object, thus furnishing the greatest amount of 
luminosity. Between the condenser and the reflector is placed the iris 
diaphragm. 
Focusing.-Focus the body tube down by means of the coarse adjust- 
ment until the objective approaches very near to the cover-glass, being 
careful not to touch it. Then with the eye at the eye-piece focus up 
carefully with the coarse adjustment until the specimen comes plainly 
into view. Be careful not to pass this focal point. It is easily unnoticed 
if the light be too intense and the specimen thin and transparent. If 
the sliding tube coarse adjustment is used, focus carefully by giving 
the tube a spiral movement. 
When the object is brought fairly well into focus by means of the 
coarse adjustment, use the fine adjustment to focus on the particular 
spot desired, for if this spot is in the centre of the field of the low power 
it should be somewhere in the field of the higher power. It is too much 
to ask of the maker that the lenses be made absolutely parfocal and 
centred. The delicacy of the centring can be appreciated when the 
magnification and the extremely small portion examined are considered. 
When the objectives are not thus fitted to the nose-piece, refocusing 
and again hunting up the object are necessary. In so doing we repeat 
the caution always to focus up before turning the nose-piece. When 
no revolving nose-piece is used the change of objectives means the 
unscrewing of one and the screwing of the other into its place, and 
refocusing. 
The beginner should always use the low-power objectives and oculars 
first. The low-power objectives have longer working distances and 
are not so apt to be injured. They always show a larger portion of the 
specimen and thus give one a better idea of the general contour. After 
obtaining this general idea the higher powers can be used to bring out 
greater detail in any particular part. Generally speaking, it is best to 
use a high-power objective and low-power eye-piece in preference 'to 
a low-power objective and high-power eye-piece. In the latter case any 
imperfections in the objective are magnified unduly by the eye-piece, 
giving, as a rule, poor definition. 
Tube Length and Cover-glass.-All objectives are corrected to a 
certain tube length (160 mm. by most makers-Leitz, 170 mm.) and 70 
PRINCIPLES OF MICROBIOLOGY 
all objectives in fixed mounts of over 0.7 N. A. are corrected to a 
definite thickness of cover-glass as well (Zeiss, 0.15 mm., 0.2 mm.; 
Leitz, 0.17 mm.; Bausch & Lomb and Spencer, 0.18 mm.). These 
objectives give their best results only when used with the cover-glass 
and tube length for which they are corrected. As indicated in Fig. 8 
the tube length extends from the eye lens of the eye-piece to the end of 
the tube into which the objective or nose-piece is screwed. If a nose- 
piece is used the draw tube must be correspondingly shortened. If the 
cover-glass is thinner than that for which the objective is corrected, the 
tube must be lengthened to obtain best results; if thicker, shortened. 
The more expensive objectives are provided with adjustable mounts 
by which the distances between the lens systems may be changed to 
compensate for difference of thickness of cover. They are successively 
used only in the hands of an expert. One of them out of adjustment 
is worse than an ordinary objective. 
Fig. 11 
Virulent diphtheria bacilli. Cultures two days old. Unstained. X 2400. 
(After Siebert.) 
Dark Ground Illumination and the Examination of Ultramicro- 
scopic Particles.-The apparatus constructed by Siedentopf and 
Zsigmondy makes visible, even in solutions otherwise apparently 
homogeneous, very minute particles, which heretofore could not be 
seen even with the highest magnifications. Particles ljuju (a milli- 
micron = one-millionth of a millimeter) are thus rendered visible. 
This increased power in microscopic analysis is made possible by 
intense (electric arc lamp) focal lateral illumination of the objects 
examined, and by shutting off the rays passing in the usual way through MICROSCOPIC METHODS 
71 
the tube of the microscope. The greater the difference between the 
refractive index of the objects colloidally dissolved or otherwise held 
in suspension and the fluid which surrounds them, the brighter will be 
the appearance of the objects, and therefore the more readily visible. 
The microscopic field, as will be seen by the photogram herewith 
(Fig. 11), is dark; the objects which refract the light show as brightly 
illuminated, sharply defined pictures, in which the black margin cor- 
responds to the contour of the object. The illuminated portion is sur- 
rounded by a fine dark zone, this in turn by alternate bright and dark 
zones, in which the illumination rapidly decreases. 
Reichert, of Vienna, has recently simplified this apparatus by devising 
a new condenser. The light which illuminates the object has a greater 
refraction than the cone of light entering the objective which produces 
the image. Its advantages over the first method are: (1) it utilizes 
the source of light better; (2) any dry objective can be used without 
alterations; (3) small particles are seen without the disturbing refraction 
rings. With this apparatus such living organisms as the Spirocheta 
pallida, and the flagella on certain bacteria, which can scarcely be seen 
by ordinary microscopes on account of their low refractive indices, 
may be demonstrated with great clearness. 
The use of microphotography with ultraviolet light (according to 
A. Kohler) makes visible particles that cannot be seen by ordinary 
light, because of the inability of the violet rays to pass through certain 
substances, e. g., chromatin. 
This method is said to increase 40,000 times our present limit of 
vision. The few discoveries claimed by these means for diseases of 
unknown origin have so far lacked sufficient corroboration to constitute 
them proved. 
Double Microscopes.-These have been devised by Metz and others 
for the purpose of making comparative studies of two objects. 
MICROSCOPIC METHODS. 
The direct microscopic examination of suspected substances for 
microorganisms can be made either with or without staining. Unstained, 
the microbes are examined living in a hanging drop or on transparent 
solid media, under daylight, or better, artificial light, to note their 
number, their motility, their size, form, and spore formation, their 
general arrangement, their reactions to specific serums and to vital 
stains, etc. But for more exact study of their structure they must be 
stained in a dried film preparation on a glass slide or a cover-glass or 
when in tissues, in sections. 
Elimination of Foreign Organisms from Preparations.-Since germs 
are present in the air, in dust, in tap-water, on our bodies, clothes, 
and on all surrounding objects, it follows that when we begin to examine 
substances for microbes the first requisite is, that the materials we use, 
such as staining fluids,' cover-glasses, etc., should be practically free 
from organisms, both living and dead, otherwise we may not be able to 72 
PRINCIPLES OF MICROBIOLOGY 
tell whether those we detect belong originally in the substances examined 
or only in the materials we have used in the investigation. Therefore 
all solutions are filtered and all apparatus thoroughly cleaned and when 
necessary sterilized. 
Examination of Bacteria in the Hanging Drop. For this examination 
special slides and methods are desirable. The slide used is one in which 
there is ground out on one surface a hollow having a diameter of about 
half an inch (Fig. 12). According to the purpose for which the hanging 
drop is to be studied, sterilization of the slide and cover-glass may or 
may not be necessary. 
Fig. 12 
Hollow slide with cover-glass. 
The technique of preparing and studying the hanging drop is as follows: 
The surface of the glass around the hollow in the slide is smeared with a little 
vaselin or other inert oil. This has for its purpose both the sticking of the 
cover-glass to the slide and the prevention of evaporation in the drop placed 
in the little chamber, which is to be formed between the cover-glass when placed 
over the hollow, and the slide. 
If the bacteria to be studied are in a fluid we place a large platinum loopful 
upon the centre of the cover-glass and, to avoid drying, immediately invert 
it by means of a slender pair of forceps over the hollow in the slide, being very 
careful to have the drop over the centre of the cover-glass. The cover-glass 
is then pressed on the slide so as to spread the vaselin and make a perfect seal. 
If the bacteria are growing on solid media, or are obtained from thick pus or 
tissues from organs, they are mixed with a suitable amount of bouillon or 
sterile physiologic salt solution1 either before or after being placed upon the 
cover-glass. 
In studying living bacteria to determine only their grouping and 
motion we may use less magnification than for studying other charac- 
teristics. In studying unstained bacteria and tissues we shut off as 
large a portion of the light with our diaphragm as is compatible with 
distinct vision, and thus favor contrasts which appear as lights and 
shadows, due to the differences in light transmission of the different 
materials under examination. It is necessary to remember that they 
are seen with difficulty, and that we are very apt, unless extremely 
careful in focusing, to allow the lens to go too far, and so come upon the 
cover-glass, break it, destroy our preparation, and, if examining patho- 
genic bacteria, infect the lens. This may be avoided by first finding 
the hanging drop with a low-power lens and then centring it. The edge 
of the drop is focused more easily than the centre. The lens of higher 
magnification is now very gradually lowered, while at the same time 
gently moving the slide back and forth to the slightest extent possible 
with the left hand. If any resistance is felt the lens should be raised, 
for it has gone beyond the point of focus and is touching the cover-glass. 
1 Physiologic salt solution is usually 0.8 per cent. NaCl in distilled water. MICROSCOPIC METHODS 
73 
Hanging Mass or Hanging Block Cultures.-In order to study the 
morphology and manner of multiplication of individual microorganisms 
to better advantage than in the hanging drop, we have used hanging 
masses of agar, made by placing a large platinum loop full of melted 
agar on a sterile cover-glass and allowing it to harden, protected from 
dust. The organisms are placed on the free surface of this mass which 
is then inverted over a hollow slide and studied as in a hanging drop. 
Hill devised the following procedure: Melted nutrient agar is poured into 
a Petri dish to a depth of about one-eighth to one-quarter of an inch. When 
cool a block is cut out about one-quarter of an inch square. The block is placed, 
under surface down, on a slide and protected from dust. A very dilute sus- 
pension of the growth to be examined is then made in sterile bouillon and 
spread over the upper surface of the block. The slide and block are then put 
in the incubator for ten minutes to dry slightly. A clean cover-slip is now placed 
on the agar block in such a way as to avoid large air-bubbles. The slide is then 
removed. With the aid of a platinum loop a drop or two of melted agar is run 
along each side of the block to fill any angles between it and the cover-glass. 
After drying in the incubator for five minutes it is placed over a hollow slide 
and sealed with paraffin. 
We consider the hanging-mass method better than that of the hanging block 
in many instances, because in the former method no pressure is exerted on the 
bacteria, and more oxygen is allowed them. 
Film Preparation (spread, smear).-Film preparation is made as 
follows: A very small amount of the blood, pus, discharges from mucous 
membranes, cultures from fluid media, or other material to be examined 
is removed, usually by means of a sterile swab or platinum loop, and 
smeared undiluted in an even, thin film over a perfectly clean,1 thin 
cover-glass or slide. From cultures on solid media, however, on account 
of the abundance of organisms in the material, a little of the growth 
is diluted by adding it to a small loopful of filtered or distilled water, 
which has been previously placed on the glass slide. It is best to add 
to the drop just enough of the culture to make a perceptible cloudiness. 
Blood films may be made either by the cover-glass or the slide method. 
To make a cover-glass preparation, two square, very thin (hence flexible) 
cover-glasses are cleaned. Holding one with thumb and index fingers by oppo- 
1 To render new cover-slips clean and free from grease, the method recommended by 
Gage is useful: Place in following solution over night. 
Bichromate of potash (J&CnO?) 200 grams 
Water, tap or distilled 800 c.c. 
Sulphuric acid 1200 c.c. 
The bichromate is dissolved in the water by heating in an agate kettle; the sulphurous 
acid is added very slowly and carefully on account of great heat developed. After cool- 
ing, it is kept in a glass vessel. It may be used more than once. 
Glasses are removed the next morning and cleansed in running tap-water until the 
yellow color disappears. They are then placed in ammonia alcohol until used. When 
used wipe with soft, clean linen or cotton cloth. If old cover-slips are used, boil first in 
5 per cent, sodium carbonate solution. 
Another procedure is, after washing with soap and water and rinsing in water, to soak 
the cover-glasses in alcohol, then wipe with soft linen, then place in a Petri dish, and heat 
in the dry sterilizer for one. hour at 200° C. to burn off fatty substances. The heating 
may be done by holding the cover-glass in the flame sufficiently to heat thoroughly with- 
out softening. A cover-glass is not clean when a drop of water spread over it does not 
remain evenly distributed, but gathers in droplets. 74 
PRINCIPLES OF MICROBIOLOGY 
site corners, the tip of a drop of blood obtained by needle puncture of finger 
or lobe of ear is made to touch the centre of the cover-glass, and the second 
clean cover-glass held similarly is allowed to fall upon the first one in such a 
manner that the corners do not coincide. The blood droplet spreads by capil- 
larity into a thin film, which is a sign to pull the two covers apart in the plane 
in which they lie; good results depend upon cleanliness, rapidity, and success 
in sliding the two covers apart. 
To make a slide film the tip of the exuded blood drop is made to touch one 
slide near one end and the edge of the second slide, held at an acute angle to 
the first one, is made to bisect the drop, which will spread at the point of con- 
tact by capillarity across the slide. Upon pulling the second or spreading slide 
over the first slide, never changing the angle and applying gentle pressure, 
a thin layer of blood suitable for examination will be formed. A slide made 
in this manner should be dried immediately by agitation in the air. It may 
then be fixed and stained in various ways. 
Milk films, after fixation, are cleared of fat by means of ether or 
alkaline solutions.1 
The film either is dried thoroughly in the air and then fixed with 
heat or any chemical fixative, or it may be placed in any of the fixatives 
while still moist. The usual fixatives- are methyl alcohol, absolute 
ethyl alcohol, Zenker's solution, etc. (see p. 84). 
When fixed with heat the glass is held by any one of the several kinds 
of forceps commonly used, and is passed three times by a rather slow 
movement through the Bunsen or alcohol flame. 
The film thus prepared is usually stained either by the simple addi- 
tion of a solution of an aniline dye, for from a few seconds to five minutes, 
or by one of the more complicated special stains described later. When 
the stain is to be hastened or made more intense the dye is used warm. 
The cover-glass or slide, with the charged side uppermost, may either rest 
on the table or be held by some modification of Cornet's forceps. When the 
solution is to be warmed the cover-glass may be floated, smeared side down, 
upon the fluid contained in a porcelain dish resting on a wire mat, supported 
on a stand; or the solution may be poured on the glass which may then be held 
over the flame in the Cornet forceps. If a slide is used it is simply inserted in 
the fluid or covered by it. The fluid both in the dish and on the slide should 
be carefully warmed so as to steam without actually boiling. The slide should 
be kept completely covered with fluid. 
After staining the film, the cover-glass or slide is grasped in the 
forceps and thoroughly but gently washed in clean water and then dried, 
first between layers of filter paper and then in the air or high over a 
flame. If a cover-glass has been used a drop of balsam or water is placed 
on a glass slide and the cover-glass put upon it with the film side down. 
Films made on slides are usually unmounted. Cedar oil is added at 
the time of examination with the oil-immersion lens and washed off with 
xylol immediately after. 
Burri's India-ink method of demonstrating bacteria. In 1907, 1908, 
and 1909 Burri recommended the following method for isolating and 
studying single bacterial cells. A solution of India ink (fliissige Perl- 
1 One-half to 1 per cent, sodium hydrate. MICROSCOPIC METHODS 
75 
tusche) in water 1 to 10 [better 1 to 4] is sterilized in test-tubes in the auto- 
clave for fifteen minutes. A small drop of this ink is mixed carefully 
with a drop of the fluid to be examined. If cultures from isolated cells 
are desired the fluid should first be diluted so that a drop contains 
presumably a single organism; then drops of the mixture are placed in 
rows upon nutrient agar plates. If the bacteria are to be examined 
immediately a drop of the mixture (ink plus undiluted bacterial fluid) is 
allowed to dry upon a glass slide and then examined under an oil- 
immersion lens. The bacteria appear a brilliant white upon a dark 
field, particles of the ink surrounding the organisms like a capsule. 
This method is especially applicable for the demonstration of such 
organisms as the Tr. pallidum which have poor staining qualities and 
a low index of refraction. 
Stains and Staining Methods for Microorganisms.-Protozoa stain 
in general as do animal cells. The protoplasm of mature bacteria 
reacts to stains much as does the nuclear chromatin of animal cells, 
though the intensity of the staining varies somewhat with the condition 
of growth, such as the age, the species, the media, and so on. 
The best bacterial stains are the basic aniline dyes, which are com- 
pounds derived from the coal-tar product aniline (C6H5NH2).1 
Aniline Dyes.-The aniline dyes which are employed for staining pur- 
poses are divided into two groups. In one the basic part of the molecule 
acts and the stains are spoken of as nuclear stains, since they color the 
nuclear chromatin of both cells and bacteria. In the other the staining 
act depends upon the acid part of the molecule, and the bacteria and 
cytoplasm of the higher cells are stained faintly. The stains in the latter 
group are used chiefly for contrast coloring. The basic dyes are usually 
employed as salts of hydrochloric acid, while the acid dyes occur as 
sodium or potassium salts. 
The following are the most commonly used basic aniline stains: 
Blue stains-methylene blue, thionin blue 1 (give the best differentiation; 
Red stains-basic fuchsin, safranin J difficult to overstain). 
Brown stain-Bismarck brown (weak, may be used as counter-stain). 
Green stain-methyl green. 
Pink stain-eosin (weak, may be used as counter-stain). 
Violet stains-methyl violet, gentian violet, crystal violet (most intense 
stains; may overstain). 
These dyes are all more or less crystalline powders, and while some are 
definite chemical compounds, others are mixtures. For this reason various 
brands are met with on the market and the exact duplication of stains is not 
always possible. Dyes should be obtained from reliable houses only. 
It is advisable to keep on hand stock saturated alcoholic solutions from which 
the staining solutions are made. These stock solutions are made by pouring 
into a bottle enough of the dye in substance to fill it to about one-quarter of its 
capacity. The bottle should then be filled with 95 per cent, ethyl alcohol, 
tightly corked, well shaken, and allowed to stand twenty-four hours. If at 
the end of this time all the staining material has been dissolved, more should 
be added, the bottle being again shaken and allowed to stand for another twenty- 
1 For a good, description of the composition and action of the various stains, see A. B. 
Lee's Microtomist's Vade-Mecum, 7th edition, 1913. 76 
PRINCIPLES OF MICROBIOLOGY 
four hours. This must be repeated until a permanent sediment of undissolved 
coloring matter is seen upon the bottom of the bottle. This bottle will then 
be labelled ''saturated alcoholic solution," of whatever dye has been employed. 
The dilution for use in staining is made by filling a small bottle three-fourths 
with distilled water, and then adding the concentrated alcoholic solution of 
the dye, little by little, until one can just see through the solution. It is some- 
times desirable to use a more concentrated solution with dyes such as methylene 
blue. Care must be taken that the color does not become too dense; usually 
about one part to ten is sufficient. 
General Observations on the Principles of Staining Bacteria.- 
The staining of bacteria is not to be considered simply as a mechanical 
saturation of the cell body with the dye, in which the latter is dissolved 
in the plasma. It is rather a chemical combination between the dye 
substance and the plasma. 
The dependence of the staining process upon the solvent condition of the 
dye is shown in the following observations: 
1. Entirely water-free, pure alcoholic dye solutions do not stain well. 
2. Absolute alcohol does not decolorize bacteria, while diluted alcohol is 
an active decolorizing agent. The compound of dye substance and plasma is 
therefore insoluble in pure alcohol. 
3. The more completely a dye is dissolved the weaker is its staining power. 
For this reason pure alcoholic solutions are inactive; and the so-called weak 
dye solutions to which strong dye solvents have been added are limited in their 
action on certain bacteria in which the dye substance is closely united. This 
is the principle of Neisser's stain for diphtheria bacilli, viz., acetic- acid- 
methylene-blue solution. 
On the other hand, the addition of alkalies to the dye mixture renders the 
solvent action less complete and the staining power more intense. According 
to Michaels, however, in Lbffler's methylene-blue solution the role of the 
alkali is purely of a chemical nature, by which it converts the methylene blue 
into methylene azure (azure II). 
The dependence of the staining process upon the nature of the microbe is 
exhibited in the following facts: 
Certain microbes stain easily, others with difficulty. To the latter belong, 
for example, the tubercle bacillus and lepra bacillus. Spores and flagella also 
stain with difficulty. The easily stained objects require but a minimum of 
time to be immersed in a watery solution, while the others must be stained by 
special dyes with or without the aid of outside influences (heat, mordants, 
etc.). The difficultly stained objects are at the same time not easily decolorized. 
The explanation of the resistance which these bacteria show to staining as well 
as to decolorizing agents is to be sought in two ways: either on the assump- 
tion that they possess a difficultly permeable or a resisting envelope, or that 
they have a special chemical constitution. The latter hypothesis holds good 
only, if at all, in regard to flagella and spores; while the assumption of the 
resisting envelope has reference more particularly to the tubercle bacillus, 
and is probably correct. The presence of fatty and waxy bodies in the envelope 
of these microorganisms is capable of demonstration. Moreover, after extrac- 
tion of these bodies by ether the tubercle bacillus loses its power of resisting 
acids, which peculiar resistance can also be artificially produced in other bacteria 
having normally no such resisting power. In many instances, doubtless, both 
of these causes, viz., resistant envelope and chemically different constitution, 
work together to produce the above-mentioned results. 
Elective staining properties, whereby certain species of organisms are exclu- 
sively or rapidly and intensely stained by certain dyes, have repeatedly been 
observed. Of the greatest practical importance in this respect is the Gram stain 
(see p. 78, and Chapter VI), used for the differential diagnosis of many species MICROSCOPIC METHODS 
77 
of bacteria; although a distinct classification of bacteria into those which are 
stained and those which are not stained by Gram's solution has been shown 
to be impracticable. There are some bacteria, however, which act uniformly 
toward Gram under most conditions; as, for example, the anthrax bacillus and 
the pyogenic cocci are always positive, the cholera and plague bacilli and 
gonococci are always negative to Gram. Other species again are at one time 
stained and at another decolorized by Gram; thus pyocyaneus is stained only 
in young individuals. Previous heating or extraction with ether does not 
prevent the action of Gram's stain, but treatment with acids or alkalies renders 
it impossible. Bacteria so treated, however, after one hour's immersion in 
Loffler's mordant regain their property of staining with Gram. 
As to the nature of Gram's staining solution, it may be mentioned that only 
the pararosanilines (gentian violet, methyl violet, and Victoria blue) are suit- 
able for the purpose, whereas the rosanilines (fuchsin and methylene blue) 
give negative results. The reason for this is that the iodine compounds with 
the pararosanilines are fast colors, while those with the rosanilines are unstable. 
These latter compounds when treated with alcohol break up into their con- 
stituents, the iodine is washed out, and the dye substance remaining in the 
tissues stain them uniformly; that is, without differentiation. But iodine- 
pararosaniline compounds are not thus broken up and consequently stain 
those portions of the tissue more or less, according to the affinity which they 
have for the dye substance. The parts stained by Gram are thus distinguished 
from those stained violet, not only quantitatively, but qualitatively; it is not 
a gentian violet, but an iodine-pararosaniline staining which occurs. 
Mordants and Decolorizing Agents.-We have already noted that the 
protoplasm of unrelated microbes may respond differently to the several 
dyes. There is, however, seldom any difficulty in selecting a dye which 
will stain sufficiently to make microbal cells in pure cultures distinctly 
visible. When the microbes are imbedded in tissue or mixed in a film 
with blood or pus, it is frequently difficult to prevent the stain from 
so acting on the tissue or pus elements as to obscure the organisms. 
Various methods are then employed to stain the germs more intensely 
than the tissues or to decolorize the tissue more than the organisms. 
Heating, the addition of alkali to the staining fluid and prolonging the 
action of the dyes increase the staining properties. We regulate 
these so as to give the best results. We also use mordants; that is, 
substances which fix the dye to the bacterial cell, such as aniline oil or 
solutions of carbolic acid on metallic salts. The decolorizing agents 
used chiefly are mineral acids, vegetable acids, diluted alcohols, various 
oils, and hot water. 
Formulae of the More Generally Used Staining Mixtures.-Loffler's 
Methylene-blue Solution.-Concentrated alcoholic solution of methylene 
blue, 30 c.c.; caustic potash in a 0.01 per cent, solution, 100 c.c. (The alkali 
not only makes the cell more permeable, but also increases the staining power 
by liberating the free base from the dye.) 
Films stained two to five minutes, heated if more intense stain is desired. 
Sections stained one-quarter to several hours and decolorized until faint blue; 
contrast stain eosin; washed, dehydrated, cleared, and mounted. 
Carbol-Fuchsin, or Ziehl-Neelsen Solution.- Distilled water, 100 
c.c.; carbolic acid (crystalline), 5 gm.; alcohol, 10 c.c.; fuchsin, 1 gm.; or it 
may be prepared by adding to a 5 per-cent, watery solution of carbolic acid 
the saturated alcoholic solution of fuchsin until a metallic lustre appears on 
the surface of the fluid. The carbolic acid, like the alkali, favors the penetration 
of the stain. 78 
PRINCIPLES OF MICROBIOLOGY 
The last two methods, combined with heating, are used to stain spores and 
certain resistant bacteria as the tubercle bacilli and other "acid resisters," 
so that they retain their color when exposed to decolorizing agents (see below). 
Carbol-Gentian Violet.-One part of saturated alcoholic solution of gentian 
violet to 10 parts of a 5 per-cent, solution of carbolic acid. 
Carbol-methylene blue, first used by Kiihne, consists of 1.5 gm. of methylene 
blue, 10 gm. of absolute alcohol, and 100 c.c. of a 5 per-cent, solution of carbolic 
acid. Carbol-thionin consists of 10 parts of a saturated alcoholic solution of 
thionin and 100 parts of a 1 per-cent, solution of carbolic acid. 
Polychrome Methylene Blue (Goldhorn).-To prepare the stain dissolve 
1 gm. lithium carbonate in 200 c.c. clean water and add 1 gm. methylene blue. 
Shake and dissolve. Pour into porcelain dish over water-bath, stirring fre- 
quently until blue color changes to a rich purple. Run through cotton in 
funnel; make up to 200 c.c. To 100 c.c. add 5 per cent, acetic acid until a 
faint pink is just visible on litmus paper above level of point discolored by 
the dye. Now add the remaining 100 c.c. of dye and allow to stand in open 
dish for forty-eight hours. Run once more through cotton into clean bottle. 
It is not necessary to use distilled water, and satisfactory results are ob- 
tained with all the different forms of methylene blue tried. B-X (Griiebler) 
is preferable. 
Fix the smear by immersion in commercial wood alcohol for fifteen to 
thirty seconds; wash well and stain for about ten to fifteen seconds in poly- 
chrome; wash and stain for from fifteen to sixty seconds in per cent, 
aqueous eosin. Wash again in water and dry in air without heat. Body of 
parasites blue; chromatin is red to purple. 
Results may be varied by using polychrome or eosin for different lengths 
of time. Admirable preparations may be obtained, even when there is pre- 
cipitation, by just rinsing the smear a little in 50 per cent, ethyl alcohol. 
This will remove any precipitation. 
Koch-Ehrlich aniline-water solution of fuchsin or gentian violet 
is prepared as follows: To 98 c.c. of distilled water add 2 c.c. aniline oil, or, 
more roughly but with equally good results, pour a few cubic centimeters of 
saturated aniline oil into a test-tube, then add sufficient water nearly to fill it. 
In either case the mixtures are thoroughly shaken and then filtered into a beaker 
through moistened filter paper until the filtrate is perfectly clear. To 75 c.c. 
of the filtrate (aniline-oil water) add 25 c.c. of the concentrated alcoholic solu- 
tion of either fuchsin, methylene blue, or gentian violet, or add the alcoholic 
solution until the aniline water becomes opaque and a film begins to form on 
the surface. 
Gram's Stain.-Another differential method of staining which is employed 
is that known as Gram's method. In this method the objects to be stained 
are floated on or covered with the aniline or carbolic gentian-violet solution 
described above. After remaining in this for a few minutes they are rinsed 
in water and then immersed in an iodine solution (Lugol's), composed of iodine, 
1 gm.; potassium iodide, 2 gm.; distilled water, 300 c.c. In this they remain 
for from one to three minutes and are again rinsed in water. They are then 
placed in strong alcohol until most of the dye has been washed out. If the 
cover-glass as a whole still shows a violet color, it is again treated with the 
iodine solution, followed by alcohol, and this is continued until no trace of 
violet color is visible to the naked eye. It may then be washed in water and 
examined, or before examination it may be counter-stained for a few minutes 
by a weak solution of a contrasting dye, such as eosin, fuchsin, carmine, or 
Bismarck brown. This method is useful in demonstrating the capsule which 
is seen to surround some bacteria-particularly the pneumococcus-and also 
in differentiating between varieties of bacteria; for some do and others do not 
retain their stain when put in the iodine solution for a suitable time (see Chapter 
VI, for further remarks upon Gram's stain; see also pp. 76-77). 
The Modifications of Gram's Stain are Many.-One only is given here. MICROSCOPIC METHODS 
79 
Nicolle's Modification.-Stain cold in carbol-gentian violet one minute; 
wash in tap-water; stain cold in the iodine mixture 1 minute; wash in tap-water; 
decolorize ten seconds in acetone (1 part) and alcohol (3 parts); wash in tap- 
water; counter-stain ten seconds in dilute carbol-fuchsin (1 to 10). 
Staining of Capsules.-Many methods of demonstrating the cap- 
sule has been devised. Two only will be given here. 
Welch's glacial acetic acid method is as follows: (1) Cover the prepara- 
tion with glacial acetic acid for a few seconds; (2) drain off and replace with 
aniline gentian-violet solution; this is to be repeatedly added until all the acid 
is replaced; (3) wash in 1 to 2 per cent, solution of sodium chloride and mount 
in the same. Do not use water at any stage. The capsule stains a pale violet. 
(.See Plate III, Fig. 15.) 
Fig. 13 
Hiss' Copper Sulphate Method (Fig. 12).-The organisms are grown, 
if possible, on ascitic fluid or serum media. If not, the organisms should be 
spread on the cover-glass mixed with a drop of serum, or better, with a drop 
of one of the diluted serum media. Dry in the air and fix by heat. 
The capsules are stained as follows: A 5 per cent, or 10 per cent, aqueous 
solution of gentian violet or fuchsin (5 c.c. saturated alcoholic solution gentian 
violet to 95 c.c. distilled water) is used. This is placed on the dried and fixed 
cover-glass preparation and gently heated for a few seconds until steam arises. 
The dye is washed off with a 20 per cent, solution of copper sulphate (crystals). 
The preparation is then placed between filter paper and thoroughly dried 
(Plate III, Figs. 14 and 16). 
Capsule stain by Hiss' method. Rhinoscleroma bacillus. X 1000. (Thro.) 
Staining Spores and Acid-fast Bacteria.-We have already noted 
that during certain stages in the growth of a number of bacteria spores 
are formed which refuse to take up color when the bacteria are stained 
in the ordinary manner. Special methods have been devised for causing 
the color to penetrate through the resistant spore membrane. 
In the simplest method a cover-slip after having been prepared in the usual 
way is covered with Ziehl's carbol-fuchsin solution and held over the Bunsen 
flame until the fluid steams. This is continued for one or two minutes. It 
is then washed and dipped in a decolorizing acid solution, such as a 2 per cent, 
alcoholic solution of nitric acid, or a 1 per cent, solution of sulphuric acid in 
water, until all visible color has disappeared, then it is washed and dipped for 80 
PRINCIPLES OF MICROBIOLOGY 
one-half minute in a saturated watery solution of methylene blue. The bodies 
of the bacilli are blue and the spores red. This same method is used for stain- 
ing acid-fast bacilli. Sometimes the spores refuse to take the stain in this 
manner. We then can adopt Moeller's method, which is designed still further 
to favor the penetration of the coloring matter through the spore membrane. 
The prepared cover-slip is held for two minutes in chloroform, then washed 
off in water, and placed from one-half to three minutes in a 5 per cent, solution 
of chromic acid, again washed off in water, and now stained by carbolic fuchsin, 
which is steamed for several minutes. The staining fluid is then washed off 
and the preparation decolorized in a 3 per cent, solution of hydrochloric acid 
or a 5 per cent, solution of sulphuric acid. The preparation is finally stained 
for a minute in methylene-blue solution. The spores will be red and the body 
of the cells blue. The different spores vary greatly in the readiness with which 
they take up the dyes, and we have, therefore, to experiment with each variety 
as to the length of time it should be exposed to the maceration of the chromic 
acid. Even under the best conditions it is almost impossible to stain some 
spores. Plate III, Fig. 22, show stained spores. 
Staining Flagella.-For the demonstration of flagella, which are 
possessed by all motile bacteria, we are indebted first to Loffler. The 
staining of flagella satisfactorily is one of the most difficult of bacterio- 
logical procedures. In all methods young (twelve-to-eighteen-hour) 
cultures of agar should be chosen. Enough of the culture to produce 
slight cloudiness is placed in a few cubic centimeters of filtered tap- 
water in a test-tube. This may be used immediately, or allowed to stand 
in the thermostat at blood heat for from one to two hours to permit 
slight development. A tiny drop of this rather thin emulsion is allowed 
to spread with as little manipulation as possible over the cover-glass 
so that it may dry quickly. This latter point seems to be the important 
one since slow drying allows the bacteria to shed their flagella. 
Bunge's modification of Lofiler's method is carried out as follows: Cover- 
glasses which have been most carefully cleaned are covered by a very thin 
smear. After drying in the air and passing three times through the flame the 
smear is treated with a mordant solution, which is prepared as follows: To 3 
parts of saturated watery solution of tannin add 1 part of a 25 per cent, solu- 
tion of ferric chloride. This mordant should be allowed to stand for several 
weeks before using. After preparing the cover-slip with all precautions neces- 
sary to cleanliness, the filtered mordant is allowed to act cold for five minutes, 
after which it is warmed and then in one minute washed off. After drying, 
the smear is stained with the carbol-fuchsin or carbol-gentian violet solution, 
and then washed, dried, and mounted. (Plate III, Figs. 18-21.) 
Frequently the flagella appear well stained, but often the process has to be 
repeated a number of times. Overheating of the film prevents the staining of 
the flagella. The cell membrane may also show by this method. 
Van Ermengen's method gives good results. It is as follows: The films are 
placed for one hour at room temperature, or are heated for five minutes over 
a water-bath at 100° C. in the following solution: 
Solution A. 
Osmic acid, 2 per cent, solution 1 part 
Tannin, 10 to 25 per cent, solution 2 parts 
Wash successively with water, absolute alcohol, and water, then place in 
the following solution for a few seconds: 
Solution B. 
0.5 per cent, solution of AgNOs in distilled water. MICROSCOPIC METHODS 
81 
Without washing transfer them to a third solution: 
Solution C. 
Gallic acid 5 grams 
Tannin 3 grams 
Fused potassium acetate 10 grams 
Distilled water 350 c.c. 
After keeping in this for a few seconds, place again in Solution B until film 
begins to turn black. Then wash and examine. 
Eosin-methylene-blue Stains.-These are polychrome staining mixtures 
introduced by Nocht, Romanowsky and others chiefly for the staining 
of animal cells, but they are also useful in differentiating bacteria and 
other germs in tissues and exudates. They are fine differential stains 
for chromatin. Many modifications have been proposed. 
In a study of the essential constituents of the Romanowsky stain, 
MacNeal says, both methylene azure and methylene violet are 
present and participating in the nuclear staining. The preparation 
of solutions directly from the pure dyes, methylene azure, methylene 
violet, methylene blue and eosin, has been recommended as the best 
manner of preparing these staining solutions as the proportion of the 
various constituents may be varied at will to obtain various kinds of 
differentiation. As a routine blood-stain for study of leukocytes and 
staining of hematozoa, the following is recommended by MacNeal: 
Solution A. 
Methylene azure 0.3 
Methylene violet (Bernthsen's, insoluble in water) 0.1 
Methylene blue 2.4 
Methyl alcohol, pure 500.0 
Solution B. 
Eosin, yellowish, water soluble 2.5 
Methyl alcohol, pure 500.0 
These solutions keep for at least a year. They are mixed in equal parts and 
diluted by the addition of 25 c.c. of methyl alcohol to each 100 c.c. of the mixture. 
This final mixture is employed in the same manner as Leishman's stain. It keeps 
for a few months. 
Giemsa's Method.-Smears are fixed in neutralized methyl alcohol for 
one minute. The staining solution is one of several recommended by Giemsa. 
Azur II-Eosin 3.0 grams. 
Azur II 0.8 gram. 
Glycerin (Merck, chem. pure) 250.0 c.c. 
Methyl alcohol (chem. pure) 250.0 c.c. 
Both glycerin and alcohol are heated to 60° C. The dyes are put into the alcohol and 
the glycerin is added slowly, stirring. The mixture is allowed to stand at room tem- 
perature overnight, and after filtration is ready for use. 
The solution is prepared ready for use by Griibler, Leipzig. 
1 drop of the stain to every c.c. of distilled water made alkaline by the previous 
addition of 1 drop of a 1 per cent, solution of potassium carbonate to 10 c.c. 
of the water is poured over the slide and allowed to stand for one-half to three 
hours. The longer time brings out the structure better, and in twenty-four 
hours well-made smears are not overstained. After the stain is poured off, 
the smear is washed in running tap-water for one to three minutes, and dried 82 
PRINCIPLES OF MICROBIOLOGY 
with filter paper. If the smear is thick, the organisms may come out a little 
more clearly by dipping in 50 per cent, methyl alcohol before washing in water; 
then the washing need not be as thorough. By this method of staining, the 
cytoplasm of protozoa stains blue and the nuclear substance a blue red or azure. 
Wright's Stain.-One per cent, methylene blue (alcohol rectified), and 0.5 
per cent, sodium carbonate are mixed and placed in a steam sterilizer for one 
hour. When cold 0.1 per cent, solution of extra B-A eosin (500 c.c. eosin to 
600 c.c. methylene-blue solution), is added until the mixture becomes purplish, 
and a finely granular black precipitate appears. This precipitate is filtered 
off and dried without being washed. A saturated solution of this is made in 
pure methyl alcohol. This is filtered and then diluted by adding to 40 c.c. of 
it 10 c.c. of methyl alcohol. In using, a few drops are placed on the film for a 
minute; then water is dropped on till a greenish irridescence appears. The 
stain then remains on for two minutes; then is washed off with distilled water, 
allowing a little to remain on until differentiation is complete. Dried with 
filter paper. 
Leishman's Stain-This is a modification of Jenner's stain which is simply 
a solution of eosin and methylene blue in methyl alcohol. Instead of ordinary 
methylene blue Leishman used the active constituents formed in this stain. 
Solution A.-To a 1 per cent, solution of medicinally pure methylene blue 
in distilled water add 0.5 per cent, sodium carbonate and heat at 65° C. for 
twelve hours, then allow it to stand ten days at room temperature. 
Solution B.-Eosin extra B-A (Griibler) 0.1 per cent, solution in distilled 
water. 
Mix Solution A and B in equal amounts and allow to stand six to twelve 
hours, stirring at intervals. Filter and wash the precipitate thoroughly. Collect, 
dry, and powder it. 0.15 gram is dissolved in 100 c.c. of pure methyl alcohol 
to form the staining solution. It keeps perfectly for at least five months. To 
stain, cover the dried but unfixed film of blood with the staining solution. 
After thirty to sixty seconds add about an equal amoun't of distilled water. 
Allow this mixture to act for five minutes. Wash in distilled water for about 
one minute, examining the specimen mounted in water under the microscope. 
Blot, dry thoroughly, mount in balsam, or preserve the specimen as an un- 
mounted film. 
Goldhorn's Method of Staining Spirochetes.-Dye; water, 200 cm.; lithium 
carbonate, 2 grams; methylene blue, 2 grams. (Merck's medicinal or a similar 
preparation.) This mixture is heated in a rice boiler with a moderate amount 
of heat until a rich polychrome has formed. This is determined by examining 
a sample against artificial light and noting the appearance of a distinctly red 
color. The solution is allowed to cool and the residue is removed by filtering 
through cotton. To one-half of this filtrate 5 per cent, acetic acid is gradually 
added until a strip of litmus paper shows above the line of discoloration a 
distinct acid reaction. The remaining half of the dye is now added, so as to 
carry the reaction back to a low degree of alkalinity. A one-half per cent. 
French eosin solution is now added gradually, while the mixture is being stirred 
until a filtered sample shows a pale bluish color with slight fluorescence. 
The mixture is allowed to stand for one day and filtered. The precipitate is 
collected on a double filter paper and dried at a temperature not exceeding 
40° C. It is then removed from the filter paper and dissolved in commercial 
wood alcohol. It is allowed to stand for one day in an open vessel and then 
filtered. 
To use the stain on smears sufficient dye to cover the smear is dropped on 
an unfixed preparation and allowed to remain for three or four seconds; the 
excess is then poured off. The slide is now introduced slowly into clean water 
with the film side down, is held there for four or five seconds and is then 
shaken in the water to wash off the excess of dye. It is then allowed to dry 
and is ready for examination. The pallidum stains violet. 
Until recently the demonstration in smears of the syphilis spirochete by MICROSCOPIC METHODS 
83 
the silver impregnation method, so successfully used by Levaditi in section, 
has been unsatisfactory. Stern, however, and Flexner corroborating him, 
have gotten beautiful results by the following simple method: 
(a) Air-dried into 37° incubator for some hours. 
(6) Ten per cent, aqueous silver nitrate for some hours (Flexner thinks three 
to four days' exposure better) in diffuse daylight. 
(c) When the brownish color reaches a certain tone (easily recognized after 
experience) and when a metallic sheen develops the slide is washed well in 
water, dried, and mounted. 
The blood cells are well preserved, they have a delicate dark brown contour, 
and contain fine light brown granules. The spirochetes are deep black on a 
pale brown and in places a colorless background. 
Other spirochetal organisms may be silvered by this method, but as they 
may be differentiated with greater difficulty than with Giemsa's stain, the 
latter should always be used as well. 
These organisms may also be demonstrated by the India-ink method 
(see p. 74). 
The flagella are brought out by Loffler's method or by the stain recom- 
mended by Goldhorn. 
Sections are prepared by the silver impregnation method of Levaditi as follows: 
Fix small pieces of tissue one-half mm. in thickness for twenty-four to forty- 
eight hours in formalin, 10 per cent. Wash in 95 per cent, alcohol twelve to 
sixteen hours. Wash in distilled water till the pieces sink. Impregnate two 
to three hours at room temperature and four to six hours at 50° C. in the follow- 
ing fluid: Nitrate of silver, 1; pyridine, 10 (added just before using); aq. dest., 
100. Wash rapidly in 10 per cent, pyridine. Reduce the silver by placing in 
the following mixture for several hours: Pyrogallic acid, 4; acetone, 10 (added 
just before using); pyridine, 15; aq. dest., 100. Harden in alcohol; xylol; 
paraffin. Levaditi's first method is longer but more reliable. Fix small pieces 
in formalin, 10 per cent. Harden in 95 per cent, alcohol. Wash in distilled 
water several minutes. Impregnate three to five days at 37° C. in 1.5 per cent, 
solution silver nitrate. Reduce twenty-four hours in: Pyrogallic acid, 4; 
formalin, 5; water, 100. Embed in paraffin. By these methods the spirochetes 
appear densely black. 
Giemsa's method gives excellent results. 
Ross' Method of Examining a Large Quantity of Malarial Blood in One 
Film.-A large drop of blood (about 20 c.mm.) is placed on a glass slide and 
is slightly spread over an area which can be covered by an ordinary cover- 
glass. This is allowed to dry in the air or it is warmed over a flame without 
heating it more than enough to fix the hemoglobin. The dry film is then 
covered with an aqueous solution of eosin (10 per cent.) and allowed to remain 
about fifteen minutes. This is then gently washed off and a weak alkaline 
methylene-blue solution is run over the film and left for a few seconds, when 
the preparation is again gently washed. After drying it is ready for examina- 
tion. 
The Eosin-methylene-blue Method Recommended by Mallory for Tissues 
may be Used for Smears as Follows:-The smears are fixed in Zenker's solution 
for one-half hour; after being rinsed in tap-water they are placed successively 
in 95 per cent, alcohol + iodine, one-quarter hour; 95 per cent, alcohol, one- 
half hour; absolute alcohol, one-half hour; eosin solution, twenty minutes, rinsed 
in tap-water; methylene-blue solution, fifteen minutes, differentiated in 95 per 
cent, alcohol from one to five minutes; and dried with filter paper. 
Staining Method for Negri Bodies (Williams' Modification of Van Gieson's 
Method).-Smears partially air-dried are fixed for ten seconds in neutral methyl 
alcohol to which 0.1 per cent, picric acid has been added. Excess of fixative 
removed by filter paper. Smears then stained in following solution: Saturated 
alcohol solution fuchsin, 0.5 c.c.; saturated alcohol solution methyene blue, 
10 c.c.; distilled water, 30 c.c. The stain is poured on the smear and held over 84 
PRINCIPLES OF MICROBIOLOGY 
the flame until it steams. The smear is then washed in tap-water and blotted 
with fine filter paper. The Negri bodies are magenta, the nerve cells blue, and 
the red-blood cells yellow, or salmon color (Plate X, Fig. 1). 
Heidenhain's Iron-hematoxylin Stain.-(a) Mordant and differentiating 
fluid: Iron oxydammonium sulphate, 2.5 grams; distilled water, 100 c.c. 
(6) Staining fluid: Hematoxylin, 1 gram; alcohol, 10 c.c.; distilled H2O, 90 c.c. 
(To be kept in a red bottle and allowed to stand for about four weeks before 
using.) For use see chapter on Ameba?. 
Preservation of Specimens.-Dry-stained preparations of bacteria 
keep indefinitely, but if mounted in Canada balsam, cedar oil, or 
dammar lac they tend gradually to fade, although many preparations 
may be preserved for many months or years. Dry unstained spreads 
should be kept in the ice-box until stained. 
Examination of Microorganisms in Tissues.-Occasionally it is of impor- 
tance to examine the organisms as they occur in the tissues themselves. The 
tissues should be obtained soon after death, so as to prevent as much as pos- 
sible postmortem changes, with consequent increase or decrease in the number 
of microbes. Selected pieces of tissues can be frozen by ether or carbon dioxide 
and sections cut, but the best results are obtained when the material is imbedded 
in paraffin or in celloidin. 
Fixing and Hardening Tissues.-From the properly selected spots small 
portions, not larger than one-quarter inch by one-eighth inch, are removed and 
placed in one of the following fixatives: 
1. Absolute alcohol for from four to eight hours, and longer if thicker. For 
the larger pieces it is better to change the .alcohol after eight hours. The pieces 
of tissue should be kept from falling to the bottom as the higher layers of alcohol 
remain nearer absolute. If along with the microorganisms one wishes to study 
the finer structure of the tissue, it is better to employ one of the other fixatives. 
2. Formalin.-For fixing in formalin the tissue is put in 4 to 10 per cent, 
formalin solution for three to twenty-four hours, and then in successive 
strengths of alcohol. 
3. Corrosive sublimate (saturated solution in 0.75 per cent, sodium chloride 
solution) is an excellent fixing agent. Dissolve the sublimate in the salt solu- 
tion by heat, allow it to cool; the separation of crystals will show that satura- 
tion is complete. For pieces of tissue one-eighth inch in thickness four hours' 
immersion is sufficient; for larger, twenty-four hours may be necessary. They 
should then be placed in pieces of gauze and left in running water for from twelve 
to twenty-four hours, according to the size of the pieces, to wash out the excess 
of sublimate. 
4. Hot sublimate alcohol (50° C.) (Schaudinn), or saturate sublimate, to 
which 5 per cent, glacial acetic acid may be added. The preparation should 
remain in it a few seconds, then should be washed for one-half hour in 60 per 
cent, iodine-alcohol, and then placed in 70 per cent, alcohol. They may 
remain here for an indefinite time, until they are to be stained, when they are 
rinsed in distilled water and then placed in the staining fluid. 
5. Two per cent, osmic acid (to be kept in a red glass with a ground-glass 
stopper). Moist smears are exposed to its fumes for a few seconds, small 
pieces for sections, four to eight hours, then carried through the various 
alcohols and xylol and mounted or imbedded in the usual way. 
6. Hermann's Fluid.-Platinum chloride, 15 c.c.; a 1 per cent, solution 
osmic acid, 4 c.c.; a 2 per cent, solution glacial acetic acid, 1 c.c. Moist 
spreads may be fixed for several minutes; very small pieces of tissue for 
twenty-four hours. 
7. Zenker's Fluid.-Add to a solution of Muller (bichromate of potash, 
2 to 2| parts; sulphate of soda, 1 part; water, 100 parts) 5 per cent, of saturated MICROSCOPIC METHODS 
85 
sublimate solution and, when ready to use, 5 per cent, of glacial acetic acid. 
Moist spreads arc fixed for one to five minutes, small pieces of tissue for three 
to twelve hours. They are then washed with water or put immediately into 
successive alcohols. 
To harden they are placed successively for twenty-four hours each in the 
following strengths of ethyl alcohol: 30 per cent., 60 per cent., and 90 per cent. 
Finally they are placed in absolute alcohol for twenty-four hours which de7 
hydrates them and they are then ready to be imbedded in paraffin. 
To imbed in paraffin, the pieces are put in (1) cedar oil until translucent, 
(2) cedar oil and paraffin, equal parts, at 52° C. for two hours, (3) paraffin 
52° C. two hours in each of two baths. They are then boxed ready for sec- 
tions. Sections are cut at 3 to 6y, and are dried at 36° C. for about twenty- 
four hours, protected from dust. 
The paraffin sections of tissue having been prepared and cut, they are ready 
for staining after the paraffin is removed. If all of the sublimate has not been 
removed by the water the sections may be immersed in iodine-alcohol for ten 
minutes. 
Loffler's Staining Method for Sections.-The section is placed in 
Loffler's alkaline methylene-blue solution for five to thirty minutes, decolor- 
ized for "a few seconds in 1 per cent, acetic acid. It is then placed in absolute 
alcohol, xylol, and Canada balsam. The number of seconds during which the 
preparation remains in the acetic acid must be tested by trials. 
References. 
Burri. Das Tuscheverfahren als Einfaches Mittel, etc., Jena, 1909. 
Giemsa. Deutsche med. Woch., 1905, xxxi, 1026. 
Kohler. Ztsch. f. wiss. Mikrosc., 1904, xxi, 129. 
Lea. Microtomist's Vade-mecum, 1913, 7th ed. 
Leishman. Brit. Med. Jour., 1901, p. 635- and 1902, p. 757. 
Metz. Ztschr. f. wiss. Mikroskop., 1913, xxx, 188. 
Reichert. Jour. Roy. Mier. Soc., 1907, 364. 
Wright. Jour. Med. Res., 1902, ii (New Series), 138. 
MacNeil. Jour. Infect. Dis., 1906, iii, 412. 
Williams. Am. Jour. Pub. Hyg., 1908, xviii, No. 1. CHAPTER IV. 
GENERAL METHODS USED IN THE CULTIVATION OF 
MICROORGANISMS. 
CULTURE MEDIA, METHODS OF ISOLATION AND CULTIVATION, 
CULTURAL CHARACTERISTICS. 
The methods employed for the artificial cultivation of microorganisms 
are of fundamental importance. By their use we can obtain one variety 
growing apart from all others, namely, in pure culture. This pure 
culture may be planted on various media and the morphologic, bio- 
chemic, and cultural characteristics studied for classification and identi- 
fication. It is evident that all glassware and instruments used must 
be free from other microorganisms; that is, they must be sterile. 
CULTURE MEDIA. 
Preparation of Glassware.-Various types of glassware, such as test-tubes, 
flasks, bottles, beakers, pipettes, etc., are used (see Figs. 14 to 22.) New glass- 
ware, as a rule, only requires mechanical cleansing with soap and water, loosen- 
ing adherent dirt with test-tube or bottle brushes. Old glassware containing 
cultures should be sterilized either in the autoclave or in a covered boiler. In 
the latter case the tubes are covered with water, about 5 per cent, of washing 
soda or soap added, and boiled for one hour. Any solid medium present is 
melted and the glassware can then be washed according to the directions already 
given. After washing, drain and allow to dry. 
Neutralization of Glassware.-Where slight changes of reaction are important 
the glassware should be neutralized after washing. Soak in 1 per cent, hydro- 
chloric acid for several hours or boil for one-half hour, wash free of acid, and rinse 
in distilled water. New glassware, especially the cheaper grades, is most likely 
to give off free alkali. 
The clean glassware is plugged loosely with ordinary non-absorbent cotton. 
The cotton should not be twisted in, as creases will form along the glass, leaving 
channels for contamination. Either fold into a plug or take a square of cotton, 
fold one corner, place a rod against the fold, and push the cotton into the neck 
of the container or tube. A sufficient amount of cotton should project for 
handling and the plug should be just tight enough to allow one to lift the con- 
tainer by means of the plug. Several thicknesses of filter paper may be used to 
cover beakers and other wide-mouth containers. 
Sterilization of Glassware.-All glassware is sterilized by dry heat. This is 
done after plugging. Some type of hot-air sterilizer is used (Fig. 24). Heat 
to 160° C. for one hour. Leave the sterilizer closed when the sterilization is 
finished, so that cooling is gradual or the glassware may crack. This heating 
not only sterilizes but sets the shape of the cotton plugs. 
Composition of Culture Media.-Most microorganisms causing 
disease require complex foodstuffs similar in constitution to those in 
the animal body. The general basis of media for these types is an CULTURE MEDIA 
87 
extract or infusion of meat. To this may be added peptone and sodium 
chloride. Some may require uncoagulated proteins, such as serum or 
Fig. 14 
Fig. 15 
Test-tubes can be also used without lip. 
Average size, 6 x f inches. 
Beakers. 
blood, or even fresh tissues. Carbohydrates may be added. The non- 
pathogenic microorganisms vary in their ability to grow on these more 
complex media, and some will only grow on simple media containing 
inorganic salts. Media may be either fluid or solid. In the latter case 
Fig. 16 
Fig. 17 
Fig. 18 
Globe flask. 
Volumetric flask. 
Erlenmeyer flask, 
there is added some jelly substance such as gelatin or agar or an 
albumin which is coagulated by heating. 88 
PRINCIPLES OF MICROBIOLOGY 
Certain technical methods are employed in the preparation of nearly 
all culture media, such as adjustment of reaction, clearing, filtering, 
Fig. 19 
Fig. 20 
Fig. 21 
Type of bottle com- 
monly used for dilutions, 
etc., or as substitutes for 
flasks. 
Blake bottle. Laid 
on its flat side it gives 
a large surface of broth 
or agar. 
Petri dish. Commonest size 
is 10 c.e. in diameter. 
etc. These details must be understood before the actual preparation 
of media is given. 
Fig. 22 
Types of fermentation tubes. 
Reaction of Media.-Titration and Adjustment.-A moderately alkaline reac- 
tion to litmus is satisfactory for the growth of most pathogenic microorganisms. 
For ordinary work the usual acid reaction of the media in preparation may be 
reduced by the addition of a 4 per cent, sodium hydrate solution until red litmus 
paper is turned slightly but distinctly blue and blue litmus paper remains the 
same color. Litmus, however, is not a delicate indicator and phenolphthalein 
is generally used, giving us more accurate knowledge of the reaction. The 
neutral points of the two indicators are different, so that media which are 
alkaline to litmus are still acid to phenolphthalein. The materials necessary 
for titration and adjustments of the reaction to phenolphthalein are normal 
and twentieth normal solutions of sodium hydrate and of hydrochloric acid, CULTURE MEDIA 
89 
a 0.5 per cent, solution of phenolphthalein in 50 per cent, alcohol, burettes, 
casserole, and stirring rod. 
Five c.c. of medium to be tested, 45 c.c. of distilled water, and 1 c.c. of the 
phenolphthalein solution are mixed in the casserole and boiled for two minutes. 
If no color is present the medium is acid and while hot the twentieth normal 
solution of sodium hydrate (^ NaOH solution) is added from a burette until 
a faint but distinct pink color appears. This color must remain on reheating, 
otherwise more alkali is needed. From the amount added we determine the 
acidity of the medium and estimate how much normal ($) solution of NaOH 
must be added to obtain the reaction desired, for example: 
Fig. 23 
Hot-air sterilizer. Lautenschlager form. 
Five c.c. required 2.4 c.c. of NaOH to neutralize, therefore 100 c.c. (twenty 
times as much) would require 2.4 c.c. of NaOH (twenty times as strong), 
in other words, the medium is 2.4 per cent, acid to phenolphthalein (+2.4 per 
cent.). Assuming we desire a reaction of +1 per cent., we must then add 2.4 
c.c. -1 c.c. or 1.4 c.c. of NaOH to every 100 c.c. of medium or 14 c.c. to a 
liter. 
Should, on the other hand, the mixture in the casserole show a pink color 
then the medium is alkaline and HC1 is used, heating as above, until only 
a faint pink color persists. If we use 0.5 c.c. then the medium is 0.5 per cent, 
alkaline (-0.5 per cent.), and it will require 0.5 c.c. of ? HC1 for every 100 c.c., 
or 5 c.c. for every liter to bring it to the neutral point. But we want the reaction 90 
PRINCIPLES OF MICROBIOLOGY 
to be +1 per cent, or 1 per cent, acid, we therefore add 5 c.c. plus 10 c.c. or 
15 c.c. of £ HC1 to each liter. 
After the first correction of the reaction by the addition of normal alkali or 
.acid the medium should be again titrated and further correction will usually 
be necessary. This may have to be repeated several times before the reaction 
desired is obtained. Various combinations, precipitation, etc., occur which 
make this necessary. 
Fig. 24 
Fig. 25 
Graduated Volumetric 
pipette. pipette. 
Obtainable in various sizes and graduations. 
Burettes. Most convenient type has a 
blue line against a white background, on 
back giving sharper readings. 
The reaction of media should always be adjusted before filtration, and after 
adjustment the medium must be heated for three to five minutes or precipita- 
tion will occur on subsequent sterilization, especially if sterilized in the auto- 
clave, due to the fact that the temperature is higher than that employed during 
the adjustment of the reaction. 
Titration of media may be done with the mixture in the casserole at about 
45° C. instead of the standard method given. The method used is not so impor- 
tant as the reproduction of a reaction which is satisfactory for the purpose in 
view. Where reactions are given it is assumed that they have been obtained 
according to the standard method. Any variations in procedure should be 
stated. CULTURE MEDIA 
91 
Clearing Media.-This is done by coagulation of an albumin, which, as it 
coagulates, enmeshes the fine particles. In certain methods the coagulation 
of the soluble albumins in the watery extract of the meat clears the medium when 
heated. Under other conditions an albumin such as eggs is added. If the medium 
is hot it must first be cooled to below 60° C. One or two eggs arc used for each 
liter. They are broken in a small pan and mixed with a small quantity of water 
by means of an egg-beater. This is then added to the medium, stirring thoroughly 
The medium is then heated to coagulate the egg albumins. 
Filtering Media.-For fluid media, paper or cotton is used. For media which 
solidify on cooling, cotton is preferable. Where media are cleared the coagulated 
albumins which settle on the paper or cotton act as a filtering medium. If 
paper is used it should be folded or a corrugated funnel employed. If cotton is 
used a spiral of wire or a perforated porcelain plate is placed in the funnel to 
support the cotton and two strips of cotton laid crosswise in the funnel, the 
torn ends extending up the side of the funnel. Before filtration the paper or 
cotton is wet with water so that any fat in the 
medium will not pass through. In wetting the 
cotton, moisten the torn ends so they adhere to the 
glass and then carefully moisten the rest. The 
medium is poured into the funnel along a glass rod, 
which is helpful in holding the cotton in place when 
this is used. The filtrate is poured back until 
it comes through clear. In the case of media which 
solidify on cooling the funnel should be heated before 
adding the medium and the medium awaiting filtra- 
tion kept hot. The use of a hot-water funnel to keep 
the funnel and its contents hot is a great advantage. 
Failing this the flask and funnel may be placed in an 
Arnold sterilizer. 
Preparation of Media for Use.-After filtration 
the medium can be either placed in flasks for storage 
or placed in various containers for immediate use 
and sterilized. In filling, care should be taken not 
to wet the necks of the containers or the cotton 
plugs will stick. Test-tubes and other small con- 
tainers are best filled from a funnel with a stop- 
cock or fitted with rubber tube and glass tube with 
pinch-cock. The amount in each container will de- 
pend on the use to which it is to be put. For the 
ordinary 15 cm. (6-inch) test-tube a depth of media 
of about 3 to 5 cm. is sufficient. If plates are to be 
poured at least 10 c.c. should be placed in a tube. If 
the medium is to be slanted in the tube, less is required. The slants may be 
made immediately after sterilization or the media melted and slanted as re- 
quired. To make the slants the tubes should be laid in a row on a table with 
a glass rod or strip of wood to raise the upper end. Care should be taken that 
the slant does not touch the cotton and sufficient media be present in the 
bottom of the tube ("butt") so that on raising the tube the slant will not fall 
down. The medium should be well set before raising. 
Sterilization of Media.-Sterilization may be accomplished by heat, by filtra- 
tion through various types of filters which do not allow the passage of bacteria, 
and occasionally by chemicals, as chloroform. Sterilization by heat may be 
accomplished in several ways-namely, fractional sterilization, intermittent 
sterilization, and sterilization by steam under pressure. 
Filtration- This method is resorted to where the application of heat will 
injure the solution, as in the case of some sugars, or where the necessary amount 
of heat would cause coagulation, as in the case of tissue extracts or serum. 
Various types of filters made of unglazed porcelain or compressed diatomaceous 
Fig. 26 
Hot-water funnel: a, point 
heat; b, inlet for water. 92 
PRINCIPLES OF MICROBIOLOGY 
earth are used. Types of these filters are the Berkefeld, Pasteur, Chamberland, 
and Doulton filters. The bacteria are held back because of the fineness of the 
Fig. 27 
Fig. 28 
Fig. 29 
Fig. 30 
Types of filters. 
pores. Various grades of fineness are procurable in some brands and on this 
depends the rapidity of filtration. In any case the permeability of the filter 
should be tested before use by filtering a broth culture of some small micro- 
organism, such as Bacillus prodigiosus. Be- 
fore filtration the fluid to be filtered should 
be rendered as clear as possible by filtration 
through paper or if necessary through paper 
pulp. The latter is prepared by soaking torn- 
up filter paper in water and then placing a 
layer in a porcelain filter, over a perforated 
porcelain plate, draining off the water and 
packing the layer tight. The depth of the 
layer will depend on the fluid to be filtered, 
its density, and the fineness of the particles 
to be removed. This should be fastened in 
the neck of a filtering flask and suction 
applied to hasten the filtration. Similar 
results may be obtained by filtration through 
sand. 
The filtration through a filter candle (the 
size selected will depend on the amount to 
be filtered) is accelerated by the use of suction 
or pressure. Suction may be secured by the 
use of a filter pump attached to the hydrant 
or other types of suction apparatus. Pres- 
sure may be obtained by air under pres- 
sure from an installed system or by a hand pump and cylinder to equalize the 
pressure. A manometer is employed to determine the pressure. The accom- 
panying cuts show the method of setting up the apparatus. 
Fig. 31 
Filter pump for attachment to 
water faucet. CULTURE MEDIA 
93 
The filter candle and all attachments with which the fluid will come in contact 
after passage through the candle must be sterile. The glassware can be sterilized 
in the hot-air oven (see below) and the filter candle and rubber connections can 
be sterilized by boiling for one hour or by steam under pressure. (See Autoclave.) 
After use the filter candle should be freed of all soluble material, especially 
coagulable matter, by running through it an abundance of clear water. If used 
for infective matter it can then be sterilized by boiling. In any case the surface 
should be lightly scrubbed with a fine brush after use. 
A new filter should be cleansed before use by filtering clear water. It should 
then be placed in cold water and boiled thoroughly. 
After continued use the candles gradually become clogged. They can be 
renewed in some degree by careful heating to glowing in an oven. This is 
apt to produce fissures, hence the filter should be retested before use. 
Fig. 32 
Fig. 33 
Arnold steam sterilizer. Two types. 
Fractional Sterilization.-This term is usually applied to sterilization at tem- 
peratures from 60° to 80° C. on successive days. The principle underlying 
this method and that of intermittent sterilization to be described is as follows: 
All bacteria when free from spores are killed by the exposure for one or two hours 
to the above temperatures. If the material be left overnight, at about 22° C., 
the spores, if present, will develop to bacteria and be destroyed by the second 
heating. Some of the bacteria may produce spores before the second heating 
or some of the spores may be slow in developing and escape. For this reason a 
third heating or with these low temperatures five or six heatings may be neces- 
sary. The method of fractional sterilization has only a limited application, viz., 
to material very slightly contaminated. It is applied to albuminous material 
before coagulation such as sera or tissue extracts and the temperature selected 
should be below the coagulation-point of the material, usually about 60° C. 
Coagulated media, where overheating will spoil the medium or render it too 
opaque, may be sterilized in the same way. All material to be treated in this 
manner should be as free from contamination as possible. The method has its 94 
PRINCIPLES OF MICROBIOLOGY 
best application to sterile material which, because of necessary manipulation, 
may become slightly contaminated. The heating may be done in a water bath 
or in a water-jacket oven. 
Fig. 34 
Fig. 35 
Upright type. Horizontal type. 
Autoclaves may be heated by direct application of heat, or by steam under 
pressure, when available. 
Intermittent Sterilization.-This term is applied to the heating of material to 
or close to the boiling-point of water on three successive days. The reasons for 
the repetitions are the same^as in fractional sterilization. In this method, how- CULTURE MEDIA 
95 
ever, only the very resistant spores escape. An Arnold sterilizer is usually 
employed (Figs. 32, 33). The material is heated in the flowing steam. The time 
of heating will depend on the size of the container employed; test-tubes of media 
usually require twenty minutes, whereas liter flasks of media would require 
forty-five minutes at least to allow the heat to penetrate sufficiently. 
Sterilization by Steam under Pressure.-This is done by means of an autoclave. 
Various forms are available which may be heated by gas or by steam (Figs. 34,35). 
All air should be displaced by the steam before closing the vent. Too rapid 
reduction of pressure will cause the media to boil up and wet or even blow out 
the plugs. The time and pressure employed will depend on conditions. As a 
rule, fifteen pounds' pressure is employed and twenty minutes are sufficient for 
media in test-tubes. Flasks should be heated one-half to one hour, depending on 
their size. Lower pressures are at times desirable where overheating is a factor, 
and the heating may be repeated on two successive days. The heating should 
be timed from the time the pressure desired is reached. The temperature will 
vary with the pressure thus: 
5 pounds' pressure = temperature 108.8° C. 
10 " " = " 115.6° C. 
15 " " = " 121.3° C. 
Storage of Media.-Media after sterilization may deteriorate for two reasons, 
namely, contamination (especially by molds penetrating the cotton stoppers), 
and by drying. 
The former may be avoided by keeping the media at a low temperature, 
40° to 45° C., in a dry refrigerator. Where media are used in small amounts 
it is best kept in flasks and tubed as needed. Evaporation of the tubed media 
can be lessened by dipping the plugs in paraffin or by the yse of rubber caps 
made for this purpose. The stock flasks after removal from the sterilizer may 
be sealed with paraffin or sealing wax. The plug should be cut off and pushed 
in slightly and melted paraffin poured on it, care being taken that it does not 
run through the plug into the media. This can be avoided if the paraffin is 
nearly cold enough to set. Sealing wax may be applied in the same way or 
rubber caps or rubber tissue tied over the neck. If the stock flasks are capped 
a small pledget of cotton moistened with bichloride of mercury solution may be 
placed between the plug and the cap to discourage the development of molds 
which may adhere during manipulation. The neck of the flask should be wiped 
free of the bichloride before pouring out the media. This precaution is specially 
useful for media stored in flasks at room temperature. The necks of all con- 
tainers may be protected from dust by covering with paper before sterilization 
or inverting tumblers over the necks of flasks. 
Preparation of Meat Infusion.-One pound (500 grams) of finely chopped 
meat, usually beef or veal, is macerated with 1000 c.c. of water and placed in the 
ice-chest for eighteen to twenty-four hours, or it may be extracted by heating 
to a temperature not exceeding 60° C. for one hour. The infusion is then strained 
through cheese-cloth and the meat squeezed out by twisting the cloth or by 
means of a meat press. The fluid contains the soluble albumins, extractives, 
salts, carbohydrates, and coloring matter of the meat. This forms the basis 
for the various media to be described. If this infusion is to be stored as such 
it must be boiled to coagulate the albumins (which also clears it), filtered, 
placed in flasks, and sterilized. Many advise the neutralization to phenol- 
phthalein1 before heating, on the ground that less of the foodstuff is precipitated 
than by heating the very acid infusion. 
As a substitute for the meat 2 to 5 grams of Liebig's extract of beef may be 
used for each liter of water. When media are made with this basis they are 
spoken of as meat-extract media in contradistinction to meat-infusion media. 
Nutrient Bouillon.-Meat infusion 1000 c.c., peptone 10 grams, and sodium 
chloride 5 grams. Warm the meat infusion to about 50° C., add the peptone 
1 See Reaction of Media. 96 
PRINCIPLES OF MICROBIOLOGY 
and salt and stir until dissolved. A convenient way is to use one's hand, as in 
this way the fluid will not get too hot, and the lumps of wet peptone are easily 
crushed. The mixture is then boiled over the free flame to coagulate the albu- 
mins, preferably after neutralizing the excess of acid. Evaporation should be 
made good by the addition of water. Correct the reaction and boil again for 
five minutes, filter, and place in the appropriate containers for sterilization. 
This medium may also be made from meat extract. 
Gelatin Media.-Meat infusion 1000 c.c., peptone 10 grams, sodium chloride 
5 grams, and gelatin ("gold label") 1001 grams. Dissolve as for nutrient broth. 
The acidity may then be reduced and the mixture then boiled or heated in the 
Arnold sterilizer for a half-hour. The reaction is now set and the media again 
heated for fifteen minutes and filtered. The clearing is done in this way by the 
albumins in the meat infusion. It should be tubed at once and sterilized in 
the Arnold sterilizer for twenty minutes on three successive days. Reheating 
and resterilization is to be avoided as the media will not set after too much 
heating. If the gelatin media is made from meat extract or with meat infusion 
which has been heated it must be cleared with eggs before filtration. In this 
case the ingredients are dissolved by heat and the reaction set. The mixture 
is then cooled below 60° C. and the eggs added; and then it is heated as 
above and filtered. 
Agar Media.-Two methods may be employed. The simplest method is to 
take 1000 c.c. of infusion or extract broth and add 1.5 per cent, of shredded 
agar. This is then dissolved by boiling over the free flame for one-half to three- 
quarters of an hour, making up the volume lost by evaporation. A better way 
is to place the mixture in the autoclave and heat for one-half to one hour at fifteen 
pounds' pressure, depending on the bulk. The reaction is then set, and the 
mixture cooled to below 60° C. and the eggs added. The mixture is again heated 
to coagulate the egg, either over the free flame or in the autoclave, and filtered. 
Another method of preparing agar is to make a double strength meat infusion, 
in which is dissolved double the amounts of peptone and salt and the excess 
acidity neutralized. To an equal quantity of water add 3 per cent, of agar 
and dissolve. Cool the agar below 60° C. and mix the two solutions. Set the 
reaction. Then heat for one-half to three-quarters of an hour to coagulate the 
meat albumin, which also clears the medium, correct the reaction if necessary, 
and filter. 
Peptone Solution (Dunham's).-Water 1000 c.c., peptone 10 grams, and sodium 
chloride 5 grams. Dissolve by heating and filter. The reaction needs no adjusts 
ment for ordinary use. 
Nitrate Broth.-Water 1000 c.c., peptone 1 gram, and nitrate-free potassium 
nitrate 0.2 gram. 
Milk.-The milk used should be as fresh as possible and preferably the best 
grade obtainable. Steam the milk in the Arnold sterilizer for a half-hour and 
place on ice for several hours or overnight to allow the cream to rise. By means 
of a siphon remove the milk from below the cream layer. This may be tubed 
or litmus solution added (litmus milk). If a good grade of milk is used no 
change of reaction is necessary; if acid, sufficient sodium hydrate solution 
should be added to render it slightly alkaline to litmus; if very acid, the milk 
should be discarded. 
Potato Media.-Large potatoes should be used and scrubbed with a nail 
brush under running water. Cylinders are then cut by means of an apple corer. 
The skin is cut off the ends of the potato cylinder and by an oblique cut, wedge- 
shaped pieces are obtained. A good butt should be left or the pieces will coil. 
While preparing the potatoes they'should be kept submerged in water or the 
cut surfaces will discolor. As the potato is acid the reaction must be changed. 
This is done by soaking in running water overnight or by soaking several hours 
in a 1 to 1000 solution of sodium carbonate. The pieces arq then placed in test- 
1 Use 120 grams in warm weather. ' CULTURE Al ED IA 
97 
tubes, a little water added to avoid drying, and sterilized. The test-tubes 
used should be one inch in diameter or over. If the pieces of potato are too 
small and thin they will curl on sterilizing. 
Glycerin potato for the cultivation of tubercle bacilli should be cut as above, 
soaked overnight in 1 to 1000 sodium carbonate solution, drained, and covered 
with a 5 per cent, glycerin solution for twenty-four hours. When tubed the 
glycerin solution or water is added for maintaining the moisture. 
Fig. 36 
Fig. 37 
Fig. 38 
Potato borer. 
Cylinder of potato, line of cut. 
Finished medium. 
Glycerin-potato Agar.-To extract agar add 5 per cent, potato juice and 5 
per cent, glycerin. (Potato juice: 1 pound of grated potato to 1000 c.c. of water. 
Soak overnight, strain, and boil. Clear with eggs and filter.) 
Semisolid Media.-Various types of semisolids are in use. The amount of 
agar will vary according to the use to which it is put. For slants, 0.75 per cent, 
to 1 per cent, of agar is necessary; for stab cultures, 0.5 per cent, or even less is 
sufficient. If the agar is to be diluted by the addition of serum or other enriching 
substance this must be allowed for in preparing the medium. The following 
is an example: Meat infusion (1 pound of meat to 500 c.c. of water) is warmed 
and 20 grams of gelatin and 20 grams of peptone dissolved in it. In 500 c.c. of 
water dissolve 10 grams of agar and cool to below 50° C. Mix the two, adjust 
the reaction, and heat to coagulate the albumin and clear the medium. Readjust 
the reaction and reheat if necessary; filter. A very satisfactory medium can be 
made by simply using 0.5 per cent, of agar instead of the usual 1.5 per cent, 
employed. This can be diluted by the addition of one-third of its bulk of an 
enrichment fluid and will still set sufficiently for stab cultures. 
Carbohydrates in Media.-Carbohydrates are added to media to determine 
whether acid or gas or both are produced or because of its utilization as a food- 
stuff to cause a more abundant growth on the part of the inoculated organisms. 
7 98 
PRINCIPLES OF MICROBIOLOGY 
Only the purest carbohydrates obtainable should be used. The following is a 
partial list of substances which may be used: 
Mono-hexoses-destrose, levulose, galactose, and mannose. 
Bi-hexoses-saccharose, maltose, and lactose. 
Tri-hexoses-raffinose. 
Polyhydric-alcohols-erythrite (tetra), adonite (penta), and mannite, sorbite, 
dulcite, and glycerin (hexa). 
Pentoses-arabinose, xylose, and rhamnose. 
Polysaccharids-glycogen, dextrin, and inulin. 
Carbohydrates are usually added to media in 1 per cent, amounts, with 
the exception that 5 per cent, of glycerin is used. Besides the true carbo- 
hydrates, various glucosides, such as salicin, coniferin, etc., are used. They 
may be used in any medium, either solid or fluid. Where meat extract or 
meat infusion is used as a basis it must be remembered that this contains 
fermentable sugar in muscle. If the media is to be used to determine whether 
a sugar is acted upon or not the muscle sugar must be removed before 
preparing the media. The meat extract or infusion to be used should be 
neutralized to phenolphthalein, and for each liter add a broth culture of 
B. coli or one of its allies. This is incubated for forty-eight hours. The bacillus 
ferments the sugar present. The infusion is now sterilized and used in the prepa- 
ration of media. Media made in this way are called "sugar-free media." To 
the sugar-free media the sugars may then be added. Serum water (Hiss) (see 
Serum Media) may also be used as a basis for carbohydrate media. 
Many of the carbohydrates are very susceptible to heat. They are split 
into simpler compounds which may be fermented by an organism unable to 
ferment the unaltered sugar and thus lead to error. The usual method is to 
add sufficient sugar to sterile water to make a 10 per cent, or 20 per cent, solu- 
tion and heat this in small containers in the Arnold sterilizer. This solution 
is then added to the media in sufficient amounts to give a final 1 per cent, 
content. 
Certain sugars, especially some samples of maltose, are so easily split by heat 
that they must be sterilized by filtration. 
In routine work with glucose, lactose, saccharose, mannite, and dulcite it 
is usually sufficient to add the sugar to the media and sterilize by intermittent 
sterilization. Although there may be a slight destruction of the glucose it is not 
enough to be important. Inulin is an exception. Because of the resistant 
spores commonly present the inulin solution should be sterilized in the autoclave. 
Fluid sugar media, with the exception of serum-water media, are usually 
filled into fermentation tubes, where gas production may be determined. Three 
types are used (Fig. 22, a, b, c, p. 88). The type (c) can only be used where 
sterilization is done in the Arnold sterilizer or autoclave. To fill these tubes, 
the small tube is first filled, and with as little inversion as possible, slipped mouth 
down into the large tube. Sufficient media is then added to allow the small 
tube to fill after expulsion of the air contained and leave the proper amount of 
media in the outside tube. The air in the under tube is driven out during 
sterilization. An indicator may be added to show the changes in reaction. 
If litmus is used the media should be very slightly alkaline to this indicator, 
or slight acid production will not become evident. In careful work, titration 
is the best method of determining the changes in reaction. 
Litmus and Other Indicators.-The ordinary litmus of commerce is not a very 
delicate indicator because of the impurities present which give it a reddish color. 
A purified litmus (Merck or Kahlbaum), or a solution such as that known as 
"Kubel and Thiemann," should be used. The purified litmus comes in the dry 
form and should be pulverized and added to distilled water in 5 per cent, 
amounts. This is steamed in the Arnold sterilizer for two hours, shaking 
the mixture every twenty minutes. The solution is then filtered and the 
filtrate sterilized. The solution must be kept sterile. Usually 5 per cent, 
of the litmus solution is added to the media. This may be varied to suit CULTURE MEDIA 
99 
personal preferences. Media to which litmus is added should be carefully ad- 
justed to a slightly alkaline reaction to litmus or slight changes will be obscured. 
(In place of litmus a 1 per cent, solution of Kahlbaum's azo-litmus may be 
employed.) Litmus media may be decolorized by the growth of bacteria which 
rob it of its oxygen and reduce it to the colorless leukobase. The color will 
return on exposure to the pir. Where stab cultures are used it may be necessary 
to melt the medium and pour it into Petri dishes to get sufficient air exposure 
to cause the color to reappear. Where the color change of individual colonies 
is to be observed, the diffusion of any acid produced may be prevented by using 
3 per cent, agar instead of 1.5 per cent. 
Various aniline dyes which are reduced to their colorless leukobase by .the 
action of sodium sulphite may be used as indicators. When the sugar is split 
by the bacteria, aldehydes are produced, which cause the color to return. Free 
acids do not make any change. The decolorized dye must be freshly prepared 
and the medium prepared with it used at once as exposure to the air causes the 
color to reappear. An example of such an indicator is the use of decolorized 
fuchsin in Endo's media. (See Typhoid Media.) Media to be used with this 
indicator should be slightly alkaline to litmus. 
Powdered insoluble carbonates may be used in solid plating media. If the 
powder is evenly distributed throughout the media, acid-producing colonies 
will be surrounded by a clear area. 
Phenolphthalein is also employed. The medium should be faintly alkaline 
and sufficient 1 per cent, alcoholic solution added to give a distinct pink color. 
The color is discharged when acid is produced. 
Media containing indicators are preferably sterilized in the Arnold sterilizer. 
The autoclave may be used in emergencies, but the indicator is somewhat 
injured. 
Use of Insoluble Carbonates.-Either crushed marble or calcium carbonate 
powder may be used. About 1 per cent, by bulk is sufficient. Their main 
application is in fluid media, especially as carbonate broth for the streptococcus- 
pneumococcus group. 
The value of the carbonates is that they neutralize the acids that may be 
produced by the growth of the bacteria, and which might prevent further devel- 
opment or even injure the viability of the culture. Sugars may be used, there- 
fore, in media containing carbonates, and the growth and viability of the cul- 
ture thus increased. The exact manner in which the calcium helps is not known, 
but it probably has some value. Where the medium is used in large amounts 
the containers should be shaken from time to time. 
Fig. 39 
Serum inspissator (Park). Ringstands are used for support, and the tilt is easily 
varied as needed. 
Media Solidified by Coagulation of Albumin.-Loffler's Bloodserum.-Mix 
3 parts of calf or sheep serum and 1 part of neutral nutrient broth to which 1 
per cent, of dextrose has been added. The mixture is then run into tubes and 
the tubes slanted in an apparatus where the temperature can be slowly raised to 
between 80° and 90° C. The heating Jn any case should not exceed 95° C. 100 
PRINCIPLES OF MICROBIOLOGY 
until the medium is coagulated or bubbling will occur and the surface will be 
spoiled. A Koch serum coagulator may be used for this purpose or a small 
water-oven or even in an Arnold sterilizer, covering the top with a cloth instead 
of the usual cover. A convenient inspissator we have devised is shown (Fig. 39). 
It has the advantage that the temperature can be controlled and each tube 
equally heated. After coagulation the tubes should be sterilized in the Arnold 
sterilizer for twenty minutes on two successive days. The medium may be 
coagulated and sterilized in the autoclave by allowing the temperature to rise 
very slowly until 110° C. is reached. This is rapid and convenient, but it has 
seemed to us that the high temperature injured the medium somewhat. 
The serum is obtained at the slaughter-house in tall cylindrical vessels. These 
should be disturbed as little as possible till coagulation is complete. If the 
coagulum adheres to the sides of the vessel, it should be loosened with a glass 
rod. After twenty-four hours on ice the serum is pipetted or siphoned off. If 
bloody it can be placed on ice to allow the corpuscles to settle out. 
Coagulated Blood-serum Media.-Serum from the cow, horse, sheep, or dog 
may be coagulated in the form of slants. This type of medium has been used with 
and without the addition of glycerin for cultivation of tubercle bacilli, but a 
more satisfactory medium can be prepared from eggs. The serum used should 
be sterile and, after tubing, heated to 70° C. for one hour. If a clear medium is 
desired the best way is to remove a tube from time to time for observation and 
cease heating when set sufficiently. Higher temperatures or too long an exposure 
render serum opaque. 
Serum may be coagulated in high columns in test-tubes especially for culti- 
vation of spirochetes. The tubes should be warmed to 65° C. and a few at a 
time placed in water at 75° C. These are tilted every few seconds and removed 
as soon as they start to set. The heat retained in the tube will complete the 
coagulation. In this way a soft almost transparent coagulum is formed. 
The serum may be diluted with saline broth, etc., for special purposes and 
treated in the same way. 
Satisfactory media of the above types require serum free from red cells or 
dissolved hemoglobin. 
Ftg. 40 
Tin pie plate 
-Cheese cloth 
Funnel 
- Rubber tubing 
, - Pinch code 
.Glass tube 
-Perforated cork 
Glass tube, large enough 
to admit test tube 
Straining and filling apparatus. (Krumwiede) The lower tube is plugged with cotton 
and the top wrapped. It is necessary to loosen the pinch-cock before heating. The whole 
is sterilized in the autoclave. 
Egg Media.-Freshly laid eggs are sterile and should be employed for the 
preparation of media. Egg media are usually coagulated. 
Plain Egg (Dorset).-The eggs are thoroughly cleansed with water of any 
adherent dirt and then washed with 5 per cent, carbolic solution and allowed CULTURE MEDIA 
101 
to partially dry. The ends are then gently dried in the flame and pierced with 
a burned sharp forceps. The hole at one end should be about three-eighths 
inch in diameter and the membrane broken; the other which is to be blown into 
should be smaller and the membrane left unbroken if possible. The eggs are 
then blown into a sterile Erlenmeyer flask, the blowing being done from the 
cheeks, which will help avoid spilling saliva and leakage of air around the out- 
side of the egg. To the egg is then added 10 per cent, of water by volume of the 
weight of the eggs. This is mixed by twirling the flask or by gently stirring 
with a glass rod. Bubbling must be avoided. The mixture is then filtered 
through cheese-cloth by gravity and tubed. (See Apparatus, Fig. 40.) The 
tubes are then slanted and coagulated by heating to 70° C. for two to two and a 
quarter hours on two successive days (see Fig. 36 for apparatus). No further 
sterilization is employed. The medium is incubated to test its sterility. 
Glycerin Egg (Lubenau).-Ten eggs are blown into a flask and 200 c.c. of 
glycerin broth (5 per cent, glycerin, neutral or slightly alkaline to litmus) added. 
The further preparation is the same as the preceding. 
If the media requires it a drop or two of water can be added after coagulation 
to supply the necessary moisture. To conserve the moisture the plugs should 
be paraffined or cut off and burned and a charred cork used to seal the tube. 
Addition of Serum or Other Enriching Substances.-Serum, blood, albuminous 
body fluids, or tissue extracts containing uncoagulated albumins may be added 
to media. Usually 1 part to 2 or 3 parts of medium are used. If agar is used 
it must be cooled to 50° C. after melting before mixing. If slants are to be made 
they should be allowed to set thoroughly preferably over night before raising 
them or they may slip down. For certain work it may be necessary to use a 2 
per cent, agar to get sufficient stiffness so that the surface is not easily broken; 
this holds especially for media for plating where surface inoculation is done. 
Media thus prepared is spoken of as blood-agar or broth, ascitic agar or broth, 
serum-agar or broth, etc. 
Watery extracts of tissues, especially of placenta, are used in the same manner; 
500 grams of tissue to a liter of water are used and extracted as for meat infusion, 
but no heat applied. After preliminary filtration through paper or sand, the 
fluid is sterilized by filtration through a filter candle. 
Nutrose may be used in media in 1 per cent, amounts to aid growth. 
Serum Media.-For special purposes serum or other albuminous fluids, such 
as ascitic fluid, are used. They' may be used as such or diluted with saline or by 
the addition of nutrient media. For these special purposes, sterilization by 
heat or even filtration should be avoided. 
Serum-water Media.-If serum is dialyzed to remove the salts it can be 
heated to 100° C. without coagulation. Serum diluted with two to three times 
its volume of distilled water can be sterilized in the Arnold sterilizer. This is 
so-called "serum-water medium" (Hiss) to which 1 per cent, of sugar is added for 
fermentation tests. Acid production is shown by the change in the litmus 
added and by coagulation of the serum. Gas production is shown by the 
bubbles in the coagulum. 
Inulin serum-water medium is the most commonly used preparation of this 
type. (See under Carbohydrates.) 
A similar medium is prepared as follows: 
Inulin (or other carbohydrate) 4 grams 
Peptone 4 grams 
Water 200 c.c. 
Litmus 5 per cent, solution. Sufficient to give a deep blue color. 
Dissolve and tube (2 to 3 c.c. to a tube) and sterilize. The method depending 
on the sugar used and precautions already given. After cooling add to each 
tube an equal quantity of sterile ascitic fluid or serum. 
Blood-media.-Blood for media is most easily obtained from larger animals 
by introducing a trocar into the jugular. From smaller animals anesthesia 102 
PRINCIPLES OF MICROBIOLOGY 
is used and the carotid is dissected out for the introduction of the trocar or 
cannula. Small quantities of blood may be obtained by heart puncture with a 
syringe. The steps must be aseptic as the blood cannot be sterilized. The 
fluid blood may be mixed with the medium at once or it is defribrinated or mixed 
with citrate solution when it is to be stored. One part of a 10 per cent, sodium 
citrate solution is diluted to nine parts of blood. 
The blood may be mixed with solid or fluid media as already described. 
For streptococci and its allies a good medium is obtained by placing a drop 
of blood on the surface of an agar slant. For certain purposes the agar is used 
in Petri dishes with a drop of blood on the surface. The blood is streaked out 
during inoculation. 
Hemoglobin Media.-Certain bacteria, like the influenza bacillus, require 
hemoglobin for their growth. This may be supplied by the addition of whole 
blood or by dilutions of the blood which has been laked in distilled water. A 
final dilution of 1 to 500 still gives a good growth. For agar slants the blood 
should be mixed with the agar at 80° to 90° C. For plate work the mixing 
should be below 55° C. 
Pancreatin Broth.-Hottinger makes the statement that the greater part 
of the nutritive elements of meat are lost in the usual process of making broth 
for laboratory use. To avoid this a process of slow digestion is recommended, 
with the addition of pancreatin, and it is claimed that in this way a much more 
favorable medium can be obtained for the growth of bacteria, with so much 
peptone as a result of the meat digestion that no addition of commercial peptone 
is required. Sodium chloride is also omitted. 
As carried out in this laboratory the slightly modified Hottinger process is 
as follows: 
Meat, 750 gm. 
Water, 1500 c.c. 
Meat, carefully freed from fat and fascia, cut in finger-thick pieces. 
Water heated to boiling. 
Meat dropped in piece by piece, with constant stirring. 
Boil up strongly and take from fire. 
Take out meat and put through cutting machine. 
Cool water to 370° C. 
Add sodium carbonate 1| gm. per liter to water. 
Put chopped meat in flasks (2-liter Erlenmeyer), 550 gm. per flask. 
' Flasks filled with water up to narrow necks. 
Add pancreatin siccum, 3 gm. per flask; chloroform, 10 c.c. per flask; toluol, 
10 c.c. per flask. 
Cork tightly and shake well. 
Incubate at 37° C. overnight. 
_ Shake next day and add more pancreatin unless the fluid shows a yellow color 
and particles of meat look smaller, showing that digestion is taking place. 
The process of digestion should continue for forty-five days at room tempera- 
ture or for two to three days in incubator, shaking well each day. At the end 
of this time the meat has become a finely divided mass, giving off a very offensive 
odor. 
According to Hottinger the medium may be stored in the ice-box at this point 
without again heating; after testing with litmus paper and acidifying with a 
slight amount of dilute HC1, if found to be alkaline. In our experience this 
plan has not worked well, and we have found it best to proceed at once as 
follows: 
Decant liquid through cheese-cloth. 
Add an equal amount of water to the residue in flask. 
Shake well. 
Allow meat to settle and again decant. 
Finally place meat on cheese-cloth and allow to drain. 
Boil the filtrate for a few minutes, then filter through absorbent cotton and 
paper until clear. CULTURE MEDIA 
103 
Store in flasks as pancreatin stock broth after autoclaving at 115 pounds' 
pressure for one-half hour. Use diluted to desired strength. 
Diluted one-half (one part stock broth, one part water) this broth gave excel- 
lent results with typhoid, paratyphoid, coli staphylococcus, dysentery Flexner 
and dysentery Mt. Desert. Diphtheria did not grow well in this medium. 
After successive transfers in different varieties of broth, cultures of typhoid, 
paratyphoid, and staphylococcus plated out showed a generally higher percent- 
age of colonies from the pancreatin broth one-half strength than from the regular 
veal broth. 
Pancreatin Agar.-Add sufficient water to pancreating stock broth to make 
the required dilution. 
Add agar 1.5 per cent. 
Heat in autoclave at 15 pounds' pressure for one hour. 
Cool to 55° C. and add eggs well beaten. 
Heat in autoclave one-half hour. 
Filter through cotton in hot-water funnel until clear. 
Titrate at a temperature as near as possible to 40° C. not above 50° C. 
Adjust to desired reaction and store in tubes or flasks after again autoclaving. 
Agar made with pancreatin broth of the 1 to 1 dilution used for successive 
slant agar cultures with typhoid, paratyphoid, coli, dysentery Flexner, dysentery 
Mt. Desert, staphylococcus, streptococcus, pneumococcus, and meningococcus 
gave equally good results as ordinary veal agar, but not as good growth with 
diphtheria and gonococcus. 
Dilutions of Media in Milk Examinations.-Berry has found that a steadily 
increasing ratio of colonies is obtained in milk plates made from both pancreatin 
and plain agars up to a broth dilution of 1 to 15. The size of the colonies 
varies, being on the whole slightly smaller with the higher degree of dilution, 
but the difference is not marked. Beyond 1 to 15 dilution of broth up to 1 to 
30, nearly as many colonies are obtained, but these are increasingly smaller 
and less distinct. 
Used Agar.-Tests have been made with used agar in order to ascertain 
whether it would be possible to utilize such agar a second time for culture 
purposes. 
The tests have been made with veal agar which had been used in Blake 
bottles for the preparation of various antigens and vaccines. 
After the surface growth is removed as far as possible, by scraping, or by wash- 
ing with salt solution, or with distilled water, the agar is melted and the different 
lots poured together as they come. The whole amount is then made neutral 
to phenolphthalein, tubed, sterilized, and again planted with various organisms. 
Except for a loss of transparency which can probably be restored by fresh 
clearing and filtering, the previous use of the agar seems in no way to affect its 
value as a culture medium. Typhoid, paratyphoid, coli, staphylococcus, strep- 
tococcus, pneumococcus, gonococcus, diphtheria, and other varieties of the 
ordinary laboratory organisms grow as vigorously as on new agar, and in some 
cases the growth is heavier on the old than on the new medium. Abundant 
cultures of amebse are also obtained on used agar. 
Special Media.-Bordet-Gengou.-Water 1000 c.c., glycerin 40 c.c., potatoes 
(sliced) 500 grams. Heat in an autoclave at 15 pounds' pressure for a half- 
hour. Pour off the liquid. To 500 c.c. of potato extract add salt solution 
(0.6 per cent.) 1500 c.c., and agar 60 grams. Autoclave to dissolve, filter and 
tube. When used an equal quantity of defirbrinated blood is added. 
Use of Fresh Tissue in Media.-Sterile fresh tissue may be added to media. 
This not only adds nutrient material but aids the production of anaerobic 
conditions. Smith used it in fermentation tubes for the growth of anaerobes, 
Williams uses it on agar for the growth of pure cultures of amebae, and Noguchi 
uses it in special media for spirochetes and other feeble-growing organisms. 
In the latter medium rabbit's kidney is most commonly used. Small pieces 
are added to fluid media or placed in test-tubes and serum or ascitic agar added. 
In the case of coagulated serum the tissue is pushed to the bottom of the tube. 104 
PRINCIPLES OF MICROBIOLOGY 
Synthetic Media.-For certain work it is an advantage to know the exact 
chemical constitution of the medium. Then, too, some bacteria, especially 
certain species in soil, refuse to grow on the more complex media. Pigment pro- 
duction is very easily observed on these media. 
Uchinsky's (Frankel's Modification).-Water 1000 c.c., asparagin 4 grams, 
ammonium lactate 6 grams, Na2HPO4 2 grams, NaCl 5 grams. 
Modified Formula for Pyocyaneus Used in Disinfection Tests.-Water 1000 c.c., 
asparagin 6 grams, sodium phosphate (ortho) 2 grams, sodium chloride 5 grams. 
Dissolve and if necessary render alkaline to litmus by addition of NaOH. 
Sterilize in small tubes and test for color production with B. pyocyaneus. 
Ringer's Solution.-Sodium chloride 10 grams, potassium chloride 0.2 gram, 
calcium chloride 0.2 gram, sodium bicarbonate 0.1 gram, glucose 1 gram, 
water 1000 c.c. 
For broth add 1 per cent, or 2 per cent, peptone. 
For agar add 1.5 to 2 per cent, agar and 1 to 2 per cent, peptone. 
Dissolve, clear, and filter. 
Use of Aniline Dyes in Media.-Various basic aniline dyes show a differential 
restraining action on the growth of bacteria. Gentian violet and allied dyes 
when present in a dilution of 1 to 100,000, preferably in agar, inhibits the growth 
of the Gram-positive group of bacteria but has no effect on the growth of the 
Gram-negative bacteria. This is a general rule. Exceptions are found and 
variations are noted with different dyes. Gentian violet may be employed to 
prevent growth of Gram-positive germs or for isolation of Gram-negative germs 
from contaminated material where the contamination is Gram-positive. An 
example of this is the use of crystal violet in Conradi medium. 
Certain dyes have a differential action on closely allied bacteria inhibiting 
completely or partially the growth of one, but allowing another to grow freely. 
An example of this is the action of brilliant green on the typhoid, paratyphoid, 
and colon bacilli. The colon types are completely or nearly completely restrained, 
typhoid less so, and paratyphoid least of all. This is applied in isolation of 
typhoid or paratyphoid from stools. 
The use of dyes as indicators has been already noted. 
Special Media for Typhoid, Paratyphoid, Dysentery, and Colon.-Numerous 
media are used. The most commonly employed plating media are Endo and 
Conradi-Drigalsky. 
Endo-medium (Kendall's Modification).-The basis is ordinary meat-extract 
agar, slightly alkaline to litmus. This should be sterilized in bottles in 100 c.c. 
amounts. When needed 1 gram of lactose is poured into a bottle and the agar 
melted which dissolves and sterilizes the lactose. To each bottle, after melting, 
is added 1 c.c. of decolorized fuchsin prepared as follows: To 10 c.c. of a 10 
per cent, watery solution of sodium sulphite add 1 c.c. of a saturated alcoholic 
solution of fuchsin and heat in the Arnold sterilizer for twenty minutes. Plates 
are then poured and allowed to harden without the covers, and dried in the 
incubator for thirty minutes, protecting the plates from dust. The medium 
must be mixed each time when needed and the plates used. The color returns 
gradually and the plates are then useless. 
Conradi-Drigalsky Medium.-Water 1000 c.c., agar 20 grams, sodium chloride 
5 grams, peptone 20 grams, nutrose 10 grams, beef extract (Liebig's) 4 grams, 
normal NaOH 50 c.c. Dissolve the ingredients in an autoclave, cool, and clear 
with eggs. Adjust reaction to a moderate but distinct alkalinity to litmus. 
To each liter of agar thus prepared are added 130 c.c. of Kubel and Thiemann 
litmus solution, crystal violet (1 to 1000 solution) 10 c.c., and 15 grams of lactose. 
Heat in an Arnold sterilizer ten minutes to obtain thorough mixing and fill in 
tubes or bottles and sterilize in Arnold sterilizer. Omit the crystal violet if 
to be used for dysentery. 
Russell's Double Sugar Medium.-To ordinary extract agar adjusted neutral 
to litmus add 1 per cent, of lactose and 0.1 per cent, of glucose and sufficient 
litmus to give a good color. Tube and slant, leaving a generous "butt" at bot- 
tom of tube for stab inoculation. CULTURE MEDIA 
105 
Brilliant Green Broth.-To extract broth neutral to phenolphthalein add 1 
per cent, of glucose and sufficient of a 0.1 per cent, solution of the dye to give 
dilutions of 1 to 500,000 down. The mixing is done immediately before use. 
Neutral Red Agar.-To extract agar add 1 per cent, of glucose and I per 
cent, of a saturated aqueous solution of neutral red. 
Neutral Red Lactose Peptone.-To peptone-water add 1 per cent, of lactose 
and 1 per cent, of a saturated aqueous solution of neutral red. Tube in fermen- 
tation tubes. 
Liver Broth.-Beef liver 500 grams, peptone 10 grams, dextrose 10 grams, 
di-potassium phosphate (K2HPO4) 1 gram, water 1000 c.c. The liver is boiled 
for two hours for extraction and strained. Then dissolve the other ingredients 
and adjust the reaction and sterilize. 
Bile Media.-For Typhoid Blood-cultures.-To ox bile add 10 per cent, glyc- 
erin and 2 per cent, of peptone. For Water Analysis. To ox bile add 1 per 
cent, of peptone and 1 per cent, of lactose. Tube in fermentation tube, and 
sterilize. In place of ox bile a 10 per cent, solution dried fresh ox gall can be 
used. 
Special Media for Cholera.-Peptone Solution (see p. 96). 
Saccharose Peptone Water (Bendick).-To 1000 c.c. peptone solution, neu- 
tralized to phenolphthalein add 1 gram of anhydrous sodium carbonate. Boil 
and filter. Add 5 grams of saccharose and 5 c.c. of a saturated solution of 
phenolphthalein in 50 per cent, alcohol. Tube and sterilize in Arnold 
sterilizer. 
Diendonne's Medium.-Mix equal parts of defibrinated beef-blood and normal 
sodium or potassium hydrate solution and steam in the Arnold sterilizer for a half- 
hour; 3 parts of this are added to 7 parts of 3 per cent, agar (neutral to litmus) and 
poured into Petri dishes (15 c.c. to a 10 cm. dish). Allow to harden uncovered, 
but protected by paper. Place strips of filter paper between the dish and cover 
to aid in the absorption of the moisture and ammonia and place in the incubator 
for twelve to fifteen hours. Nothing will grow on the medium when first made. 
The plates are good for about ten to fourteen days. Various modifications of 
this medium have been suggested. 
Pilon.-Substitute 12 per cent, sodium carbonate (crystalline) solution 
for the sodium hydrate. The plates can be used after thirty minutes' drying. 
Alkaline Egg Medium (Krumwiede).-Make an egg-water mixture, using 
equal parts of egg and water. Mix equal parts of the egg-water and 12 per 
cent, sodium carbonate (crystalline) and steam in the Arnold sterilizer for 
twenty minutes. Mix while hot 30 parts to 70 parts of 3 per cent, agar (the 
meat extract may be omitted) and pour on plates and dry for twenty minutes. 
If the meat extract is omitted the colonies are small but very characteristic. 
The colonies are much increased in size when extract is present or if 0.5 per 
cent, of glucose or saccharose is added. The reaction of the agar need not be 
corrected. Goldberger claims that the alkalinity is unnecessarily high, and 
makes the egg mixture as given with a 6.5 per cent, solution of anhydrous 
sodium carbonate and mixes 1 part of the egg with 5 parts of glucose extract agar. 
Special Media for Toxin Production, etc.-For Diphtheria Toxin (Banzhaf).- 
Lean veal free from fibrous tissues, minced, 1 pound to the liter of water. Soak 
overnight and then heat to between 45° and 55° C. for one hour. Then bring 
to a boil. Strain. To fluid add peptone 2 per cent, and sodium chloride 0.5 
per cent. Boil for a half-hour. Adjust reaction to 1.2 per cent, acid to phenol- 
phtalein. Place about 800 c.c. in 2-liter Erlenmeyer flasks and add 3 gram's 
of calcium carbonate. Sterilize in an autoclave at 5 pounds' pressure for a 
half-hour on two successive days. 
For Tetanus Toxin.-(1) One pound of lean veal per liter of water. Soak over- 
night at 37° C. If meat rises to top it is ready for use; if not, allow to stand 
another day. Heat to 45° to 55° C. for one hour, then boil. Strain, add peptone 
1 per cent., salt 0.5 per cent. Boil for half an hour, adjust reaction to + 1.6 per 
cent. Sterilize in globe flasks, medium reaching into neck, in Arnold sterilizer 106 
PRINCIPLES OF MICROBIOLOGY 
for one hour at a time for two days. Immediately after sterilization fill the 
neck of the flask with boiling paraffin or albolene. 
The Hygienic Laboratory (Washington, D. Cd) Method for Making Tetanus 
Toxin.-Media.-Chopped round steak, 500 grams; tap-water, 1000 c.c. Mix. 
Place in ice-box overnight, strain, and press out juice through cheese-cloth 
until 1000 c.c. are obtained. Boil to coagulate albumins, filter through paper, 
making up loss of evaporation. Add 1 per cent, peptone (Witte's), 0.5 per cent, 
sodium chloride, and 1 per cent, anhydrous glucose. Heat until peptone is in 
solution; titrate and adjust reaction to 1. Filter through paper and put in one 
liter flask. Sterilize in Arnold for ninety minutes. 
Preliminary Cultivation.-Fill some 7x1 inch test-tubes with glucose broth 
to within 2 inches of top of tube and sterilize. When still warm (about 35° to 
40° C.) inoculate and place in thermostat at 37° C. From the first tube inocu- 
late a second in twenty-four hours and so on for five to 
six days. 
Inoculation for Toxin.-From the last tube the 1000- 
c.c. flask of glucose broth (previously heated in the 
Arnold for one hour and cooled to 35° or 40° C.) is 
inoculated and placed in the thermostat at 37° C. 
for fifteen days. 
The culture is now passed through a Berkefeld filter 
and 750 grams of ammonium sulphate are added per 
1000 c.c. of filtrate. The brownish scum is skimmed 
off, placed between hard filter papers and the moisture 
pressed out, dried over sulphuric acid in vacuum for 
about three days, pulverized, and stored in U-shaped 
tubes with P2O5 at 5° C. 
Broth for Production of Mallein.-Meat infusion 2000 
c.c., potato juice (see under Potato) 2000 c.c. Mix and 
add peptone 80 grams and sodium chloride 20 grams. 
Boil ten minutes, adjust reaction to +1 per cent, to 
phenolphtalein. Boil, filter, add 4.5 per cent, glycerin. 
Put about 250 c.c. to flask and sterilize. For eye mal- 
lein concentrate to one-tenth volume. 
Special Media for Yeasts and Molds.-Most of the 
pathogenic yeasts and molds can be cultivated on the 
media already described. Growth is much improved 
if glucose or maltose is added. The reaction should, 
however, be acid or at most slightly alkaline to litmus. 
For molds, especially those infecting the skin, an 
agar containing no meat but 1 to 2 per cent, peptone 
and 2 per cent, of glucose or maltose and 0.5 per cent, 
of glycerin with no change of reaction gives a very 
favorable medium. 
For yeasts, beerwort media are especially favorable. Hopped beerwort is 
obtained at a brewery. Autoclave, cool, and filter, and then tube (this avoids 
subsequent precipitation). For solid media add 10 per cent, gelatin or 1.5 
per cent. agar. Various carbohydrates may be added in 2 per cent, amounts. 
Special Media for Protozoa.-For Blood Flagellates (Novy and MacNeal).- 
Equal parts of nutrient agar and fresh defibrinated blood (rabbit or rat). The 
medium is allowed to stiffen slanted, so more water of condensation may 
gather at bottom of slanted surface. The medium should be planted while fresh 
with blood or other infected material containing the living flagellates (e. g., 
trypanosomes, Leishmania). 
Medium for Malarial Organisms (Bass and Johns).-10 c.c. of blood drawn 
from a malarial patient, and carefully defibrinated. Tins is placed in smaller 
test-tubes in 1 c.c. amounts. To each small test-tube before adding the blood, 
1 per cent, of a 50 per cent, solution of dextrose is added. The red corpuscles 
settle so that J cm. layer of serum is left about them. The parasites grow in a 
Fig. 41 
Platinum needle and 
loop. For most purposes 
finer wire is used. CULTIVATION OP MICROORGANISMS 
107 
thin layer near the top of the cell sediments. Beneath tins zone the parasites 
die. Bass says leukocytes should be gotten rid of because they destroy the 
parasites, but the Thompsons say this is unnecessary. Most favorable tem- 
perature 39° to 40° C. 
Medium for Pure Cultures of Amebce (Williams').-Fresh sterile brain, liver, 
or kidney is cut in small pieces and placed on the surface of alkaline, slightly- 
nutrient tissue agar. 
CULTIVATION OF MICROORGANISMS 
Microorganisms can seldom be identified by their microscopic and 
staining characteristics alone. By these methods only their individual 
form, arrangement, and motility or lack of motility can be studied. 
To go beyond this we grow the organism in pure culture, that is apart 
from all other organisms, and determine its cultural characteristics 
on various media, and in some instances its immunity reactions and its 
action in the animal body. In cultivating microorganisms it is not only 
necessary to supply the appropriate foodstuff, but also to have the 
appropriate conditions as to temperature, moisture, access of oxygen, etc. 
When we make cultures of any material we are apt to find that 
instead of only one variety of microorganism, there are a number 
present. If we grow the material in fluid media we find that as the 
different varieties develop they spread through the medium and become 
hopelessly mixed; furthermore, the more vigorous varieties outgrow 
the feebler ones, which are thus lost. If, however, the microorganisms 
in the material be scattered through or on a solid medium, each individual 
will develop at the spot where it lights and produce in its growth a 
colony which is visible to the naked eye or can be seen with a low-power 
lens. This is an example of how pure cultures are obtained without 
which the study of microorganisms would be impossible. 
Methods of Isolating Pure Cultures.-Plating Methods.-Two plating methods 
are employed, pour plates or streak plates. Agar or gelatin media are used. 
The medium is first rendered fluid: gelatin, by placing the tubes in warm 
water (35° to 40° C.); agar, by immersing the tubes in boiling water until 
fluid and then cooling the tubes to between 40° and 42° C. Agar only melts 
completely when heated to the boiling-point of water or thereabouts and 
hardens as the temperature falls below 40° C. The cooling of the agar is neces- 
sary, otherwise the organisms would be injured by the heat. If enriching fluids 
are to be used they are added to the agar when between 45° and 50° C. The 
material from which we wish to obtain pure cultures is then inoculated into the 
fluidified media. The method of inoculation is in general as follows: A loop 
of the material is carried into a tube of the medium and thoroughly mixed with 
the medium by tilting and rolling the tube. Agitation sufficient to produce 
bubbles must be avoided. From this tube three loops are carried over to a 
second tube which is mixed in the same manner. From the second tube five loops 
are carried to a third tube and mixed. The lips of the tubes are flamed and the 
contents of each poured into a Petri dish, which is if necessary tilted to spread 
the agar evenly over the bottom of the dish. In pouring, the lid of the dish is 
raised just sufficiently to pour the contents of the tube into the dish, the lid acting 
as a protection against falling dust. In diluting the material with culture media, 
it is our object to separate the organisms so that when colonies develop from 
them they will be well separated, that is, discrete. If they were not well sepa- 
rated, we would touch several colonies in attempting to transfer the growth of one 108 
PRINCIPLES OF MICROBIOLOGY 
and thus fail to obtain pure cultures. As we have no way of telling what 
dilution will give us well-separated colonies a series of dilutions is necessary. 
The method will have to vary with the probable number of organisms in the 
material. If they are in great numbers further dilutions of the material will be 
necessary, as the first tube will give overcrowded plates. A preliminary dilution 
or two may be made in saline solution or broth. Where the material contains 
very few organisms more material must be inoculated into the first tube. The dilu- 
tions may be made with a sterile pipette instead of a loop. The tip of the pipette 
is touched to the material and carried over to the first tube of medium, mixing 
being done by sucking the medium up and down in the pipette; about to c.c. of 
the contents of this tube is carried to a second tube, to c.c. of this to a third, etc. 
Streak Plates.-In this method the medium (agar) is poured into Petri dishes 
and allowed to harden. The material is then streaked over the surface of the 
medium either with a platinum loop or spatulum or a bent-glass rod. Two or 
more plates are inoculated in succession without further sterilization of the loop 
or spatulum. This dilutes the material so that discrete colonies are obtained. 
A difficulty that is apt to arise in the use of streak plates, and to a less degree 
with pour plates, is collection of water in condensation, which finding its way 
to the surface of the plate will cause a spreading of the growth over the whole 
surface. This can be avoided to a large extent by cooling the agar before pouring 
on the plates. Special covers made of porous earthenware can be obtained, which 
will absorb the moisture. Incubating the plates upside down is a good precau- 
tion. Motile bacteria are most likely to spread over the surface of media. The 
special media used for the isolation of fecal bacteria must, because of the great 
numbers of motile bacteria in the stool, be comparatively dry. As most of these 
media restrain the ordinary contaminating bacteria from growing they may be 
allowed to harden and dry in the Petri dishes with the covers left off, and the 
drying continued by placing them in the incubator partly open. 
Fishing Colonies.-This is the term used for the process of transferring the 
microorganisms from the colony to a tube of medium which after growth 
has taken place is our pure culture. This is usually done with a straight 
platinum wire, which is less likely to touch any colony other than the one 
we wish to fish. Fishing is done either by selecting the colony with the 
unaided eye or by the aid of a microscope. The advantage gained by the use 
of the microscope is that we can be sure that the colony to be fished has no 
microscopic colonies in the immediate neighborhood likely to be touched by the 
platinum wire. Furthermore, if the fishing is done under the microscope the fact 
that one and only one colony has been touched is visually proved. Opaque 
media are fished by eye. In fishing under the microscope the colony to be fished 
is centred under a No. 2 or AA objective and the wire introduced between the 
objective and the agar until the tip of the wire touches the colony. The progress 
of the wire is watched through the microscope to be sure that nothing is touched 
but the colony, and to observe by the broken appearance of the colony that it 
has been touched. The sense of touch is relied on to tell whether the objective is 
touched. In fishing by eye the plate is held against the light and the colony 
touched with the wire. In either case the organism is transferred to a tube of 
medium-usually an agar slant-by rubbing the tip of the wire over the surface 
of the agar. 
Possible Sources of Error in Plating Methods.-Theoretically each colony is the 
progeny of one single organism, or in the case of organisms that adhere to form 
pairs, chains, etc., the progeny of such an aggregate of one variety of organism. 
Practically it not infrequently happens that dissimilar varieties cling together 
and a colony develops containing both. A second plating from such a colony 
is usually sufficient to yield pure cultures. In exceptional instances there is the 
greatest difficulty in separating such "dingers" and it is sometimes impossible. 
Special Methods of Isolation.-Dilution Method.-It is possible by gradual 
dilution of material to reach a point where some of the loops of the material 
may only contain one organism which when carried to tubes of medium will CULTIVATION OF MICROORGANISMS 
109 
yield pure cultures. This method was employed before the introduction of 
solid media. The uncertainty of the method is evident. For certain purposes 
modified dilutions are still employed, starting usually with cultures obtained 
from colonies on plates, the method being employed not so much to obtain 
pure cultures as to obtain cultures known to be. the progeny of one cell. 
Hanson's Method for Yeasts.-Cover-slips are given a thin coating of paraffin, 
through which lines are drawn dividing the cover into small squares and each 
numbered. The marking is done with a needle. The covers are now exposed 
to hydroflouric acid to etch the markings into the glass. They are then cleaned 
and sterilized. Dilute the yeast-containing material with fluidified gelatin 
until one loop averages one yeast cell as determined by microscopic examination. 
The gelatin is then spread over the surface of the sterile cover-slip, hardened, 
and inverted over a Bottcher moist chamber, sealing with vaselin. This is 
placed under the microscope and the position of the single cells determined, 
making a sketch for later reference. Incubate at room temperature and when 
colonies have developed fish those developing from single cells. The advantage 
of this method over plating is that on plates a proportion of the colonies will 
develop from adhering cells. The purity of yeasts in industries is of enormous 
financial importance. 
India-ink Method (Burri).-The use of india-ink for microscopic examination 
has already been described. By making successive dilutions of small amounts 
of culture in drops of ink it is possible to make small spots of ink containing 
only one organism. This is determined microscopically. This is the starting- 
point for the pure culture from one cell. The reader is referred to the article 
by Burri for the technical details. The method has only a very limited 
application (see page 74). 
Capillary Method (Barber and others).-Reference is made to the original 
articles for the details of this method for isolating one cell. 
Preliminary Enrichment Methods.-Very often it is impossible to isolate 
certain organisms from material because they are too few in number. With 
certain organisms it is possible to inoculate the material in special media which 
will allow free multiplication of this organism, but be unfavorable for the others 
contained in the material. Examples of this are the use of Lbffler's medium 
for mixed cultures from the throat to find diphtheria, or the peptone method 
for the enrichment of cholera and other vibrios. Another method is the use of 
ascitic broth for the isolation of diphtheria from mixed material. The last 
two methods take advantage of the surface growth of the bacteria. The same 
end is attained by the use of the following factors: blood-media for hemo- 
globinophilic bacilli; increase of the alkalinity, as in selective media for 
vibrios; increased acidity, as with tartaric acid, for yeasts; dyestuffs, gentian 
violet, to inhibit Gram-positive bacteria; brilliant green to inhibit the colon 
types in mixed cultures from stools; use of heat, spore-containing mixed cultures 
being heated to 80° C., to kill the non-spore-bearing types; motility, the motile 
organism spreading from the point of inoculation of the mixed material, as with 
amebae and spirochete-containing material; filtration through filters, spiro- 
chetes passing filters, most bacteria being held back (see Filtrable Viruses); 
germicidal agents; antiformin for separation of acid-fasts from other bacteria; 
finally, the use of animals (see p. 122). 
Special Methods for Anaerobes.-(See Anaerobic Methods.) 
Applications of the Plating Methods.-Study of Colony.-The appearance of 
the colony is one of the points used in identifying bacteria, or the colony appear- 
ance being known the isolation of a certain organism is greatly simplified. In 
plates showing two or more types of colonies it is an aid in determining the rela- 
tive proportion of the subsequently identified varieties in the material examined. 
In examining colonies both the macroscopic and microscopic characteristics 
are determined. The age at which a colony will be most characteristic depends 
on the rapidity of growth of the organism. Daily observations are most suited 
for full information. As a rule young colonies are most characteristic, although 110 
PRINCIPLES OF MICROBIOLOGY 
such characteristics as pigment production and gelatin liquefaction may only 
appear on longer incubation. With the naked eye the following points should 
be observed: moist or dry transparent, translucent or opaque, edge sharply 
defined or indefinite, regular or irregular outline, fringe-like margin or thread- 
like outgrowths, color, flat, raised, or umbilicated, etc. On microscopic exami- 
nation the presence or absence of granules, whether fine or coarse, their arrange- 
Fig. 42 
Fig. 43 
Irregular fringed colony (B. malignant edema). 
(From Kolle and Wassermann.) 
Colonies of typhoid and colon bacilli 
in rather soft gelatin. 
ment and distribution, should be observed, as well as the finer details of the 
characteristics mentioned under the naked-eye examination. The observations 
are usually made with a low-power objective, No. 2 or No. 3. The fine details 
may be observed with a higher lens, and the arrangement of the bacteria espe- 
cially at the edge of the colony can be observed. The accompanying figures 
illustrate the colony characteristics mentioned. 
Fig. 44 
Fig. 45 
Colony of colon bacilli grown in soft 
gelatin. 
One large irregular colony of colon and 
two smaller colonies of typhoid bacill in 
soft gelatin. (Figs. 41 to 43 from photo- 
graphs by Dunham.) 
Determination of the Number of Bacteria.-If a measured amount of fluid 
is mixed with a plating medium we should theoretically be able to determine 
the number of bacteria by counting the number of colonies that develop. This 
would be true if each bacterium was completely separate, if each was able 
to multiply, if the medium was satisfactory for the development of each variety 
contained in the material, and if the oxygen access, moisture, and temperature CULTIVATION OF MICROORGANISMS 
111 
of incubation were favorable for the growth for all the varieties. This is an 
impossibility. Most nearly accurate results are obtained where only one or at 
Fig. 46 
Fig. 47 
Fig. 46.-Moist raised colonies with no visible structure, looking like a drop of water 
Fig. 47.-Deep colonies, usually either light brown, gray, or yellow in color, opaque 
with little marking. (Figs. 46-53 from Lehman and Neumann.) 
Fig. 48 
Fig. 49 
Fig. 50 
Fig. 48.'--The colony very finely granular. 
Fig. 49.-Colonies opaque in centre with lighter borders. The margin is coarsely 
granular, or has twisted threads. 
Fig. 50.-Colony in gelatin. The centre is coarsely granular in partly fluid gelatin. 
The borders are formed of wavy bands of threads. 
Fig. 51 
Fig. 52 
Fig. 53 
Fig. 51.-Colonies circular in form, composed of radiating threads. 
Fig. 52.-Colonies with opaque centres, with a thin border fringe. 
Fig. 53.-Colony showing a network of threads which is thicker in centre. 
most several varieties are present and the conditions for their growth main- 
tained. Although the method gives an incorrect result when mixed material 112 
PRINCIPLES OF MICROBIOLOGY 
is used the enumeration of the colonies that develop is of a great deal of practical 
value, as in the examination of milk and water. If the methods employed are 
always the same, the comparison of the results gives us valuable information 
as to the bacterial content. 
The method employed is in general as follows: To 9 c.c. of water is added 1 c.c. 
of the material to be examined; this is thoroughly shaken to separate the 
organisms; 1 c.c. of this is then added to 9 c.c. of water thoroughly shaken, and 
1 c.c. of this added to 9 c.c. of water and so on. This gives a dilution of 1 in 10, 
1 in 100, 1 in 1000, etc. How far to dilute the material must be determined from 
the character and probable bacterial content. One c.c. of each of these dilutions 
is placed in a Petri dish and the melted plating medium poured into the dish, 
the two being mixed by tilting the dish. Mixing can be done by adding the 
dilutions to the tubes of plating medium and pouring the content into the dishes. 
Though in this way all the medium cannot be poured out of the tube and some 
bacteria are left behind, it is easier to obtain even distribution of the colonies 
in this way and for comparative work the method gives reliable results. The 
counts by this method average about 10 per cent, lower than where mixing is 
done in the dish. 
Fig. 54 
Photograph of a large number of colonies developing in a layer of gelatin contained in a 
small Petri dish. Some colonies are only pinpoint in size; some as large as the end of a 
pencil. The colonies here appear in their actual size. 
In place of dilutions, made by pipettes, loops or rings on a platinum needle 
can be employed which hold a known quantity (small) of the material to be 
examined. 
To obtain a satisfactory count the colonies should be about 100 to 200 to a 
plate. Lower numbers than this are too few to give a fair average, higher numbers 
are difficult to count because the colonies have not sufficient space for full 
development, some of the colonies become confluent, and some of the feebler 
bacteria are checked in their development by the crowding and do not develop 
colonies. The plate that shows about 100 colonies is chosen, therefore, for count- 
ing. Where no one plate is completely satisfactory in number and even distribu- 
tion of the colonies, two of the nearest satisfactory plates are counted and the 
results averaged. Wherever possible all the colonics on a plate should be counted. 
Where the colonies are crowded it is necessary to divide the plate into sections 
to facilitate counting. This is best done by placing the Petri dish on a Wolff- 
hiigel counting plate, which is a glass plate ruled in square centimeters, some 
of the squares being still further divided into 9 small squares. A row of squares CULTIVATION OF MICROORGANISMS 
113 
is counted and then a row at right angles to the first, to obtain a fair average. 
The number of colonies is divided by the number of squares counted. If the 
usual 10 c.c. Petri dish is used the number per square centimeter is multiplied 
by 63, the number of square centimeters on the dish to give the total number. 
This is multiplied by the dilution used in this dish which gives the colonies devel- 
oping from 1 c.c. of the material used for plating. If dishes of another size are 
used the area in square centimeters is determined by multiplying the diameter 
by 3.1416. 
Fig. 55 
Fig. 56 
Well-distributed colonies in agar 
in portion of Petri dish. 
Wolffhugel's apparatus for counting colonies. 
Methods of Inoculating Culture Media.-The inoculation of plating media 
has been described under the methods of isolating pure cultures. Platinum 
wire is usually employed for transferring the growth from one medium to another. 
The wire is either attached to the end of a glass rod or to special aluminum holders 
which are made for this purpose. Straight wire or wire with the ends bent 
into a loop is used. The wire is sterilized by heating it in the flame of a Bunsen 
burner until red-hot. Care must be taken that the wire is cool before it is used. 
The straight wire is used for fishing colonies or where tubed solid media is to 
be inoculated by long puncture through the media (stab culture). The loop is 
used for ordinary transfers. In transferring from solid media the loop is drawn 
upward over the medium filling the loop with the growth; this is rubbed over 
the surface of solid media or in the case of fluid media against the glass at upper 
level of the fluid. In transferring fluid cultures a loop of the fluid is used. Where 
the growth on fluid media is in the form of a pellicle it may be necessary to 
inoculate this pellicle so that it floats or growth will not take place. In trans- 
ferring the tubes or other containers it must not be kept open any longer than 
necessary or contamination is likely to occur. In transferring from and 
to tubed medium the tubes are held by placing them against the palm and fin- 
gers of the left hand, grasping the tubes by the butt between the thumb and the 
palm, low enough so that the contents of the tube are not covered by the thumb. 
The plugs, if dusty, are passed quickly through the flame and if necessary the 
resulting flame blown out. The right hand holding the sterilized platinum 
wire is used to remove the cotton plugs, one being grasped between the small 
finger and the other between the fourth and fifth finger. After drawing out the 
plugs the necks of the tubes are flamed and the growth then transferred and the 
plugs replaced; the platinum wire is then sterilized. 
Apparatus and Methods of Incubation.-The selection of the temperature 
for incubation depends on the organism to be grown and the medium employed. 
Although the optimum temperature for growth may vary between 5° and 50° 
C. or more, the great majority of organisms will grow at either 22° or 37° C. 
These are the temperatures usually employed. For general purposes the former 
8 114 
PRINCIPLES OF MICROBIOLOGY 
is obtained by incubation at room temperature. Where a more exact and uni- 
form temperature is necessary an incubator must be employed. This is a double- 
walled oven, the space between the walls being filled with water; heat is supplied 
by gas or electricity and cooling by the addition of cold water. The latter is 
only necessary where the outside temperature rises above 22° C. The heating 
or the addition of water is controlled by an automatic regulator. To maintain 
a temperature of 37° C. a similar incubator is employed, the heating, however, 
being continuous, an automatic regulator controls the temperature. The 
Fig. 57 
Incubator. 
heat-controlling devices are of varied construction, the expansion of a fluid or 
the change in shape of a bar of two unequally expanding metals is used to make 
and break the electric contact or open and close the gas vent. In the former 
mercury is used alone or at the point of control. Where a large incubating- 
room is required a room can be heated with a large gas stove, a funnel being- 
placed over the stove and connected with this a radiator-like arrangement of 
four- to six-inch pipes ending in a chimney-like outlet for the products of combus- 
tion. The radiator distributes the heat more evenly through the room. In CULTIVATION OF MICROORGANISMS 
115 
spite of this there will be a variation in temperature in different parts of the 
room, the shelves near the radiator or nearest the ceiling being the warmer. 
This is an advantage rather than a disadvantage, as a range of about 5° is 
available for special purposes. In using gas, all connections are best made of 
metal; flexible metal tubing is available. Small burners are preferably of the 
safety type, which, if extinguished, turn off the gas automatically. In the case 
of gas stoves there should be an independent pilot flame not influenced by the 
regulator, which will relight the stove if turned off. 
Fig. 58 
Fig. 59 
Fig. 60 
Safety-burner. 
Heat regulator. (Dunham.) 
Heat regulator. (Roux.) 
Bimetallic. 
Oxygen Requirement Methods.-Where aerobic conditions are required the 
access air to the growth is all that is required. For anaerobic conditions the 
oxygen must be removed. This is done (1) by exhaustion of the air, (2) by chem- 
ical absorption of the oxygen, (3) by displacement of the air as by hydrogen, 
or (4) by growing in the depths of solid media, or in fluid media under a layer 
of alboleneto prevent the reabsorption of air driven off during sterilization; (5) 
or, lastly, by a combination of these methods. The addition of fresh animal 
tissues to media for the enhancement of the growth of anaerobic organisms has 
been mentioned under Media. Occasionally reducing substances are added 
directly to the medium, or the organism is grown in symbiosis with an aerobic 
bacillus to absorb the oxygen. 
A group of organisms spoken of as " micro-aerophilic " will only grow when 
the oxygen is partly removed. This is done by exhaustion of the air to measured 
degree, by growing the organism in a sealed jar containing also a stated number 
of cultures of an organism that absorbs oxygen in its growth, as B. subtilis, 
or by mixing the material with melted agar in tubes and allowing the agar to 
set, the development taking place at the depth that has the favorable oxygen 
content. In the second method the optimum number of square centimeters 
of B. subtilis culture per liter must be known. 116 
PRINCIPLES OF MICROBIOLOGY 
Anaerobic Methods.--For exhaustion a pump is required; the filter pumps for 
attachment to the faucet are the most convenient where water under pressure 
is available. Any type of pump, however, 
may be employed. The extent exhausted 
can be measured by a manometer. Any 
type of jar with a tightly fitting cover and 
an opening with a stop-cock and of appro- 
priate dimensions for purpose can be used. 
The Novy jar is a satisfactory type. 
Museum jars with a side opening are very 
satisfactory substitutes. The jar must be 
absolutely tight or the vacuum will not be 
held. Absorption can be added to exhaus- 
tion by placing pyrogallic acid and a stick 
of sodium hydrate in the bottom of the jar. 
After exhaustion is completed a small 
amount of water is allowed to be sucked 
back into the jar. This dissolves the sodium 
hydrate and the resulting reaction of the 
sodium hydrate with the pyrogallic acid is 
accompanied by absorption of the oxygen 
from the air remaining in the jar. 
Hydrogen may be used to replace the 
exhausted air. After exhaustion, hydrogen 
from a Kipp apparatus is sucked back into 
the jar diluting the remaining air; the jar is 
again exhausted arid then filled with hydro- 
gen, again exhausted and filled. Only traces 
of air will remain, and if absolute anaerobia- 
sis is required pyrogallic acid may be used 
to absorb this last trace as above, allowing 
the water to be sucked back before the 
negative pressure is completely relieved by 
the hydrogen. In relieving the negative 
pressure it may be necessary to use a 
pneumatic trough to collect the hydrogen, 
as the generation of hydrogen will be too 
Fig. 62 
Fig. 61 
Buchner's anaerobic tube. The 
fluid consists of pyrogallic acid dis- 
solved in 10 per cent. NaOH solution. 
By Wilson's method the tubes are 
charged with pieces of caustic potash 
covered with pyrogallic acid. 
Novy jar for anaerobic cultures. 
slow and air will be drawn through the Kipp apparatus. It is advisable not 
to relieve the negative pressure completely, as this helps to hold the lid 
tightly in place, and furthermore the subsequent expansion when the jar is CULTIVATION OF MICROORGANISMS 
117 
incubated will tend to loosen the lid and allow inward diffusion of air. When 
the pyrogallic method is used the tubes or plates must be raised above the level 
of the fluid. Of the methods given the second and third are the most satis- 
factory. The absorption method may be used alone, either using a jar to 
hold the tubes or plates, or individual plates or tubes may be handled as in the 
following: 
Buchner's Method- The culture tube is placed in a larger tube, at the bottom 
of which is placed the pyrogallic acid, on which some sodium hydrate is poured 
and the tube quickly closed with a rubber stopper, or the NaOH may be added 
dry and water added. 
Wright's Method.-The tubes are plugged with absorbent cotton; after inocu- 
lation of the medium the plug is pushed into the tube, pyrogallic placed on the 
stopper, sodium hydrate solution added, and the tube quickly corked. 
Zinsser's Method for Plates.-The dishes employed must be deeper than the 
ordinary Petri dishes. The agar is poured in the smaller dish in the ordinary 
manner, and as soon as hard inverted over moistened filter paper to protect it 
from contamination. In the larger dish is placed the pyrogallic acid and the 
smaller dish placed in it. One side of the smaller dish is raised and the hydrate 
solution poured in the space between the two dishes and the inner dish dropped 
back. As rapidly as possible albolene is run in the space between the dishes 
from a pipette previously filled so that no time is lost. 
The methods for exclusion of oxygen are simple and can be, in most instances, 
substituted for the more cumbersome methods described. Plating may be done 
with the ordinary Petri dishes as follows: The bottom part of the dish, open 
side up, is placed in the cover and sterilized in this shape and protected from 
dust until used. The plating medium is inoculated as though pour plates were 
to be made in the ordinary way. The inoculated medium is then poured into 
the covers and the bottom of the dish laid on the fluid medium, tilting it so 
that the air can escape as it falls into place. With a little practice this can be 
done so that no bubbles are present. After the medium has set the edge is 
painted over with melted paraffin to prevent drying and contamination. If 
any of the medium flows over the edge it is wiped away by carbolized cotton 
(see Fig. 63). After incubation the parts of the dish are carefully pulled apart 
Fig. 63 
Bottom of dish. 
Agar. 
•• Cover of dish. 
Anaerobic plating method. 
and the colonies may be fished. As a rule the agar remains in the cover, if it 
should adhere to the other part of the dish this can be laid in the cover to 
allow of fishing under the microscope. If gas-producing bacteria are present 
fishing should be done as soon as sufficient growth has taken place or the gas 
will disrupt the medium and cause the growth to spread over the plate. If an 
actively motile organism is present the method may fail because of spreading 
between the medium and the glass. 
Another method for the isolation of pure cultures is the shake culture. The 
material is diluted with a plating medium in the usual way, although more medium 
is used in the tubes. Instead of pouring the medium into plates the medium 
is allowed to solidify in the tubes. The colony desired can be obtained by 
scratching the tube with a file at the level of the colony, then wet the tube with 
alcohol, and burn this off and press on the scratch with a very hot piece of metal 
or glass rod. The tube will crack and the column of agar can be cut through 
to allow the separation of the two parts of the tube and the colony fished. This 
method has a limited application, but is especially useful where the organism 118 
PRINCIPLES 'OF MICROBIOLOGY 
to be isolated is a partial anaerobe and grows at a certain level in the agar. 
Pure cultures of these organisms may not be obtained at the first attempt, but 
the level of thickest growth may then be used, the first culture serving in this 
way as an enrichment. 
For the cultivation of pure cultures the use of stab cultures in agar 
or gelatin may be used. The most satisfactory medium for this purpose 
is a semisolid agar. Because of the softness of the medium the material for 
subinoculation is easily obtained; the stab inoculation closes better than if 
made in stiffer medium, and the medium does not dry out and split so readily. 
For subinoculation a platinum wire bent like a corkscrew is a help in obtaining 
the material from the depths of the media. With spirochetes it is much easier 
to obtain the growth with the capillary pipette from about the tissue at the 
bottom of the tube than where the usual 1| per cent, is employed. The use 
of the capillary pipette for the transfer of cultures is open to much wider appli- 
cation with semisolid media, because if the medium has a satisfactory density 
the ordinary rubber teat exerts sufficient suction to draw up the medium and 
a syringe is not needed. 
The exclusion of oxygen for fluid medium is most simply carried out by steriliz- 
ing the medium under a layer of albolene. This can be done either in tubes or 
flasks, preferably the globe flasks. In the latter the medium is filled to the neck 
and albolene added, leaving room for expansion, after sterilization and con- 
traction of the medium more sterile albolene is added if necessary. 
Occasionally anaerobes are grown in mixed culture with an aerobic organism 
to absorb the oxygen. One of the best organisms for this purpose is the hay 
bacillus. 
As a general rule the addition of a fermentable carbohydrate to the medium 
gives much better growth of anaerobic organisms. 
The Study of Pure Cultures.-The study of pure cultures is the basis 
for classification and identification. The characteristics studied are: 
the morphology, staining reactions, and motility when grown on or in 
both solid and fluid media (see Microscopic Study); the cultural charac- 
teristics when grown on various media, its food, oxygen, and temperature 
requirements, its reaction to the various immunity tests (see chapters 
on Immunity), and the action of the organism or its products on experi- 
mental animals (see Use of Animals). The following is a short summary 
of the more important cultural reactions and the methods of eliciting 
them. 
Cultural Characteristics.-Colony Morphology.-(See Application of Plating 
Methods.) 
Growth on Agar and Other Solid Media {Surface Growth).-Moist or dry, 
flat or raised, flaky or easily emulsified, adherent to medium or not, mucoid 
or slimy, smooth, irregular, or threaded margin, color or pigment production 
(see below), extension into medium, etc. 
Growth in Solid Media.-In stab culture, depth to which growth extends, 
character of growth around the surface puncture, the spreading of the growth 
into the medium (an index of motility best shown in,semisolid media), thread- 
like extensions from the line of puncture, in gelatin, liquefaction, non-liquc- 
faction, rate, area and character if present. In shake cultures, the character of 
the colonies. 
Fluid Media.-Pellicle, tenacious or easily broken, spreading up the side of 
the tube or not, thick or thin, clear or cloudy, and degree of clouding, sedi- 
ment or deposit on sides of tube, granular, flaky, gelatinous, mucoid, or stringy, 
stalactite formation, more densely clouded at one level than another. CULTIVATION OF MICROORGANISMS 
119 
Food Requirements.-Growth on simple or complex media, albumins such as 
serum required, blood or hemoglobin necessary, carbohydrate needed for full 
development (example glycerin for tubercle bacillus). 
Oxygen Requirements.-Growth only in free oxygen obligate aerobe; growth 
only where oxygen is absent (depths of media, etc.), obligate anaerobe; growth 
under both conditions, facultative anaerobe (an available carbohydrate is neces- 
sary to elicit the facultative character); growth with a definite but lessened 
amount of oxygen (development only in one level of solid media), micro-aero- 
philic. This last characteristic may be lost after cultivation for some time on 
artificial culture media. 
Optimum Temperature.-Not often used, but may furnish absolute evidence 
of the dissimilarity of two organisms. Not only the optimum but also the range, 
especially lower, should be determined. 
Pigment Production.-The color and shade, the optimum medium and tem- 
perature for its production, limited to the area of growth or diffused through 
the medium; its solubility in extracting agents, as alcohol, ether, chloroform, 
etc.; more than one pigment present, as shown by use of different extractives. 
In the case of the coccacese, especially the presence of pigment and its color 
and shade may be determined with ordinary agar; the growth is taken up with 
a platinum loop and spread on a piece of white glazed paper. Although the 
presence of pigment may be completely obscured on the agar, its presence is 
immediately evident on the paper. 
Ferment Action (Cultural Evidence of).-The most important culturally 
are those causing fermentation of carbohydrates. Less important are proteo- 
lytic, diastatic, inverting, and rennin-like ferments. 
Fig. 64 
Stab cultures of three cholera spirilla in gelatin, showing in upper portion of growth 
considerable liquefaction of nutrient gelatin. 
Fermentation of Carbohydrates.-The sugars selected will depend on the 
organism to be identified as well as the medium selected as a basis for the addi- 
tion of the sugar. The preparation and the use of indicators or titration to 
determine the production of acid and the use of fermentation tubes to demon- 
strate gas production have been described. The amount of gas is expressed in 
terms of percentage, thus if the closed arm is half-full of gas, 50 per cent., etc. 
To determine roughly proportion of carbon-dioxide and of hydrogen, mark on 
tube quantity of gas produced, then fill the bulb with sodium hydroxide 
solution (10 per cent.) and insert a rubber stopper; the tube is inverted several 
times to mix and the gas again collected in the closed arm. The gas absorbed 
is carbon-dioxide; the remainder is usually hydrogen. In the case of media 
containing coagulable proteins acid production if sufficient is followed by 118 
PRINCIPLES OF MICROBIOLOGY 
to be isolated is a partial anaerobe and grows at a certain level in the agar. 
Pure cultures of these organisms may not be obtained at the first attempt, but 
the level of thickest growth may then be used, the first culture serving in this 
way as an enrichment. 
For the cultivation of pure cultures the use of stab cultures in agar 
or gelatin may be used. The most satisfactory medium for this purpose 
is a semisolid agar. Because of the softness of the medium the material for 
subinoculation is easily obtained; the stab inoculation closes better than if 
made in stiffer medium, and the medium does not dry out and split so readily. 
For subinoculation a platinum wire bent like a corkscrew is a help in obtaining 
the material from the depths of the media. With spirochetes it is much easier 
to obtain the growth with the capillary pipette from about the tissue at the 
bottom of the tube than where the usual 1| per cent, is employed. The use 
of the capillary pipette for the transfer of cultures is open to much wider appli- 
cation with semisolid media, because if the medium has a satisfactory density 
the ordinary rubber teat exerts sufficient suction to draw up the medium and 
a syringe is not needed. 
The exclusion of oxygen for fluid medium is most simply carried out by steriliz- 
ing the medium under a layer of albolene. This can be done either in tubes or 
flasks, preferably the globe flasks. In the latter the medium is filled to the neck 
and albolene added, leaving room for expansion, after sterilization and con- 
traction of the medium more sterile albolene is added if necessary. 
Occasionally anaerobes are grown in mixed culture with an aerobic organism 
to absorb the oxygen. One of the best organisms for this purpose is the hay 
bacillus. 
As a general rule the addition of a fermentable carbohydrate to the medium 
gives much better growth of anaerobic organisms. 
The Study of Pure Cultures.-The study of pure cultures is the basis 
for classification and identification. The characteristics studied are: 
the morphology, staining reactions, and motility when grown on or in 
both solid and fluid media (see Microscopic Study); the cultural charac- 
teristics when grown on various media, its food, oxygen, and temperature 
requirements, its reaction to the various immunity tests (see chapters 
on Immunity), and the action of the organism or its products on experi- 
mental animals (see Use of Animals). The following is a short summary 
of the more important cultural reactions and the methods of eliciting 
them. 
Cultural Characteristics.-Colony Morphology.-(See Application of Plating 
Methods.) 
Growth on Agar and Other Solid Media {Surface Growth).-Moist or dry, 
flat or raised, flaky or easily emulsified, adherent to medium or not, mucoid 
or slimy, smooth, irregular, or threaded margin, color or pigment production 
(see below), extension into medium, etc. 
Growth in Solid Media.-In stab culture, depth to which growth extends, 
character of growth around the surface puncture, the spreading of the growth 
into the medium (an index of motility best shown in,semisolid media), thread- 
like extensions from the line of puncture, in gelatin, liquefaction, non-lique- 
faction, rate, area and character if present. In shake cultures, the character of 
the colonies. 
Fluid Media.-Pellicle, tenacious or easily broken, spreading up the side of 
the tube or not, thick or thin, clear or cloudy, and degree of clouding, sedi- 
ment or deposit on sides of tube, granular, flaky, gelatinous, mucoid, or stringy, 
stalactite formation, more densely clouded at one level than another. CULTIVATION OF MICROORGANISMS 
119 
Food Requirements.-Growth on simple or complex media, albumins such as 
serum required, blood or hemoglobin necessary, carbohydrate needed for full 
development (example glycerin for tubercle bacillus). 
Oxygen Requirements.-Growth only in free oxygen obligate aerobe; growth 
only where oxygen is absent (depths of media, etc.), obligate anaerobe; growth 
under both conditions, facultative anaerobe (an available carbohydrate is neces- 
sary to elicit the facultative character); growth with a definite but lessened 
amount of oxygen (development only in one level of solid media), micro-aero- 
philic. This last characteristic may be lost after cultivation for some time on 
artificial culture media. 
Optimum Temperature.-Not often used, but may furnish absolute evidence 
of the dissimilarity of two organisms. Not only the optimum but also the range, 
especially lower, should be determined. 
Pigment Production.-The color and shade, the optimum medium and tem- 
perature for its production, limited to the area of growth or diffused through 
the medium; its solubility in extracting agents, as alcohol, ether, chloroform, 
etc.; more than one pigment present, as shown by use of different extractives. 
In the case of the coccaceae, especially the presence of pigment and its color 
and shade may be determined with ordinary agar; the growth is taken up with 
a platinum loop and spread on a piece of white glazed paper. Although the 
presence of pigment may be completely obscured on the agar, its presence is 
immediately evident on the paper. 
Ferment Action (Cultural Evidence of).-The most important culturally 
are those causing fermentation of carbohydrates. Less important are proteo- 
lytic, diastatic, inverting, and rennin-like ferments. 
Fig. 64 
Stab cultures of three cholera spirilla in gelatin, showing in upper portion of growth 
considerable liquefaction of nutrient gelatin. 
Fermentation of Carbohydrates.-The sugars selected will depend on the 
organism to be identified as well as the medium selected as a basis for the addi- 
tion of the sugar. The preparation and the use of indicators or titration to 
determine the production of acid and the use of fermentation tubes to demon- 
strate gas production have been described. The amount of gas is expressed in 
terms of percentage, thus if the closed arm is half-full of gas, 50 per cent., etc. 
To determine roughly proportion of carbon-dioxide and of hydrogen, mark on 
tube quantity of gas produced, then fill the bulb with sodium hydroxide 
solution (10 per cent.) and insert a rubber stopper; the tube is inverted several 
times to mix and the gas again collected in the closed arm. The gas absorbed 
is carbon-dioxide; the remainder is usually hydrogen. In the case of media 
containing coagulable proteins acid production if sufficient is followed by 120 
PRINCIPLES OF MICROBIOLOGY 
coagulation, as milk, serum water, serum broth; the coagulation is shown in 
solid media by precipitation or opacity which develops. The ability to attack 
a carbohydrate may differ under aerobic and anaerobic conditions. 
Proteolytic action is shown by gelatin liquefaction, liquefaction of Loffler's 
blood-serum, digestion of milk. Prolonged incubation even for a month may 
be needed. The production of peptone may also be used for determining the 
digestion of albuminous media. Note odor and, if present, its character. 
Alkali Production.-Determined by indicators or titration; note in milk 
cultures especially. An initial acidity may be noted due to traces of a fer- 
mentable carbohydrate. The cultures may have to be incubated for one to 
three weeks. Milk on prolonged incubation may become translucent whether 
due to alkali production or to a proteolytic enzyme not determined. 
Sulphuretted hydrogen, to demonstrate, use peptone water cultures to which 
is added 1 per cent, of a 1 per cent, solution of lead acetate or ferric tartrate; 
the precipitate which forms on mixing these turns black if sulphuretted hydrogen 
is produced. 
Nitrate Production.-Grow in nitrate broth. Two solutions are necessary 
for the test. 
1. Naphthylamin 0.1 gram 
Distilled water 20.0 c.c. 
Acetic acid 25 per cent 150.0 c.c. 
Dissolve the naphthylamin in the'water by means of heat, cool, filter, and 
add the acetic acid. 
2. Sulphanilic acid . 0.5 gram 
Acetic acid dil. 1 to 16 150.0 c.c. 
Keep them separate and mix in equal parts as needed. 
To about 4 c.c. of the culture add 2 c.c. of the mixed solutions. The develop- 
ment of a pink color shows the presence of nitrates, the intensity of the color 
being proportional to amount present. 
Similar reduction processes are seen in the decolorization of litmus and some 
of the aniline dyes. The bacteria utilize the oxygen of the dyes, reducing them 
to their leuco-bases. 
Aromatic Products (Indol Production).-The usual medium is peptone water. 
Two methods are available for testing: (1) Salkowski Method. To the culture 
add several drops of concentrated sulphuric acid or 1 c.c. of a 10 per cent, 
solution and then add 1 c.c. of a 1 to 10,000 of sodium sulphite solution. A 
pink color develops at the point of contact with the acid, which on shaking 
diffuses throughout. (2) Ehrlich Method. This test is more constant and 
reliable. To the culture add 1 c.c. of a 2 per cent, solution of paradimethylamino- 
benzaldehyde in 95 per cent, alcohol and then add, drop by drop, concentrated 
hydrochloric acid until a red zone at the point of contact of the alcohol and the 
peptone. Not more than 0.5 c.c. of the acid is required. On standing the 
zone deepens and widens. The color is soluble and the test should be confirmed 
by shaking with chloroform, which dissolves out the color. The tests for indol 
are made after four to six days' incubation. 
Cholera-red Reaction.-The nitrate is produced by some bacteria from the 
nitrate present as an impurity in the peptone, and the red color appears on the 
addition of acid alone. 
Both of the above tests are interfered with by the presence of a fermentable 
carbohydrate. 
Voges-Proskauer Reaction.-Grow for three days in 2 per cent, glucose-peptone 
solution in fermentation tubes. Add 2 to 3 c.c. of a strong solution of potassium 
hydrate. A pink color develops on exposure to the air. DESCRIPTIVE CHART-SOCIETY OF AMERICAN BACTERIOLOGISTS. 
Prepared by Committee on Methods of Identification of Bacterial Species.-F. D. Chester, F. P. Gorham, Erwin F. Smith. 
GLOSSARY OF TERMS. 
MAXIMUM TEMPERATURE, temperature above which growth 
does not take place. 
MEDIUM, nutrient substance upon which bacteria are grown. 
MEMBRANOUS, growth thin, coherent, like a membrane. 
MINIMUM TEMPERATURE, temperature below which growth 
does not take place. 
MYCELIOID, colonies having the radiately filamentous appearance 
of mold colonies 
NAPIFORM, liquefaction with the form of a turnip. 
NITROGEN REQUIREMENTS, the necessary nitrogenous food. 
This is determined by adding to nitrogen-free media the nitro- 
gen compound to be tested. 
OPALESCENT, resembling the color of an opal. 
OPTIMUM TEMPERATURE, temperature at which growth is most 
rapid. 
PELLICLE, in fluid bacterial growth either forming a continuous or 
an interrupted sheet over the fluid. 
PEPTONIZED, said of curds dissolved by trypsin. 
PERSISTENT, many weeks, or months. 
PLUMOSE, a fleecy or feathery growth. 
PSEUDOZOOGLE7E, clumps of bacteria, not dissolving readily in 
water, arising from imperfect separation, or more or less fusion 
of the components, but not having the degree of compactness 
and gelatinization seen in zooglese. 
PULVINATE, in the form of a cushion, decidedly convex. 
PUNCTIFORM, very minute colonies, at the limit of natural 
vision. 
RAPID, developing in twenty-four to forty-eight hours. 
RAISED, growth thick, with abrupt or terraced edges 
RHIZOID, growth of an irregular branched or root-like character, 
as in B. mycoides. 
RING, same as RIM, growth at the upper margin of a liquid culture, 
adhering more or less closely to the glass. 
REPAND, wrinkled. 
SACCATE, liquefaction the shape of an elongated sac, tubular, 
cylindrical. 
SCUM, floating islands of bacteria, an interrupted pellicle or bacteria 
membrane. 
SLOW, requiring five or six days or more for development. 
SHORT, applied to time, a few days, a week. 
SPORANGIA, cells containing endospores. 
SPREADING, growth extending much beyond the line of inocula- 
tion, i. e., several millimetres or more. 
STRATIFORM, liquefying to the walls of the tube at the top and 
then proceeding downward horizontally. 
THERMAL DEATH-POINT, the degree of heat required to kill 
young fluid cultures of an organism exposed for ten minutes 
(in thin-walled test-tubes of a diameter not exceeding 20 mm.) 
in the thermal water-bath. The water must be kept agitated 
so that the temperature shall be uniform during the ex- 
posure. 
TRANSIENT, a few days. 
TURBID, cloudy with flocculent particles; cloudy plus floccu- 
lence. 
UMBONATE, having a button-like, raised centre. 
UNDULATE, border wavy, with shallow sinuses. 
VERRUCOSE, growth wart-like, with wart-like prominences. 
VERMIFORM-CONTOURED, growth like a mass of worms or 
intestinal coils. 
VILLOUS, growth beset with hair-like extensions. 
VISCID, growth follows the needle when touched and withdrawn, 
sediment on shaking rises as a coherent swirl. 
ZOOGLEAC, firm gelatinous masses of bacteria, one of the most typical 
examples of which is the Streptococcus mesenterioides of sugar 
vats (Leuconostoc mesenterioides), the bacterial chains being 
surrounded by an enormously thickened firm covering inside 
of which there may be one or many groups of the bac- 
teria. 
NOTES. 
AGAR HANGING BLOCK, a small block of nutrient agar cut from 
a pour plate, and placed on a cover^glass, the surface next 
the glass having been first touched with a loop from a young 
fluid culture or with a dilution from the same. It is examined 
upside down, the same as a hanging drop.1 
AMEBOID, assuming various shapes like an ameba. 
AMORPHOUS, without visible differentiation in structure. 
ARBORESCENT, a branched, tree-like growth. 
BEADED, in stab or stroke, disjointed or semi-confluent colonies 
along the line of inoculation. 
BRIEF, a few days, a week. 
BRITTLE, growth dry, friable under the platinum needle. 
BULLATE, growth rising in convex prominences, like a blistered 
surface. 
BUTYROUS, growth of a butter-like consistency. 
CHAINS, 
Short chains, composed of 2 to 8 elements. 
Long chains, composed of more than 8 elements. 
CILIATE, having fine, hair-like extensions, like cilia. 
CLOUDY, said of fluid cultures which do not contain pseudo- 
zoogleee. 
COAGULATION,2 the separation of casein from whey in milk. This 
may take place quickly or slowly, and as the result either of the 
formation of an acid or of a lab ferment. 
CONTOURED, an irregular, smoothly undulating surface, like that 
of a relief map. 
CONVEX, surface the segment of a circle, but flattened. 
COPROPHYL, dung bacteria. 
CORIACEOUS, growth tough, leathery, not yielding to the platinum 
needle. 
CRATERIFORM, round, depressed, due to the liquefaction of the 
medium. 
CRETACEOUS, growth opaque and white, chalky. 
CURLED, composed of parallel chains in wavy strands, as in anthrax 
colonies. 
DIASTASIC ACTION, same as DIASTATIC, conversion of starch 
into water-soluble substances by diastase. 
ECHINULATE, in agar stroke a growth along line of inoculation, 
with toothed or pointed margins; in stab cultures growth 
beset with pointed outgrowths. 
EFFUSE, growth thin, veily, unusually spreading. 
ENTIRE, smooth, having a margin destitute of teeth or notches. 
EROSE, border irregularly toothed. 
FILAMENTOUS, growth composed of long, irregularly placed or 
interwoven filaments. 
FILIFORM, in stroke or stab cultures a uniform growth along line 
of inoculation. 
FIMBRIATE, border fringed with slender processes, larger than 
filaments. 
FLOCCOSE, growth composed of short curved chains, variously 
oriented. 
FLOCCULENT, said of fluids which contain pseudozooglese, i. e., 
small adherent masses of bacteria of various shapes and floating 
in the culture fluid. 
FLUORESCENT, having one color by transmitted light and another 
by reflected light. 
GRAM'S STAIN, a method of differential bleaching after gentian 
violet, methyl violet, etc. The + mark is to be given only 
when the bacteria are deep blue or remain blue after counter- 
staining with Bismarck brown. >. 
GRUMOSE, clotted. 
INFUNDIBULIFORM, form of a funnel or inverted cone. 
IRIDESCENT, like mother-of-pearl. The effect of very thin films. 
LACERATE, having the margin cut into irregular segments as if 
torn. 
LOBATE, border deeply undulate, producing lobes (see Undulate). 
LONG, many weeks, or months. 
(1) For decimal system of group numbers see Table I. This 
will be found useful as a quick method of showing close relationships 
inside the genus, but is not a sufficient characterization of any 
organism. 
(2) The morphologic characters shall be determined and 
described from growths obtained upon at least one solid medium 
(nutrient agar) and in at least one liquid medium (nutrient broth). 
Growths at 37° C. shall be in general not older than twenty-four to 
forty-eight hours, and growths at 20° C. not older than forty-eight 
to seventy-two hours. To secure uniformity in cultures, in all cases 
preliminary cultivation shall be practised as described in the revised 
Report of the Committee on Standard Methods of the Laboratory 
Section of the American Public Health Association, 1905. 
(3) The observation of cultural and biochemical features 
shall cover a period of at least fifteen days and frequently longer, 
and shall be made according to the revised Standard Methods 
above referred to. All media shall be made according to the same 
Standard Methods. 
(4) Gelatin stab cultures shall be held for six weeks to deter- 
mine liquefaction. 
(5) Ammonia and indol tests shall be made at end of tenth 
day, nitrite tests at end of fifth day. 
N 
(6) Titrate with - NaOH, using phenolphthalein as an indi- 
cator; make titrations at same times from blank. The difference 
gives the amount of acid produced. 
The titration should be done after boiling to drive off any 
CO2 present in the culture. 
(7) Generic nomenclature shall begin with the year 1872 
(Cohn's first important paper). 
Species nomenclature shall begin with the year 1880 (Koch's 
discovery of the pour plate method for the separation of organisms). 
(8) Chromogenesis shall be recorded in standard color terms. 
A NUMERICAL SYSTEM OF RECORDING THE SALIENT 
CHARACTERS OF AN ORGANISM. (GROUP NUMBER.) 
TABLE I. 
REPAND, wrinkled. 
100 .Endospores produced 
200 Endosporea not produced 
10 Aerobic (strict) 
20 Facultative anaerobic 
30 Anaerobic (strict) 
1 Gelatin liquefied 
2 Gelatin not liquefied 
0.1 Acid and gas from dextrose 
0.2 Acid without gas from dextrose 
0.3 No acid from dextrose 
0.4 No growth with dextrose 
0.61 Acid and gas from lactose 
0.02 Acid without gas from lactose 
0.03. No acid from lactose 
0.04 No growth with lactose 
0.001 Acid and gas from saccharose 
0.002 Acid without gas from saccharose 
0.003 No acid from saccharose 
0.004 No growth with saccharose 
0.0001 Nitrates reduced with evolution of gas 
0.0002 Nitrates not reduced 
0.0003 Nitrates reduced without gas formation 
0.00001 Fluorescent 
0.00002 Violet chromogens 
0.00003 Blue chromogens 
0.00004 Green chromogens 
0.00005 Yellow chromogens 
0.00006 Orange chromogens 
0.00007 Red chromogens 
0.00008 Brown chromogens 
0.00009 Pink chromogens 
0.00000 . Non-chromogenics 
0.000001 Diastasic action on potato starch, strong 
0.000002 Diastasic action on potato starch, feeble 
0.000003 Diastasic action on potato starch, absent 
0.0000001 Acid and gas from glycerin 
0.0000002 Acid without gas from glycerin 
0.0000003 No acid from glycerin 
0.0000004 No growth with glycerin 
The genus according to the system of Migula is given its proper 
symbol which precedes the number thus: (7) 
Bacillus com (Esch.) Mig. becomes B. 222.111102 
Bacillus alcaligenes Petr. becomes B. 212.333102 
Pseudomonas campestris (Pam.) Sm. becomes Ps. 211.333151 
Bacterium suicida Mig. becomes Bact. 222 232103 
1 See p. 73 for advantages of "hanging mass" of agar. 
2 Term also applied to the solidification of serum in media; e. g.y the Hiss inulin medium for the differentiation of pneumococci (see diplococcus of pneumonia). Source Date of Isolation Name Group No. (9 
DETAILED FEATURES. 
5. Gelatin Stab. 
Growth uniform, best at top, best at bottom. 
Line of puncture, filiform, beaded, papillate, villous, 
plumose, arborescent. 
Liquefaction crateriform, napiform, infundibuliform 
saccate, stratiform: begins in d. 
complete in d 
Medium fluorescent, browned 
6. Nutrient Broth. 
Surface growth, ring, pellicle, flocculent, membranous, 
none. 
Clouding slight, moderate, strong: transient, persistent: 
none: fluid turbid. 
Odor, absent, decided, resembling 
Sediment, compact, flocculent, granular, flaky, viscid 
on agitation, abundant, scant. 
7. Milk. 
Clearing without coagulation. 
Coagulation prompt, delayed, absent. 
Extrusion of whey begins in days. 
Coagulum slowly peptonized, rapidly peptonized. 
Peptonization begins on.. . ,d, complete on ... ,d. 
Reaction, Id...., 2d 4d...., 10d...., 20d 
Consistency, slimy, viscid, unchanged. 
Medium browned, reddened, blued, greened. 
Lab ferment, present, absent. 
8. Litmus Milk. 
Acid, alkaline, acid then alkaline, no change. 
Prompt reduction, no reduction, partial slow reduction. 
9. Gelatin Colonies. 
Growth slow, rapid. 
Form, punctiform, round, irregular, ameboid, myce- 
lioid, filamentous, rhizoid. 
Elevation, flat, effuse, raised, convex, pulvinate, crateri- 
form (liquefying). 
Edge, entire, undulate, lobate, erose, lacerate, fimbriate, 
filamentous, floccose, curled. 
Liquefaction, cup, saucer, spreading. 
10. Agar Colonies. 
Growth slow, rapid (temperature ) 
Form, punctiform, round, irregular, ameboid, myce- 
lioid, filamentous, rhizoid. 
Surface smooth, rough, concentrically ringed, radiate, 
striate. 
Elevation, flat, effuse, raised, convex, pulvinate, um- 
bonate. 
Edge, entire, undulate, lobate, erose, lacerate, fimbriate, 
floccose, curled. 
Internal structure, amorphous, finely-, coarsely-granular, 
grumose, filamentous, floccose, curled. 
11. Starch Jelly. 
Growth, scanty, copious. 
Diastatic action, absent, feeble, profound. 
Medium stained 
12. Silicate Jelly (Fermi's Solution). 
Growth copious, scanty, absent. 
Medium stained 
13. Cohn's Solution. 
Growth copious, scanty, absent. 
Medium fluorescent, non-fluor escent. 
14. Uschinsky's Solution. 
Growth copious, scanty, absent. 
Fluid viscid, not viscid. 
15. Sodium Chloride in Bouillon. 
Per cent, inhibiting growth 
16. Growth in Bouillon over Chloroform, unrestrained, 
feeble, absent. 
17. Nitrogen. Obtained from peptone, asparagin, glyco- 
coll, urea, ammonia salts, nitrogen. 
18. Best media for long-continued growth 
19. Quick tests for differential purposes 
2. Ammonia production, feeble, moderate, strong, absent, 
masked by acids. 
3. Nitrates in nitrate broth. 
Reduced, not reduced. 
Presence of nitrites ammonia 
Presence of nitrates free nitrogen 
4. Indol production, feeble, moderate, strong. 
5. Toleration of Acids, great, medium, slight. 
Acids tested 
6. Toleration of NaOH, great, medium, slight. 
7. Optimum reaction for growth in bouillon, stated in 
terms of Fuller's scale 
8. Vitality on culture media, brief, moderate, long. 
9. Temperature relations. 
Thermal death-point (10) minutes' exposure in 
nutrient broth when this is adapted to growth of 
organism) C. 
Optimum temperature for growth °C.; or 
best growth at 15° C., 20° C„ 25° C„ 30° C., 37° C., 
40° C„ 50° C„ 60° C. 
Maximum temperature for growth °C. 
Minimum temperature for growth °C. 
10. Killed readily by drying: resistant to drying. 
11. Per cent, killed by freezing (salt and crushed ice or 
liquid air) 
12. Sunlight: Exposure on ice in thinly sown agar plates; 
one-half plate covered (time 15 minutes), sensitive, 
not sensitive. 
Per cent, killed 
13. Acids produced 
14. Alkalies produced 
15. Alcohols 
16. Ferments, pepsin, trypsin, diastase, invertase, pectase, 
cytase, tyrosinase, oxidase, peroxidase, lipase, catalase, 
glucase, galactase, lab, etc 
17. Crystals formed: 
18. Effect of germicides: 
BRIEF CHARACTERIZATION 
NOTE-Underscore required terms. Observe notes 
and glossary of terms on opposite side of card. 
Mark + or O, and when two terms 
occur on a line erase the one which 
does not apply unless both apply. 
I. MORPHOLOGY (2) 
1. Vegetative Cells, Medium used 
temp age days 
Form, round, short rods, long rods, short chains, long 
chains, filaments, commas, short spirals, long spirals, 
Clostridium, cuneate, clavate, curved. 
Limits of Size 
Size of Majority 
Ends, rounded, truncate, concave. 
f Orientation (grouping) 
Agar J Chains (No. of elements) 
Hanging-block ] Short chains, long chains 
I Orientation of chains, parallel, irregular. 
2. Sporangia, medium used temp 
age days 
Form, elliptical, short rods, spindled, clavate, drum- 
sticks. 
Limits of Size Size of Majority . . 
A„nr f Orientation (grouping) 
tt„_ Went 1 Chains (No. of elements) 
g g I Orientation of chains, parallel, irregular. 
Location of Endospores, central, polar. 
3. Endospores. 
Form, round, elliptical, elongated. 
Limits of Size 
Size of Majority 
Wall, thick, thin. 
Sporangium wall, adherent, not adherent. 
Germination, equatorial, oblique, polar, bipolar, by 
stretching. 
4. Flagella, No Attachment polar, bipolar, peri- 
trichiate. How Stained 
5. Capsules, present on 
6. Zooglea, Pseudozooglea. 
7. Involution Forms, on in....days at....° C. 
8. Staining Reactions. 
1 : 10 watery fuchsin, gentian violet, carbol-fuchsin, 
Loeffler's alkaline methylene blue. 
Special Stains, 
Gram Glycogen 
Fat Acid-fast 
Neisser 
Diameter over 1M 
;* 
0 
o 
J 
o 
M 
Cm 
£ 
o 
Chains, filaments 
Endospores 
Capsules 
Zooglea, Pseudozooglea 
Motile 
§ 
Involution forms 
Gram's stain 
Cloudy, turbid 
o 
Ring 
2 
Pellicle 
Sediment 
Shining 
5 
Dull 
GQ 
Wrinkled 
W 
Ph 
Chromogenic 
P 
Eh 
Round 
Proteus-like 
(A 
ft 
Rhizoid 
<1 
s 
Filamentous 
Ph 
P 
Curled 
Eh 
1-1 
Surface growth 
P 
Q 
0m 
Needle growth 
Moderate, absent 
o 
Abundant 
o 
ft 
Discolored 
Starch destroyed 
Grows at 37° C. 
Grows in Cohn's sol. 
Grows in Uschinsky's sol. 
Gelatin (4) 
UI 
§ o 
Blood-serum 
w 
Ph 
.S? 
a a 
Casein 
£ 
H 
< 
K 
Acid curd 
Pm 
§ 
Rennet curd 
m3 
C 
Casein peptonized 
Q 
§ 
a 
Indol (5) 
Hydrogen sulphide 
M 
0 
Ammonia (8) 
o 
S 
Nitrates reduced (s) 
Fluorescent 
Luminous 
Animal pathogen, epizoon 
Plant pathogen, epiphyte 
z 
o 
Soil 
Milk 
H 
Fresh water 
S 
Salt water 
Ph 
H 
Sewage 
UI 
s 
Iron bacterium 
Sulphur bacterium 
Substance 
Method used 
Minutes 
Temperature 
Killing quantity 
r 
Amt. required to 
restrain growth 
H. CULTURAL FEATURES (») 
1. Agar Stroke. 
Growth, invisible, scanty, moderate, abundant. 
Form of growth, filiform, echinulate, beaded, spreading, 
plumose, arborescent, rhizoid. 
Elevation of growth, flat, effuse, raised, convex. 
Lustre, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verrucose. 
Optical characters, opaque, translucent, opalescent, 
iridescent. 
Chromogenesis (8) 
Odor, absent, decided, resembling 
Consistency, slimy, butyrous, viscid, membranous, 
coriaceous, brittle 
Medium grayed, browned, reddened, blued, greened. 
2. Potato. 
Growth scanty, moderate, abundant, transient, per- 
sistent. 
Form of growth, filiform, echinulate, beaded, spread- 
ing, plumose, arborescent, rhizoid. 
Elevation of growth, flat, effuse, raised, convex. 
Lustre, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verrucose. 
Chromogenesis (8) Pigment in water in- 
soluble, soluble: other solvents 
Odor, absent, decided, resembling 
Consistency, slimy, butyrous, viscid, membranous, 
coriaceous, brittle. 
Medium, grayed, browned, reddened, blued, greened. 
3. Loeffler's Blood-serum. 
Stroke invisible, scanty, moderate, abundant. 
Form of growth, filiform, echinulate, beaded, spread- 
ing, plumose, arborescent, rhizoid. 
Elevation of growth, flat, effuse, raised, convex. 
Lustre, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verrucose. 
Chromogenesis (8) 
Medium grayed, browned, reddened, blued, greened. 
Liquefaction begins in d, complete in d, 
4. Agar Stab. 
Growth uniform, best at top, best at bottom: surface 
growth scanty, abundant: restricted, wide-spread. 
Line of puncture, filiform, beaded, papillate, villous, 
plumose, arborescent: liquefaction. 
III. PHYSICAL AND BIOCHEMICAL FEATURES. 
1. Fermentation-tubes con- 
taining peptone-water or 
sugar-free bouillon and 
Dextrose 
Saccharose 
i Lactose 
Maltose 
Glycerin 
Manmt 
Gas production, in per cent. 
\CO2' 
Growth in closed arm 
Amount of acid produced Id. 
Amount of acid produced 2d. 
Amount of acid produced 3d. 
IV. PATHOGENICITY. 
1. Pathogenic to Animals. 
Insects, crustaceans, fishes, reptiles, birds, mice, rats, 
guinea-pigs, rabbits, dogs, cats, sheep, goats, cattle, 
horses, monkeys, man 
2. Pathogenic to Plants: 
3. Toxins, soluble, endotoxins. 
4. Non-toxin forming. 
5. Immunity bactericidal. 
6. Immunity non-bactericidal. 
7. Loss of virulence'on culture-media: prompt, gradual, 
not observed in months. CULTIVATION OF MICROORGANISMS 
121 
Toxin Production.-The presence of a toxin is not unusually elicited by cul- 
tural methods; one exception is the demonstration of hemolysins by plating 
with blood-agar. The hemolysis is shown by a clearing about the colony. 
For other methods of demonstrating the production of toxin, and the use of 
immunological reactions and animal tests sec the appropriate sections of this 
work. 
The accompanying chart is given as an example of a complete cultural study 
of an organism and the methods of using cultural reaction for classification. 
The characteristics to be elicited will depend on the group of organism studied. 
In the case of many of the organisms pathogenic for man and animal, the 
cultural characteristics only give us a presumptive identification, the final 
identification depending on animal and immunological tests. 
References. 
Bass and Johns. Jour. Exper. Med., 1912, xvi. 
Hottinger. Centralbl. f. Bakt., December 4, 1912. 
Krumwiede and Pratt. Jour. Inf. Dis., 1913, xii, 199. 
Williams. Jour. Med. Res., 1911, xx,'263. CHAPTER V. 
THE USE OF ANIMALS FOR DIAGNOSTIC AND TEST 
PURPOSES. 
Suitable animals are necessarily employed for many microbiologic 
purposes. (1) To obtain a growth of varieties that for any reason 
grow with difficulty on artificial culture media, as in the case of tubercle 
bacilli: hence material suspected to contain tubercle bacilli is injected 
into guinea-pigs, with the knowledge that, if present, although in too 
small numbers to be detected by microscopic or culture methods, 
they will develop in the animals' bodies, and thus reveal themselves. 
The same may be true of glanders, tetanus, and anthrax bacilli, of 
pneumococci, of other bacteria, and of protozoa. Certain microorganisms 
have not yet been grown on artificial media. This is true of few bacteria, 
of most protozoa, of many of the spirochetes, and of certain unknown 
infectious agents such as produce smallpox and Rocky Mountain spotted 
fever. (2) To cause an increase of one variety of organisms in a mixture 
and thus obtain a pure culture: An injection of sputum subcutaneously 
in rabbits may give rise to a pure pneumococcus septicemia or a pure 
tuberculosis. (3) To test virulence: Animals are used to test the 
virulence or toxin production of organisms, where, as in the case of 
diphtheria, we have very virulent, attenuated, and non-virulent bacilli 
of, so far as we know, identical cultural characteristics. Here the injec- 
tion of a susceptible animal, such as the guinea-pig, is the only way 
that we can differentiate between those capable of producing diseases 
from those that are harmless. Still another use of the animals is to 
differentiate between two virulent organisms, which, though entirely 
different in their specific disease poisons, are yet so closely allied mor- 
phologically and in culture characteristics that they cannot always be 
separated except by studying their action in the animal body both with 
and without the influence of specific serums. In this way the typhoid and 
colon bacilli may be separated, or the pneumococcus and streptococcus. 
(4) To test the antitoxic or microbicidal strength of sera: Diphtheria 
antitoxin is added to diphtheria toxin and injected into guinea-pigs, 
and streptococcus immunizing serum is mixed with living streptococci 
and injected into the vein of a rabbit. (5) To produce antitoxic, bacteri- 
cidal, or agglutinating sera. 
The Inoculation of Animals.-The inoculation of animals may be 
made either through natural channels or through artificial ones: 
1. Cutaneous. The material is rubbed into the abraded skin. 
2. Intracutaneous. The material is injected into the skin. Im- 
portant specific local reactions may be obtained by this method. THE INOCULATION OF ANIMALS 
123 
3. Subcutaneous. The substances are injected by means of a hypo- 
dermic needle under the skin, or are introduced by a platinum loop 
into a pocket made by an incision. 
4. Intravenous. The substances are injected by means of a hypo- 
dermic needle into the vein. This is usually carried out in the ear 
vein of the rabbit. If rabbits are placed in a holder, so that the animal 
remains quiet and only the head projects, it is usually easy to pass a 
small needle directly into one of the ear veins, especially those running 
along the edge of the ear. If the ear is first moistened with a 3 per 
cent, carbolic acid solution, and then supported between the finger 
inside and the thumb outside, the vein is usually clearly seen and entered 
with ease, if a small, sharp needle is held almost parallel with the ear 
surface and gently pushed into it. When no holder is present, the 
rabbit can be held by an assistant seizing the forelegs in one hand and 
the hind in another and holding the rabbit head downward, or the 
animal may be held between the knees of the operator, its body resting 
on the operator's apron. 
5. Into the anterior chamber of the eye. 
6. Into the body cavities. The peritoneal and less often the pleural 
cavities are used for microbal injection. The hypodermic needle is 
usually employed, less often a glass tube drawn out to a fine point. 
The needle or the pointed glass tube is gently pushed through the 
abdominal wall, moved about to be certain that the intestines have not 
been perforated and the fluid injected. 
7. By inhalation. This method is carried out by forcing the animal 
to inhale an infected spray or dust. 
8. By the trachea. This method is carried out by making an incision 
in the trachea and then inoculating the mucous membrane or injecting 
substances into the trachea and bronchi. 
9. Through the intestinal tract by swallowing or by the passage of 
a rubber tube. Morphine may be given to prevent peristalsis. 
10. Into the brain substance or ventricles after trephining, or when 
the parietal bones are thin, as in the guinea-pig and the rabbit, after 
making a tiny opening with the point of a small, heavy scalpel. 
Mice, which are usually inoculated subcutaneously in the body or 
at the root of the tail, are best placed in a mouse holder, but can be 
inoculated by grasping the tail in a pair of forceps, and then, while 
allowing the mouse to hang head downward in a jar, a glass plate is 
pushed across the top until only space for its tail is left. 
Monkeys and apes are used for certain infections, such as syphilis 
and smallpox, where only they and man are markedly susceptible. 
All these methods must be carried out with the greatest care as to 
cleanliness, the hair being clipped and the skin partially, at least, 
disinfected. The operator must be careful not to infect himself or his 
surroundings. After the inoculations the animals should be given the 
required food and kept in appropriate quarters. For food, rabbits and 
guinea-pigs usually require only carrots and hay. 
When possible, all animals should be anesthetized during painful 
experiments. 124 
PRINCIPLES OF MICROBIOLOGY 
If animals die, autopsies should be made at the earliest moment pos- 
sible, for soon after death some of the species of the bacteria in the 
intestines are able to penetrate through the intestinal walls and infect 
the body tissues. If delay is unavoidable, the animals should be put 
immediately in a place where the temperature is near the freezing point. 
In making cultures from the dead bodies the greatest care should be 
taken to avoid contamination. The skin should be disinfected, and any 
dust prevented by wetting with a 5 per cent, solution of carbolic acid. 
All instruments are sterilized by boiling in 3 per cent, washing-soda 
solution for five minutes. Changes of knives, scissors, and forceps 
should be made as frequently as the old ones become infected. When 
organs are examined the portion of the surface through which an incision 
is to be made must be sterilized, if there is danger that the surrounding 
cavity is infected, by searing with the flat blade of an iron spatula 
which has been heated to a dull-red heat. Tissues if removed should be 
immediately placed under cover so as not to become infected. Sterile 
deep Petri plates are useful for this purpose. 
When it is necessary to transport tissues from a distance they should 
be wrapped in sterile cloths and sent to the point of destination as 
soon as possible. In warm weather they may be kept cool by sur- 
rounding the vessels which contain them with ice. 
Animals rarely show the same gross lesions as man when both suffer 
from the same infection. The cell changes, however, are similar, and, 
also, so far as we can test them, the curative or immunizing effects 
of protective serums. 
Leukocytes for Testing Phagocytosis.-Inoculate into the pleural 
cavity of a rabbit 5 c.c. of a thick suspension of aleuronat powder in 
a boiled starch solution. The solution should be thick enough to hold 
the aleuronat in suspension. A 20 to 25 per cent, solution of peptone 
gives good results. The fluid is withdrawn eighteen to twenty-four 
hours after the injection. 
For purposes of obtaining the opsonic index the whole blood is taken. 
For description of the method see chapter on opsonins. 
Leukocytes from the horse can be readily obtained by mixing the 
blood with 1 per cent, of sodium citrate and allowing the mixture to 
stand. The red cells rapidly sink and leave the leukocytes in the super- 
natant fluid. CHAPTER VI. 
THE PROCURING AND HANDLING OF MATERIAL FOR 
MICROBIOLOGIC EXAMINATION FROM THOSE 
SUFFERING FROM DISEASE. 
A long experience has taught us that physicians very frequently 
take a great amount of trouble, and yet, on account of not carrying 
out certain simple but necessary precautions, make worthless cultures 
or send material almost useless for microbiologic study.. 
In making cultures from diseased tissues various procedures may 
be carried out, according to the facilities which the physician has and 
the kind of information that he desires to obtain. From the dead 
body culture material should be removed at the first moment pos- 
sible after death. Every hour's delay makes the results less reliable. 
From both dead and living tissues, the less the alteration that occurs 
in any substance between its removal from the body and its examination 
and inoculation upon or in culture media or animals, the more exact will 
be the information obtained. If the material is allowed to dry many 
microbes will be destroyed in the process, and certain forms which 
were present will be obliterated or, at least, entirely altered in the 
proportion which they bear to others. If possible, therefore, smears 
should be made and culture media should be inoculated directly from 
the patient or dead body. For the latter purpose a microbiologist 
should take the most suitable of the culture media to the bedside or 
autopsy table. Such a list of media, if fairly complete, would com- 
prise nutrient bouillon alone and mixed with one-third its quantity 
of ascitic fluid, slanted nutrient agar, slanted agar streaked with rabbit 
or human blood, firmly solidified slanted' blood-serum and slanted 
ascitic glucose agar. Additional media will be necessary for special 
purposes, such as the isolation of typhoid or tetanus bacilli. If only 
one variety of media is to be used the solidified blood-serum is most 
useful for parasitic bacteria, and this can be easily carried by the 
physician and inoculated by him, even if he is not very familiar with 
bacteriologic technique. In the first place some of the infected material 
should always be smeared on a couple of clean slides or cover-glasses 
and fixed moist in methyl alcohol or allowed to dry in the air. These 
can be stained and examined later, and may give much valuable 
information. 
The material must be obtained in different ways, according to the 
nature of the infection. 126 
PRINCIPLES OF MICROBIOLOGY 
For the detection of the bacteria causing septicemia we are met 
with the difficulty that there are apt to be very few organisms present 
in the blood until shortly before death. It will, therefore, be almost 
useless to take only a drop of blood for cultures, as even when present 
there may not be more than eight or ten organisms in a cubic centi- 
meter. If cultures are to be made at all, it is, therefore, best to make 
them correctly by taking from 5 to 20 c.c. of blood by means of a sterile 
hypodermic needle or a suitable glass tube armed with a hypodermic 
needle, from the vein of the arm after disinfecting the skin with tincture 
of iodine. To each of five different tubes containing bouillon we add 
1 c.c. of blood, and to a flask containing 100 c.c. we add 5 c.c. We 
have made by this mixture of blood and bouillon a most suitable 
medium for the growth of all bacteria which produce septicemia, and, 
at the same time, have added a sufficient quantity of blood to insure 
us the best possible chance of having added some of the bacteria pro- 
ducing the disease. We also add to each of several tubes of melted 
nutrient agar, at 40° C., 1 c.c. of blood and pour the mixture into Petri 
plates, so as to indicate roughly the number of organisms present by 
the number of colonies developing. When blood must be carried to a 
distance, clotting should be prevented by having in the test-tube suffi- 
cient 10 per cent, solution of sodium citrate, bile, or ammonium oxalate 
to prevent clotting. 
From wounds, abscesses, cellulitis, etc., the substance for microbio- 
logic examination can, as a rule, best be obtained by means of a syringe, 
or when the lesion is opened, by small rods armed with a little absorbent 
cotton. A number of these swabs can be sterilized in a test-tube and 
so carried. The swab is inserted in the wound, then streaked gently 
over the oblique surface of the nutrient agar in one tube, over the blood 
serum in another, and then inserted in the bouillon. Finally, either at 
the bedside or in the laboratory, material is thinly streaked over the 
surface of nutrient agar contained in several Petri dishes. We inoculate 
several varieties of media, with the hope that one at least will prove 
a suitable soil for the growth of the organisms present. From surface 
infections of mucous membranes, as in the nose, throat, vagina, etc., 
the swab, again, is probably the most useful instrument for obtaining 
the material for examination. The greatest care, of course, must be 
used in all cases to remove the material for study without contaminating 
it in any way by other material which does not belong to it. Thus, for 
instance, if we wish to obtain material from an abscess of the liver, 
where the organ lies in a peritoneal cavity infected with microbes, one 
must first absolutely sterilize the surface of the liver by pressing on it 
the blade of a hot-iron spatula before cutting into the abscess, so that 
we may not attribute the infection which caused the abscess to the 
germs which we obtained from the infected surface of the liver. From 
such an organ as the uterus it is only with the greatest care that we can 
avoid outside contamination, and only an expert microbiologist familiar 
with such material will be able to eliminate the vaginal from the uterine 
microbes. ROUTINE TECHNIQUE CARRIED OUT AT LABORATORY 
127 
A statement of the conditions under which materials are obtained 
should always accompany them when sent to the laboratory for examina- 
tion, even if the examination is to be made by the one who made the 
cultures. These facts should be noted, or otherwise at some future 
date they may be forgotten and misleading information sent out. 
The work of obtaining material for examination without contamination 
is at times one of extreme difficulty. It simply must be remembered 
that if contamination does take place our results may become entirely 
vitiated, and if the difficulties are so great that we cannot avoid it, 
it may simply mean that under such conditions no suitable examination 
can be made. Where the substance to be studied cannot be imme- 
diately subjected to cultures or animal inoculations, it should be trans- 
ferred in a sterile bottle as soon as possible to a location where the 
cultures can be made. If for any reason delay must take place, the 
material should at least be put in a refrigerator where cold will both 
prevent any further growth of some varieties of microbes and lessen 
the danger of the death of others. 
In obtaining samples of fluid, such as urine, feces, etc., the bottles 
in which they are placed should always be sterile, and, of course, no 
antiseptic should be added. It is necessary clearly to explain this to 
the nurse, for she has probably been instructed to add disinfectants 
to all discharges. Disinfected material is, of course, entirely useless 
for complete microbiologic investigations. It cannot be too much 
emphasized that materials which are not immediately used should be 
sent to the laboratory as quickly as possible, for in such substances as 
feces, where enormous numbers of various kinds of microbes are present, 
those which we seek most, such as the typhoid bacilli, frequently suc- 
cumb to the deleterious products of the other microbes present. Even 
when abundantly present, living typhoid bacilli may entirely disappear 
from the feces in the course of twelve hours, while at other times they 
may remain for weeks. These differences depend on the associated 
organisms present, the chemical constitution of the feces or urine, and 
the conditions under which the material is obtained. Water and milk 
rapidly change in their bacterial content if not kept under 40° F. 
For obtaining fluid for agglutination and other purposes, blister 
fluid is valuable. A blister can be raised quickly by placing a piece 
of blotting paper moistened with a little strong ammonia on the skin 
and covering with a watch-glass, or one may be more slowly formed by 
a cantharides plaster. 
Routine Technique Carried Out at Laboratory when Thorough 
Examination Required.-As has just been indicated, the microbio- 
logic examination proceeds somewhat differently according to the 
information needed. When, as is the case with most clinical material, 
definite knowledge in regard to the presence or absence of a particular 
organism is desired, the culture media are used which are known to 
be most suitable for the organisms sought, such as Loffler's blood- 
medium for the diphtheria bacillus. These media have been already 
partly given in the chapter on Culture Media. Others are later 128 
PRINCIPLES OF MICROBIOLOGY 
described under each microorganism. When, as is generally the case 
with autopsy material and sometimes with clinical, a complete ex- 
amination is needed in order to determine unknown organisms, the 
procedure may be as follows: 
1. At the autopsy table the routine cultures and smears are made 
as described above. 
2. Material from the different parts is secured under aseptic precau- 
tions in sterile receptacles and taken to the bacteriologic laboratory. 
The receptacles should be surrounded by ice if the laboratory is at a 
distance. 
3. A smear from each part is stained and examined in order to deter- 
mine in some measure the kind and number of microbes present, so we 
may more wisely select suitable culture media if other than those 
already used be needed, and may make the right culture dilutions if 
these be necessary. 
Gram's stain (see p. 78) gives more information, especially in regard 
to the first point, than any other one stain, so when possible this stain 
should be used. Other stains, however, may help, if for any reason 
Gram's is not at hand; and smears made from blood or from sus- 
pected syphilitic material should be stained by Giemsa's method dr an 
equivalent (see Staining Methods). 
A Gram-stained smear may show all Gram-negative or all Gram- 
positive microbes or a mixture of the two, or it may show a number 
only partially stained (gram amphophile). 
The following points must be remembered in using this stain and in inter- 
preting the results: 
(a) The smears should be thin and evenly spread. 
(b) The staining solutions should be fresh (aniline- water- gentian-violet lasts 
about three weeks). 
(c) Controls, fresh cultures (about twenty-four hours old) of a Gram-negative 
and a Gram-positive bacterium, should be used on the same slide with the 
smear to be examined. 
(d) If there is much albumin in the suspected material less heat should be 
used in fixing. 
(e) If the urine is very acid the results may not be good. 
(/) Mix urinary sediment with egg albumen, better to fix it, and wash out 
urinary salts with tap-water and stain. 
(</) Too much dependence should not be placed upon the finding of Gram- 
negative microbes in tissues, since organisms which in pure young cultures 
may be positive to Gram, show forms as they grow older both in tissues and 
in cultures, intermediate between the positive and negative, as well as a varying 
number of negative forms. 
If the smears show only Gram-negative organisms, the material 
probably contains one or more of the following: ROUTINE TECHNIQUE CARRIED OUT AT LABORATORY 
129 
B._coh group. 
B. typhosus group. 
B. dysenterise group. 
B. proteus. 
B. mucosus (capsulatus). 
B. pyocyaneus. 
B. influenza? group. 
B. pertussis group. 
B. fusiformis. 
B. mallei. 
B. edematis (malignant 
edema). 
B. of symptomatic anthrax. 
B. pestis. 
B. of Morax-Axenfeld. 
Most frequently 
from intestinal 
tract. 
Most frequently 
found, and 
some indication 
of their pres- 
ence in history. 
Gram- 
negative 
bacilli. 
Most frequently 
from chest con- 
tents. 
Less frequently found, and gen- 
erally a marked indication of 
their presence in history. 
Gram- 
negative 
cocci. 
Micrococcus intracellularis. 
Micrococcus catarrhalis. 
Micrococcus gonorrheae. 
Micrococcus melitensis. 
Generally marked indication of 
their presence in history. 
Gram- 
negative 
spirilla. 
S. cholerae and allied 
forms. 
Marked indication of presence of first 
form in history. 
Mouth spirals. 
Tr. pallidum. 
Sp. recurrentis. 
Unimportant, unless indicated in history, 
when Tr. pallidum or Sp. recurrcntis, 
respectively, should be looked for. 
Old forms of any of the Gram-positive or Gram-amphophile organisms. 
If only Gram-positive organisms are demonstrated, the material 
may contain one or more of the following: 
B. diphtherise group. 
B. tetani (not often demon- 
strated in smears from lesion). 
B. tuberculosis group. 
B. leprae group. 
B. anthracis group. 
B. welchii and some other in- 
testinal anaerobes. 
Gram- 
positive 
bacilli. 
Generally marked indication 
of their presence in history. 
Staphylococcus group. 
Streptococcus group (including 
pneumococcus and its variety, 
pneumococcus mucosus). 
Micrococcus tetragenus. 
Gram- 
positive 
cocci. 
Some indication of their pres- 
ence in history. 
Gram- 
positive 
spirilla. 
None. 
Higher 
bacteria. 
Nocardia. 
Actinomycosis. 
Yeasts and molds, certain forms. 
If organisms, partially stained by Gram's method are demonstrated, 
the material may contain one or more of the following: 
Molds. 
Yeasts. 
Protozoa. 
Slightly known cocci and bacilli. 
Ampho- 
phile. 
Older forms of Gram-positive organisms. 130 
PRINCIPLES OF MICROBIOLOGY 
Certain slightly known streptococci and bacilli, older forms of any 
of the Gram-positive organisms. 
Micrococcus intra- 
cellularis. 
Streptococcus (in- 
cluding pneumo- 
coccus) group. 
B. influenzae group. 
B. tuberculosis 
Fluid generally 
cloudy with many 
leukocytes. 
Meningeal 
(Cerebrospinal). 
Fluid generally clear. 
Fluids, 
from 
serous 
mem- 
branes. 
group. 
Streptococcus (in- 
cluding pneumo- 
coccus) group. 
B. mucosus (capsu- 
latus) group. 
B. influenzae group. 
B. tuberculosis 
Pericardial and 
pleural. 
Fluid may be cloudy. 
Fluid generally clear. 
group. 
B. coli group. 
Streptococcus group. 
B. tuberculosis. 
Peritoneal. 
group. 
Yeast, molds, amebee. 
Streptococcus (including pneumococcus) group. 
Micrococcus (including staphylococcus and diplococcus) group. 
B. mucosus (capsulatus). 
B. influenzae group. 
B. tuberculosis group. 
B. pertussis group. 
Yeasts, molds, amebae. 
Lungs. 
B. diphtheriae group. 
B. influenzae group. 
Streptococcus group. 
B. mucosus group. 
B. tuberculosis. 
Yeasts, molds, amebae. 
Tr. pallidum. 
Nose and 
throat. 
Streptococcus (including pneumococcus) group. 
Group of hemiglobinophilic bacilli. 
Gonococcus group. 
B. Diphtheriae group. 
B. Morax Axenfeld group. 
B. tuberculosis group. 
Yeasts and molds. 
Eye. 
B. coli group (including B. fecalis alcaligenes and B. acidi lactici). 
B. typhosus group. 
B. dysenteriae group. 
Gram-positive anaerobes. 
Yeasts, molds, amebae. 
Many forms whose importance has not been worked out. 
B. coli group. 
Feces. 
Urine. 
Streptococcus (kidney). 
M. gonorrheae. 
B. typhosus. 
B. tuberculosis. 
Molds, flagellates. ROUTINE TECHNIQUE CARRIED OUT AT LABORATORY 
131 
M. gonorrheae. 
Streptococcus. 
B. tuberculosis. 
Treponema pallidum. 
Certain anaerobes. 
Many other forms probably much less important. 
Pelvic 
organs. 
Streptococcus (including pneumococcus) group. 
B. typhosus group. 
Trypanosome group. 
Group of malarial organisms. 
Sp. recurrentis. 
Blood. 
Meningococcus group. 
Streptopneumococcus group. 
Group of hemiglobinophilic bacilli. 
B. tuberculosis group. 
Tr. pallidum. 
Malarial organisms. 
Rabies organisms. 
Yeasts, molds, etc. 
Brain. 
The following media should be used for the accompanying reasons: 
Nutrient broth, for motility, morphology, and arrangement (chains, groups, 
etc.). 
Potato for color and abundance of growth. 
Peptone broth for indol. 
Fermentation tube for anaerobes, acidity and gas. 
(a) Poured plates for isolated colonies (dilutions accord- 
ing to the number of organisms seen in smears). (Blood- 
agar if pneumococcus or streptococcus indicated.) 
(6) Streaked plates for surface colonies. (Blood-agar if 
influenza bacilli are indicated.) 
Nutrient agar 
and gelatin. 
Special media according to the kinds of organisms demonstrated in smears 
or indicated in histories. Such special media are described in the chapter on 
Media, and are indicated under the individual organisms. . CHAPTER VII. 
THE RELATION OF MICROORGANISMS TO DISEASE. 
In preceding chapters we have considered the growth of microorgan- 
isms for the most part in dead organic substances. Now we have to 
consider their growth and the production of their poisons in the living 
host and the results of such development. While it is true that there is 
h great difference between living and dead matter, and that, therefore, 
the living animal cannot be looked upon as merely a quantity of special 
material to be used for food for growth, still, in a very real sense, we 
are warranted in considering the infected living body as a food mass 
more or less favorable for the growth of microorganisms. The difference 
is that besides the chemical substances, temperature, and conditions 
inherent in the fluids and cells of the living body, microorganisms have 
also to reckon with the constant production of new substances by 
the living cells of the invaded organism, which may be antagonistic to 
them. In the production of lesions there are four main factors involved 
-viz., on the part of micobrganisms, the power to elaborate poison 
or proteins that may be split by ferment to yield poisons, and the ability 
to multiply; on the part of the cells of the body the degree of sensi- 
tiveness to the poisons of the microbe and the tendency of the cells 
which have absorbed the poisons or proteins of the invading germs to 
develop substances which neutralize the poisons or attack the invading 
bacteria or protozoa. No known variety of microorganisms has, in 
small numbers, the ability to produce enough poison to do appreciable 
injury in the body, nor is it probable that there is any variety which, 
if it multiplied in the body to the extent that some pathogenic bacteria 
are capable of, would not produce disease. 
To understand the germ factor in the production of disease we 
must recognize that both the body invaded and the parasitic cells which 
invade are living organisms, and that the products of the cellular 
activity of the body act on the microorganisms at the same time their 
products act upon the human cells. Just as there are different races 
and species of animals having dissimilar characteristics, there are dif- 
ferent races and species among bacteria and protozoa. The descendants 
of both under changing conditions gradually become diverse. In fact, 
the rapidity of the development of new generations of the unicellular 
organisms allow in them of much quicker changes under new conditions 
than are possible in the higher animals and plants. Considering these 
and other facts, we can readily understand how the different types of 
the microorganisms do not grow equally well in every variety of animal, 
and after discovering that there are variations in the properties of the 
blood from day to day we are not surprised that they do not find the THE RELATION OF MICROORGANISMS TO DISEASE 
133 
body of the same animal always equally suitable. The study of bacteria 
in the more simple and known conditions of artificial culture media has 
shown us how extremely sensitive many bacteria are to slight chemical 
and other changes. We have also found that conditions which are 
favorable to multiplication may still be unfavorable for the produc- 
tion of toxins. 
If we take specimens of diphtheria bacilli from three different cases 
of diphtheria, we sometimes find that on growing them for several 
days in suitable bouillon one culture will have produced poison in the 
fluid to such a degree that a single drop suffices to kill a large guinea- 
pig; the second, grown in a similar manner, will kill another animal of 
the same size with half a drop; while the third will kill with one-tenth 
of a drop. This illustrates the important fact that different varieties 
of the same bacillus have different toxin-producing powers under prac- 
tically the same conditions. 
The cultivation of the tetanus bacillus also furnishes some inter- 
esting facts which illustrate the complicated ways in which the growth 
of varieties of bacteria are hindered or assisted. The tetanus bacillus, 
when placed in suitable media, will not grow except in the absence of 
oxygen; but place it under the same conditions, together with a micro- 
organism which actively assimilates oxygen, and the two in association 
will grow in the presence of air. As a rule, when tetanus bacilli are 
driven into the flesh by a dirty nail or blank cartridge plug, aerobic 
bacteria are driven in also and so help to further infection by using up 
the free oxygen, thus introducing an anaerobic environment. 
The influenza bacillus is a striking example of the special require- 
ments of certain bacteria. On media it will thrive in pure culture 
only in the presence of hemoglobin. 
It is evident, therefore, that for each variety of organism there are 
special conditions requisite for growth, and that a temperature, degree 
of acidity, kind of food, supply of oxygen, etc., suitable for one may be 
utterly unsuitable for another; that, still further, when two organisms 
grow together one may so alter some of these conditions as to render 
unsuitable ones suitable, and vice versa. 
The pathogenic protozoa, indeed, the parasitic forms, are few in 
numbers compared with the total number of protozoa. They exert 
their harmful action mainly mechanically or by the direct destruction 
of the special host tissue which they find suitable for food. That they 
may produce specific toxic substances has been demonstrated in only 
one or two instances, the most marked of which is that of the poison 
obtained in the aqueous or glycerin extracts and the dried powder from 
mutton sarcosporidia which will be spoken of later. 
Though in general no specific toxins have been shown to exist in 
protozoan forms or to be excreted by them, the fact that there is 
spontaneous recovery from various protozoan infections and that a 
reinfection does not take place soon after, indicates that some specific 
toxins or substances are formed which help to produce immunity. 
Rossie has stated that he has obtained immune sera against infusoria; 134 
PRINCIPLES OF MICROBIOLOGY 
and antibodies have been demonstrated in animals which have re- 
ceived non-lethal doses of trypanosomes and of ameba'. 
Infection through protozoa is often accomplished by means of 
some of the lower animals acting either as intermediary hosts or as 
direct carriers of the virus (see Germ Carriers). 
Progress of Poisoning in an Infecting Disease.-When a fatal dose 
of a living virulent organism is injected into the peritoneal cavity 
of a guinea-pig Vaughan states the following effects result: For a 
period of time which usually varies from eight to twelve hours the 
animal remains apparently normal. Its temperature may fluctuate 
slightly, but not beyond the normal limits. This is the period of 
incubation and varies within certain time limits, but within these 
it is fairly constant. During this time, the bacteria are multiplying 
enormously in the animal body. They are converting animal 
proteins into bacterial proteins. This is largely a synthetic or con- 
structive process. The soluble proteins of blood and lymph are built 
into the cellular proteins of the bacteria. There is no liberation of the 
protein poison and consequently no disturbance in the well-being of the 
host. During the period of incubation of an infectious disease the 
invading organism supplies the ferment, the soluble proteins of the 
animal body constitute the substrate, the process is constructive, simple 
proteins are built into more complex ones, no protein poison is liberated, 
and no recognizable symptoms mark the progress of the infection. 
Still, in the development of the phenomena of infection, the period of 
incubation is critical, and the rate at which the infecting virus multi- 
plies during this time is an important factor in determining the final 
outcome. The more virulent the virus, the more rapidly does it mul- 
tiply and this means a larger amount of animal protein converted into 
bacterial protein. 
Somewhat abruptly there is a change in the behavior of our inoculated 
guinea-pig. The hairs behind the ears begin to stand out and soon the 
entire coat becomes rough. It no longer eats, but retires to one corner 
of the cage and seems to be in distress. Slight pressure over the abdomen 
elicits evidence of pain and the temperature begins to fall and continues 
to do so until death. In case of recovery, a rise of temperature is the 
first evidence of improvement. 
This somewhat abrupt change in the condition of the animal marks 
the end of the period of incubation and the beginning of the active dis- 
ease. The animal cells have become sensitized and are now pouring out 
a specific ferment which digests the bacterial proteins. In the active 
stage of the disease, the animal cells supply the ferment, the bacterial 
proteins constitute the substrate, complex cellular proteins are split 
into simpler bodies, the process is analytic and destructive, the protein 
poison is liberated, the symptoms of disease develop, and life is placed 
in jeopardy. 
Adaptation of Pathogenic Bacteria and Protozoa to Certain Tissues.- 
Parasitic microorganisms have gradually adapted themselves not only 
to certain species of animals, but to certain circumscribed areas of the LOCAL EFFECTS PRODUCED BY MICROORGANISMS 
135 
body. Thus the diphtheria bacilli grow chiefly upon the mucous 
membranes of the respiratory tract, but cannot develop in the blood 
or in the subcutaneous tissues. The cholera spirilla develop in the 
inflamed intestinal mucous membrane, but cannot grow in the respi- 
ratory tract, blood, or tissues. The tetanus bacilli develop in wounds 
of the subcutaneous tissues, but cannot grow on the intestinal mucous 
membranes or in the blood. The malarial parasites develop in the 
human body chiefly in or on the red-blood cells. 
Other microbes find, indeed, certain regions especially suitable for 
their growth, but under conditions favorable for them are capable of 
developing in other locations. Thus, the typhoid bacillus grows 
most luxuriantly in the Peyer patches and mesenteric glands, but also 
invades the blood, spleen, and other regions. The tubercle bacillus 
often remains localized in the apex of a lung or a gland for years, 
but may at any time invade many tissues of the body. The gonococcus 
finds the mucous membrane of the genito-urinary tract most suitable 
for its development, but also frequently is capable of growth in the 
eye and peritoneum and sometimes in the general circulation. The 
pneumococcus develops most readily in the lungs, but also invades the 
connective tissues, serous membranes, and the blood. The malarial 
protozoa grow not only in human red cells, but also in the cells of 
the stomach and the salivary glands of the mosquito. 
Most of these germs, although ordinarily increasing only in the body 
of man, can be grown on suitable dead material. 
There are a few unicellular organisms which, in so far as we know, 
find the bodies of human beings or animals the only fit soil for their 
growth. These are called strict parasites. 
Adaptation of Microbes to the Soil upon which They are Grown.- 
Those organisms which grow both in living and dead substances vary 
from time to time as to their readiness to develop in either the one 
or the other. As a general rule, bacteria grown in any one medium 
become more and more accustomed to that and other media more or 
less analogous to it, while, on the other hand, they are less easily culti- 
vated on media widely different from that in which they have developed. 
Thus we had a culture of tubercle bacilli which, after having grown 
for three years in the bodies of guinea-pigs, would grow only with great 
difficulty on dead organic matter, while a bacillus which was obtained 
from the same stock, but grown since on bouillon, will usually no longer 
increase in the animal body. From the same stock, therefore, two 
varieties have developed, the one having lost and the other gained in 
ability to develop as a parasite. 
Local Effects Produced by Microorganisms and Their Products.- 
The local effects of the microbal poisons and proteins upon the cells 
give rise to the various kinds of inflammation, such as serous, fibrinous, 
purulent, croupous, hemorrhagic, necrotic, gangrenous, and, finally, 
proliferative. Some organisms incite specific forms of inflammation 
along with those common to many organisms; others produce, so far 
as we can detect, no peculiar form of lesions. 136 
PRINCIPLES OF MICROBIOLOGY 
Thus inflammation and serous exudation into the subcutaneous 
tissues follow injections of the pneumococcus or anthrax bacillus. 
The development of the streptococcus or pneumococcus in the endo- 
cardium or pleural cavity is followed by a serous exudation, frequently 
with more or less fibrin production. The formation of pus results 
more especially from the streptococcus, pneumococcus, and staphylo- 
coccus; but nearly all forms of bacteria, when they accumulate 
in one locality, may produce purulent inflammation. The colon, 
typhoid, and influenza bacilli frequently cause the formation of 
abscesses. 
Catarrhal inflammation, with or without pus, follows the absorp- 
tion of the products of many bacteria, such as the gonococcus, pneu- 
mococcus, streptococcus, and influenza bacillus, etc. The hemor- 
rhagic exudation seen in pneumonia is usually due to the pneumo- 
coccus; it is observed also in other infections. Cell necrosis is pro- 
duced frequently by the products of the diphtheria and of the typhoid 
bacilli and by those of other bacteria. Specific proliferative inflam- 
mation follows the localization of the products derived from the tubercle 
bacillus and the leprosy bacillus. 
Not only can the poisons of one species of organisms, according to 
the tissues attacked, produce several forms of inflammation, but the 
same organism will vary as to its mode and extent of invasion, this 
depending, first, upon its own characteristics, at the time, as to viru- 
lence, etc., and, second, upon the conditions in the infected animal, 
such as its health and power of resistance, the location of infection, 
and the circumstances under which the animal is kept. Such varia- 
tions, therefore, are in no case specific, for different poisons will pro- 
duce changes which appear identical. 
Manner in which Microorganisms Produce Injury.-They produce 
serious mechanical injury only when they exist in such enormous 
numbers or bunched together as to interfere mechanically with the 
circulation or, together with fibrin, cause minute thrombi, and later 
emboli, which finally produce infarction and abscesses in different 
parts of the body. The effects are almost wholly due to the chemical 
substances contained in the cells or excreted by them, which are 
directly poisonous or after being split by ferments. Some portion of 
the protoplasm of almost every variety of bacteria acts as an irritant 
to tissues and combines with some of the substance of some of the 
body cells, and the protoplasm of most exerts a positive chemotaxis. 
The poisonous products can often be separated from the fluid in which 
the organisms have grown, or they can be extracted from the microbes. 
Injected into animals these products cause essentially the same cellular 
lesions as are produced by the organisms when they develop in the 
animal body. The substances contained in or produced by the organ- 
isms, with few exceptions, attract the leukocytes, and when great 
masses of bacteria die suppuration usually follows. The same properties 
are undoubtedly true of the poisons derived from the protozoa. VARIATION IN DEGREE OF VIRULENCE IN MICROBES 
137 
General Symptoms Caused by Microbal Poisons Absorbed into 
the Circulation.-Fever is produced, under favorable conditions, by 
all microbal proteids. A recpiisite is that sufficient proteid be absorbed; 
on the other hand, they must not be absorbed with such rapidity as 
to overwhelm the infected host, for a moderate dose may raise the 
temperature, while a very large dose lowers it, as occurs sometimes 
when a very large surface, such as the peritoneum, is suddenly involved. 
As Vaughan and others have shown, these foreign proteids have to be 
acted upon by the body ferments before they exert their specific 
effect. 
With few exceptions the microbal proteids produce an increase in 
the number of leukocytes and a lessening in the amount of hemoglobin 
in the blood. In uncomplicated infection with typhoid bacilli there 
is a leucopemia. The polynuclear leukocytes are usually increased 
in varying proportions in bacterial infections. The red-blood cells 
are directly injured by a number of bacterial substances. The 
deleterious effects on the nutrition are partly due to the direct effect 
of the poison and partly to the diseased conditions of the organs of 
the body, such as the spleen, kidney, and liver. Degeneration of the 
nerve cells is frequently noticed after infectious diseases; especially 
is this true of diphtheria. Several bacterial poisons have been found 
to produce convulsions; the best example of this is the tetanus toxin. 
In protozoan infections the mononuclear leukocytes and the eosino- 
philes are chiefly increased. 
Influence of Quantity in Infection.-With pathogenic microbes the 
number introduced has an immense influence upon the probability 
of infection taking place. 
If we introduce a few bacteria into a culture medium containing 
some fresh human blood or serum it is probable that they will all die 
because of the presence of sufficient bactericidal substance in the blood 
to destroy them; whereas if a greater number are introduced there 
will be at first a great diminution of these, those that die having com- 
bined with the bactericidal substances in the serum which neutralizes 
them; then those bacteria which survive begin to increase, and soon 
they multiply enormously. The same is true for parasitic bacteria 
in the body. A few only gaining entrance, they may die; a larger 
number being introduced, some may or may not survive; but if a still 
greater quantity is injected it is almost certain unless the animal is 
immune that there will be some surviving members, which will begin 
to proliferate and excite disease. 
Variation in Degree of Virulence Possessed by Microbes.-Microbes 
differ, as has already been stated, as to the ease and rapidity with 
which they grow in any nutritive substance and the amount of poison 
they produce. Both of these properties not only vary greatly in 
different members of the same species, but each variety of organism 
may to a large extent be increased or diminished in virulence. The 
septicemic class of bacteria when grown in the body fluids seem to 
gradually develop the power to elaborate protective substances in their 138 
PRINCIPLES OF MICROBIOLOGY 
own bodies or produce cells with less substance having affinity for the 
bactericidal bodies of the blood, and thus become less vulnerable. 
With those bacteria whose virulence is great a very few organisms 
will produce disease almost as quickly as a million, allowance only 
being made for the short time required for the few to become equal 
in number to the million. At the other extreme of virulence, however, 
many millions may have to be introduced to permit of the development 
of any of the organisms in the body. With these bacteria we are 
thus able to produce either no effect whatever, or a local, effect, or in 
some cases a general septicemia, by regulating the amount of infection 
introduced. 
Somewhat distinct, again, from that class of microorganisms which 
multiply rapidly are those like the tubercle and leprosy bacilli, which, 
while surely developing infection, increase more slowly. Here increase 
of virulence is shown, as before, by the production of disease through 
the introduction of very small numbers into the body, but increase in 
rapidity of development cannot progress except to within certain limits. 
A single streptococcus may, through its rapid multiplication, produce 
death in eighteen hours; a single tubercle bacillus, on the other hand, 
cannot produce sufficient numbers in less than two weeks. The 
virulence of the septicemic class of bacteria is not at all the same 
when measured in different animals, and it is largely for this reason 
that the virulence in test animals does not usually correspond with the 
severity of the case from which the organism was derived. 
Experimental Increase and Decrease in Toxicity and Virulence.- 
The power to produce toxin can be taken from microbes by growing 
them under adverse circumstances, such as cultivation at the maximum 
temperature at which they are capable of development. Some microbes 
are easily attenuated; others are robbed of their virulence only with 
great difficulty. Increase of toxin production is more difficult, and 
it is only possible to obtain it to a certain extent. The means usually 
employed are the frequent replanting of cultures. But with all our 
efforts we are usually only able to restore approximately the degree of 
toxin formation which the cultures originally possessed. The adap- 
tation of microbes to any nutritive substance, living or dead, so that 
they will grow more readily, is more easily brought about, provided 
they will grow at all. The streptococcus from erysipelas and the 
pneumococcus from pneumonia are typical of this class of organisms. 
Inoculate a rabbit with a few streptococci obtained from a case of 
human sepsis, and, as a rule, no result follows; inject a few million, 
and usually a local induration or abscess appears; but if one hundred 
million are administered septicemia develops. From this rabbit now 
inoculate another, and we find that a dose slightly smaller suffices to 
produce the same effect; in the next animal inoculated from this, still 
less is required, and so on, until in time, with some cultures, a very 
minute number will surely develop and produce death. With other 
cultures increase in virulence does not take place. The same increase 
in virulence can be noted when septic infection is carried in surgery or MIXED INFECTION 
139 
obstetrics from one human case to another. By allowing microbes to 
continue to develop under certain fixed conditions they become accus- 
tomed to these conditions, and less adapted to all that differ. 
Mixed Infection.-The combined effects upon the tissues of the 
products of two or more varieties of pathogenic microbes, and also of 
the influence of these different forms on each other, are of great im- 
portance in the production of disease. The infection from several 
different organisms may occur at the same time, or one may follow 
the other or others-so-called secondary infection. Thus, an abscess 
is often due to several forms of pyogenic cocci. If a fresh wound is 
infected from such a source the inflammation produced will probably 
be caused by all the varieties present in the original infection. Peri- 
tonitis following intestinal injuries must necessarily be due to more 
than one variety of organism. Thus, whenever two or more varieties 
of bacteria are transferred to a new soil, mixed infection takes place 
if more than one is capable of developing in that locality. 
Forms of infection which are allied to both mixed and secondary 
infection are those occurring in the mucous membranes of the respi- 
ratory and digestive tract. In these situations pathogenic microbes 
of slight virulence are always present even in health. Thus, in the 
upper air passages there are usually found streptococci, staphylococci, 
and pneumococci. When through the invasion of one or several 
infective agents, as the diphtheria bacillus or the virus of smallpox or 
scarlet fever, the epithelium of the mucous membrane of the throat 
is injured or destroyed, the pyogenic cocci already present are now 
enabled in this diseased membrane to grow, produce their poison, and 
even invade deeper tissues. The intestinal mucous membrane is 
invaded in a similar way by the colon bacilli and other organisms 
after injury by the typhoid or dysentery bacilli or cholera spirilla. 
Generally speaking, all inflammations of the mucous membranes and 
skin contain some of the elements of mixed infection. Blood-infection, 
on the other hand, is usually due to one form of microbe, as even when 
several varieties are introduced, only one, as a rule, is capable of 
development. The same is true to a somewhat less extent of inflam- 
mation of the connective tissue. The additional poison given off by 
the associated microbes aid infection by the primary invaders by 
causing a lowering of the vital resistance of the body. In some cases 
the secondary infection is a greater danger than the primary one, as 
pneumococcic bronchopneumonia in laryngeal diphtheria or strep- 
tococcic septicemia in scarlet fever and smallpox. 
Microorganisms are also at times directly influenced by the products 
of associated organisms. These may affect them injuriously, as, for 
example, the pyogenic cocci in anthrax; or they may be necessary to 
their development, as in the case of anaerobic bacteria. Not infre- 
quently the tetanus bacilli or spores would not be able to develop in 
wounds were it not for the presence of aerobic bacteria introduced 
with them. This is shown outside the body, where tetanus bacilli 
will not grow in the presence of oxygen unless aerobic bacteria are 140 
PRINCIPLES OF MICROBIOLOGY 
associated with them. Again, it is found that the association of one 
variety with another may increase its virulence. Streptococci are 
stated to increase the virulence of diphtheria bacilli, but here it is 
probably the loss of resistance of the tissues because of the strepto- 
coccic poison. On the other hand, the absorption of the products of 
certain bacteria immunizes the body against the invasion of other 
bacteria, as shown by Pasteur that attenuated chicken-cholera cultures 
produce immunity against anthrax. In intestinal putrefaction harm- 
less varieties of bacteria may be made to crowd out dangerous ones. 
Tissue Characteristics Influencing the Entrance and Growth of 
Microbes.-The Skin.-The skin is a poor soil for bacteria and is a 
great protection against the penetration of microorganisms. When 
they do penetrate, it is through the diseased glands, or more often 
through some unobserved wound. The bacterial toxins are, when at 
all, absorbed to a slight extent through the skin. 
There is an apparent exception to the above statements in the fact 
that the pyogenic staphylococci and sometimes the streptococci exist 
upon the skin or in it between its superficial horny cells, some excep- 
tional circumstances, such as wounds or burns, being required to 
allow the organisms to penetrate deeper. The cutaneous sweat 
glands, and the hair follicles with their appended sebaceous glands, 
may allow entrance of infection, as various incidents may lead to the 
introduction and retention of virulent microorganisms. When this 
occurs the retained products may lead to necrosis of the epithelium 
and thus allow the bacteria to penetrate to the deeper tissues. The 
secretion of the sebaceous glands appears to be little, if at all, bac- 
tericidal, but the perspiration, on account of the acidity, is slightly so. 
A species of protozoa (Leishmania) is found frequently in certain skin 
lesions in the tropics. 
Subcutaneous Connective Tissues.-Many microbes cannot develop 
in the connective tissues and others produce a milder infection there 
than elsewhere. Others develop readily. The tissue fluids have micro- 
bicidal properties similar to but less in amount than the blood. 
The Mucous Membranes.-The moist condition of the surface of the 
membranes and their frequent contact with irritating substances 
render them liable to microbal infection. Organisms, such as the pneu- 
mococci and streptococci, reproducing themselves in it become some- 
what attenuated. The mucous membranes are protected by the 
cleansing produced by the flow of the secretion and by its slight germ- 
icidal action. In infancy the membranes are readily infected by 
gonococci and later by pneumococci, by the Koch-Weeks bacillus and 
others. The mucous membranes of the nasal cavity are somewhat 
cleansed by the nasal secretion. The deeper portions of the nasal 
cavity are usually the seat of streptococci and other bacteria, while 
the extreme anterior portion contains saprophytic bacteria from the 
air. The mouth in a person in health is cleansed by the feebly bac- 
tericidal saliva. When the teeth are decayed many varieties of microbes 
abound. Many of these are difficult to cultivate. The bacteria, LOCATION OF POINT OF ENTRY OF ORGANISMS 
141 
such as the diphtheria bacilli, streptococci, etc., invade the mucous 
membrane of the tongue or mouth comparatively seldom. 
The tonsils with their crypts are usually the seat of the pyogenic 
cocci and are readily infected by the diphtheria bacilli and others. 
Whether the absolutely intact epithelium allows the passage of these 
bacteria is disputed, but the probability is that it does. With the 
slight pathologic lesions usually present it undoubtedly does. 
The Lungs.-Most inhaled organisms which pass the larynx are caught 
in the bronchi. Many of these are gradually removed by the ciliated 
epithelium. Both the alveolar epithelial cells and the leukocytes which 
enter the air sacs and bronchioles have been shown to take up bacteria. 
The normal lung is, therefore, rapidly freed of saprophytic and many 
parasitic bacteria. When subjected to deleterious influences, such as 
exposure to cold, the lung tissues seem to lose their protective defences 
and become subject to infection. 
The Stomach.-The pure gastric juice, through the hydrochloric 
acid it contains, is able to kill most non-spore-bearing organisms in a 
short time, but because of neutralization through food, or because the 
microbes are protected in the food, many of them pass into the intes- 
tines. Tubercle, typhoid, colon, and dysentery bacilli, when fed by 
the mouth with food, readily pass beyond the stomach. Certain 
acidophilic germs, as well as yeasts and torulse, seem to grow in the 
gastric secretion; these are largely non-pathogenic. Perforation of 
the stomach is usually followed by peritonitis, because of the irritant 
effect of the gastric juice and the bacteria which were temporarily pres- 
ent in the stomach. The gastric juice alters tetanus and diphtheria 
toxins. The toxicity of some poisons, such as occur in decayed meat, 
are not destroyed. The stomach is exceptionally free from bacterial 
inflammations. 
Intestines.-The bile is feebly germicidal for some organisms, but, 
on the whole, the intestinal secretions have little or no germicidal 
power. The number of microbes increases steadily from the duode- 
num to the head of the colon, and diminishes slightly from the upper 
to the lower end of the colon. The pancreatic juice destroys many 
of the toxic microbal products. The presence of the bacilli of the 
colon group, of streptococci, etc., does not often lead to any inflamma- 
tory condition in the normal intestines of healthy persons. In children 
suffering from the prostrating effects of heat they are apt to excite 
inflammatory changes. Even pathogenic bacteria, such as the 
typhoid, dysentery, and tubercle bacilli, may pass through the whole 
length of the healthy intestines without inciting inflammations. Slight 
lesions aid the passage of bacteria to the deeper structures. Tubercle 
bacilli and other pathogenic bacteria may pass through the intestinal 
wall to the lymph and cause distant infection without leaving any 
trace of their passage. Non-pathogenic protozoa are frequently found in 
the intestines, and, in tropical countries, pathogenic forms are also found. 
Importance of Location of Point of Entry of Organisms.-Most 
microbes cause infection only when they gain access to special tissues 142 
PRINCIPLES OF MICROBIOLOGY 
and must, therefore, enter through certain portals. This fact is of 
immense importance in the transmission or prevention of disease. 
Thus, for example, let us rub very virulent streptococci, typhoid bacilli, 
and diphtheria bacilli into an abrasion on the hand. The typhoid 
bacillus produces no lesion, the diphtheria bacillus but a very minute 
infected area, but the streptococcus may give rise to a severe cellulitis 
or fatal septicemia. Now place the same bacteria on an abrasion 
in the throat. The typhoid bacillus is again harmless; the diphtheria 
bacillus produces inflammation, a pseudomembrane, and toxemia, 
and the streptococcus causes an exudate, an abscess, or a septicemia. 
Finally, introduce the same bacteria into the intestines, and now it is 
the typhoid bacillus which produces its characteristic lesions, while 
the streptococcus and diphtheria bacillus are usually innocuous. 
If we tried in this way all the parasitic organisms we would find that 
certain varieties are capable of developing, and thereby exciting disease 
only on the mucous membrane of the throat, others of the intestine, 
others of the urethra; some develop only in the connective tissues or in 
the blood; .while others, again, under favorable conditions, seem able 
to grow in or upon most regions of the body. 
The Dissemination of Disease.-The spread of infection is influ- 
enced by: 1. The number of species of animals subject to infection. 
Many human infectious diseases do not occur in animals, and many 
animal infections are not found in man. Thus, so far as we know, 
gonorrhea, syphilis, measles, smallpox, typhoid fever, etc., do not 
occur in animals under ordinary conditions; while tuberculosis, an- 
thrax, glanders hydrophobia, and some other diseases are common to 
both man and animals. 
2. The quantity of the infectious material and the manner in which 
it is thrown off from the body. 
In diphtheria, typhoid fever, cholera, pulmonary tuberculosis, septic 
endometritis, influenza, and gonorrhea, enormous numbers of infec- 
tious bacteria are cast off through the discharges from the mouth, 
intestines, and genito-urinary secretions, causing great danger of infec- 
tion. On the other hand, in tuberculous peritonitis, streptococcic 
meningitis and endocarditis, gonorrheal rheumatism, and the like 
there is little or no danger of infecting others, as few or no bacteria are 
cast off. 
3. The resistance of the infectious organisms to the deleterious effects 
of drying, light, heat, etc. 
In this case the presence or absence of spores is of the greatest 
importance. The spore-bearing bacilli such as tetanus and anthrax, 
being able to withstand destruction for a long time, retain their 
power of producing infection for months or even years after elimination 
from the body. The bacteria which form no spores show great varia- 
tion in their resistance to outside influences. Some of these, such 
as the influenza bacilli and the gonococci, the virus of syphilis and 
hydrophobia, are extremely sensitive; the pneumococci, cholera spirilla, 
glanders bacilli, etc., are a little hardier; then follow the diphtheria GERM CARRIERS 
143 
bacilli, and after them the typhoid and tubercle bacilli and the 
staphylococci. Yeasts, molds, and protozoa produce resisting spores, 
which, however, are not as highly resistant as most bacterial spores. 
4. The ability or the lack of ability to grow outside of the infected 
tissues. 
Such bacteria as the pneumococcus, tubercle, influenza, diphtheria, 
glanders, and leprosy bacilli do not develop, as far as we know, outside 
of the body under ordinary conditions. Under exceptional circum- 
stances, as in milk, some may develop. Others, again, such as the 
streptococcus and staphylococcus, typhoid and anthrax bacillus, the 
cholera spirillum, and some anaerobes, may develop under peculiar 
conditions existing in water or soil. 
While for the pathogenic bacteria, as a rule, the saprophytes met 
with in the soil and water are antagonistic, yet in some cases-and 
especially is this true of the anaerobic bacteria-they are helpful. 
5. Germ Carriers.-In human carriers microorganisms develop in or 
upon some portion of the skin or mucous membrane, either after or be- 
fore disease, and without causing infection. As complete a knowledge 
as possible of this saprophytic development in man of parasitic microbes 
is necessary if we are to combat the spread of infection. In the super- 
ficial layers of the epithelium and on the surface of the skin we find 
the different pyogenic cocci, which are capable of infecting a wounded 
or injured part or causing inflammation in the glands. Acne, the 
pustules in smallpox, the pus on a burned surface, boils, etc., all 
come from these pyogenic cocci. In surgical cases the skin has to 
be as thoroughly disinfected as possible, to prevent the formation of 
stitch-hole abscesses and wound suppuration. 
In the secretion of the mucous membrane covering the pharynx 
and nasopharynx there is always an abundance of microbes. In throats 
examined in New York City, streptococci, staphylococci, and pneu- 
mococci are found in almost every instance, and even in the country 
they are often present. In the anterior nares there are fewer parasitic 
bacteria than in the posterior portions. Many other varieties of bacteria, 
such as the meningococci and the influenza bacilli, are probably often 
present in small numbers. In those constantly in contact with cases 
of diphtheria, and in those convalescent from diphtheria, virulent 
diphtheria bacilli are frequently found in the throat. 
After convalescence from typhoid fever, from one to three per cent, 
remain bacillus carriers for months or years. The bacilli continue to 
develop in the bile passages and are passed with the feces. 
Certain pathogenic protozoa may be carried for some time in the 
intestines of man. 
Lower Animals.-The lower animals, as a rule, do not retain in their 
bodies bacteria pathogenic for human beings, but, as direct carriers of 
bacterial infection, they are important factors. Flies and other insects 
may convey organisms which are simply attached to their feet or other 
surfaces of their bodies. Biting insects, especially, such as fleas, ticks, 
bed-bugs, lice, flies, and mosquitoes, are a source of danger in certain 144 
PRINCIPLES OF MICROBIOLOGY 
protozoan infections, since these insects act as intermediate hosts in 
these cases. (See Malaria and Other Protozoan Diseases.) 
Microbal Auto-infection.-When the intestinal canal is injured, or 
its circulation hindered by strangulation, etc., Bacillus coli and some 
other bacteria may penetrate through the injured walls and cause 
peritonitis or general infection. Under certain conditions,, as during 
the debility due to hot weather, the bacteria in the intestines cause, 
through their products, irritation, and in children even serious intes- 
tinal inflammation. Long after an acute gonorrhea has passed 
gonococci may remain in sufficient numbers to cause a new inflam- 
mation or produce infection in others. A cystitis may run on chron- 
ically for years, and then suddenly become acute or spread infection 
to the kidneys. A persistent gonorrheal vaginal infection may lead 
to a gonorrheal endometritis, or peritonitis or salpingitis, under 
suitable conditions. The staphylococci in the skin and the colon 
bacilli and pyogenic cocci in the fecal discharges may also be carried 
into the bladder and uterus and produce septic infection. Persons 
carrying diphtheria bacilli in their throats or typhoid bacilli in their 
gall-bladder may, under predisposing conditions, develop diphtheria 
or typhoid fever. 
In nearly all cases of infection the products of bacterial growth are 
absorbed into the blood, and along with them a few bacteria also, even 
when they do not reproduce themselves in it. The greater the extent 
of the infection and the more deep-seated it is, the greater is the amount 
of absorption. The bacteria enter the blood, according to Kruse, by 
(1) passive entrance through the stomata of the capillary walls; (2) 
carriage into the blood in the bodies of leukocytes; (3) growth of the 
bacteria through the walls of the vessels; (4) transmission of the bac- 
teria through the lymph glands placed between the lymph- and blood- 
vessels. 
When bacteria are abundant in the blood they become fixed in the 
capillaries of one or all of the organs, especially of the liver, kidneys, 
spleen, and lungs, and then directly or by means of the leukocytes, 
which penetrate the capillary walls, they pass into the tissues and 
substance of the organs. They thus reach the lymph channels and 
glands, or gain entrance into the gall-bladder, saliva, etc., or press 
through the epithelium, as in the alveoli of the lungs; more rarely 
they pass through the kidney tissue into the urine, as in typhoid 
fever, though some deny that this can happen unless there is a pre- 
vious inflammation of the kidneys. 
Microbes: Their Elimination through the Milk.-The passage of bac- 
teria through the breast is important, from the fact that milk is so 
largely used as food. Observers have reported the finding of tubercle 
bacilli in cows' milk when the gland itself was intact and the ani- 
mal tuberculous. Some authorities have put its presence in milk, 
under these circumstances, as high as 50 per cent, of the cases. This, 
in our experience, is undoubtedly too high. The fact that tubercle 
bacilli swallowed with the sputum are passed alive in the feces ex- EXTRACELLULAR TOXINS 
145 
plains the frequent occurrence of bacilli in the milk of cows without 
udder tuberculosis because of the contamination of the milk with 
manure. They are undoubtedly present, however, in the milk of 
some animals in which tuberculous disease of the gland could not be 
demonstrated. In these cases lymph glands adjacent to the udder 
are usually infected. The finding of streptococci and staphylococci 
is due probably in the majority of cases to the infections taking place 
as the milk is voided, for the epithelium at the outlet of the lacteal 
ducts is always infected with staphylococci, and frequently with 
streptococci, which have often been received from the mouth of the 
sucking infant. 
Elimination of Microbes by the Skin and Mucous Membranes.- 
Whether bacteria pass from the blood by the sweat is a mooted point. 
The skin is always the seat of the staphylococcus and frequently of 
other bacteria, so that it is difficult to determine in any given case 
the origin of the bacteria found in the sweat. Many observers have 
reported the passage of bacteria from the blood through the mucous 
membrane. These, as shown by Hess, are few in number, however. 
Bacteria are sometimes eliminated through the urine, but here, as a 
rule, when great numbers of organisms are found, it is due to devel- 
opment in the bladder. The removal of the poisonous products of 
bacteria by the kidneys, intestines, etc., on the contrary, is of great 
advantage to the organism. 
Some of the More Important Microbal Poisons.-Toxins.-Any 
poisonous substance formed in the growth of bacteria or other micro- 
organism is loosely called a toxin, but in the strict sense this term 
should be restricted to the extracellular poisons, as these alone have the 
important characteristic of causing the cells of the body affected by 
them to produce antitoxins. The different bacterial poisons vary 
greatly in their characteristics. As little is known concerning their 
chemical nature, except that they are proteins, we are not able to 
classify them. There are certain known differences among them which 
are important and which may be made use of for purposes of study 
to divide the bacteria into two groups: 
1. Those varieties of bacteria that excrete in ordinary culture media 
water-soluble, very specific, toxic products, extracelhilar toxins. Type: 
diphtheria, tetanus. These alone produce poisons. 
2. Those varieties which possess toxic protein substances, which are 
more or less closely bound to the living cell, and which are only in a 
small degree separable in unchanged condition outside of the body. 
On death of the cell they partly become free, partly remain united, or 
become secondary poisonous modifications. Type: cholera, typhoid, 
pneumococcus. These are frequently called endotoxins. Some of 
these substances are only poisonous when split up by specific ferments 
present in the serum or cell fluids. 
Extracellular Toxins.-Among the properties of the extracellular 
toxins are the following: They are, so far as known, uncrystallizable, 
and thus differ from ptomains; they are soluble in water and they 
10 146 
PRINCIPLES OF MICROBIOLOGY 
are slowly dialyzable, through thin membranes, but not through thick 
membranes such as are used in refining antitoxic globulins; they are 
precipitated along with peptones by alcohol, and also by ammonium sul- 
phate; if they are proteins they are either albumoses or allied to the 
albumoses; they are relatively unstable, having their toxicity diminished 
or destroyed by heat and freezing as well as by chemical manipulation 
(the degree of heat, etc., which is destructive varies much in different 
cases). Their potency is often altered in the precipitations practised 
to obtain them in a pure or concentrated condition, but among the 
precipitants ammonium sulphate has but moderate harmful effect. 
A remarkable characteristic of the group is that they are highly specific 
in their properties and have the power in the infected body to excite 
the production of antitoxins. The diphtheria and tetanus bacilli are 
the best known extracellular toxin producers. 
Precipitation of the Extracellular Toxins.-Ammonium sulphate crys- 
tals are added to the fluid containing the toxin until it is saturated. 
A large excess of ammonium sulphate crystals is then added and the 
whole kept at about 37° C. for twelve to eighteen hours. -The toxin 
is precipitated along with the albumoses and peptones and rises to the 
surface. This is skimmed off and dried in a vacuum or in an exsiccator 
containing strong sulphuric acid. The dried powder is placed in vacuum 
tubes and stored in the dark. Under these conditions the toxins deteri- 
orate very slowly. During the process there may be a considerable loss 
of toxin, even when every care is taken. Tetanus toxin is espe- 
cially liable to deterioration. Banzhaf has obtained a diphtheria toxin 
that is practically free from the meat extractions in the broth. He 
adds alcohol up to 65 per cent, to the slightly acidified toxin broth. 
The slight flocculent precipitate that is formed, after standing about 
one hour, is filtered off and dried in a vacuum. This dried toxin does 
not deteriorate. One gram of the powder contains 40,000 m. L. D., 
whereas the original toxin broth contained 5000 m. L. D. per gram of 
solids. 
Ferment Characteristics of Toxins.-The comparison of the action of 
bacteria in the tissues in the production of these toxins to what takes 
place in the gastric digestion has raised the question of the possibility 
of the elaboration by these bacteria of ferments, by which the process 
may be started. It would not be prudent to dogmatize as to whether 
the toxins do or do not belong to such an ill-defined group of substances 
as the ferments. It may be pointed out, however, that the essential 
concept of ferments is that of a body which can originate change without 
itself being appreciably changed, and no evidence has been adduced 
that toxins fulfil this condition. Another property of ferments is that, 
so long as the products of fermentation are removed, the action of a 
given amount of ferment is indefinite. In the case of toxins no evidence 
of such an occurrence has been found. A certain amount of a toxin is 
always associated with a given amount of disease effect. 
Intracellular Poisons.-Regarding the intracellular poisons or toxins 
which are more intimately associated with the bacterial cell and are SIMILAR VEGETABLE AND ANIMAL POISONS 
147 
produced by all bacteria we know much less. They are apparently 
proteins, and it is probable that their chemical nature is somewhat 
similar. They do not produce antibodies. These bacterial proteins 
may be poisonous originally, or only when split by a ferment. They 
have been studied by Vaughan, Friedberg and others. 
Nature of the Microbal Proteins.-Vaughan in his recent Herter 
lecture describes his theories of their nature in the following words: "(a) 
" The infective bacteria, taking the colon, typhoid, tubercle, and the 
pneumococcus as types, contain an intracellular poison. This is not a 
toxin; it isnot specific; it produces no antibody when injected into animals. 
(6) These bacteria elaborate no soluble toxin or poison. In old cultures 
there may be a trace of poison, but this results from the autolysis of 
the cells and is not a cellular secretion, (c) This poison is a group in the 
protein molecule and can be obtained in soluble form only after cleavage 
of the cellular proteins, which may be accomplished by superheated 
steam, dilute acids, or alkalis, (d) It exists in all true proteins, in patho- 
genic and non-pathogenic bacteria, and in vegetable and animal proteins, 
(e) It may result from the cleavage action of proteolytic ferments. (/) 
In most vegetable and animal proteins the poisonous group is neutral- 
ized by combination with non-poisonous groups; consequently such 
proteins have no poisonous action until they undergo molecular dis- 
ruption. (g) This primary group is poisonous because of the avidity 
with which it combines with secondary groups in the proteins of the 
animal body. (h) The specificity of proteins lies in their secondary, 
non-poisonous groups. It is in these that one protein chemically and 
biologically differs from another, (i) Biologic relationship among 
proteins is determined by the chemical structure of their molecules. 
There are as many kinds of proteins as there are kinds of cells, (k) 
The symptoms of the infections differ chiefly on account of the organ 
or tissue in which the virus accumulates and where it is split up and its 
poison liberated. (Z) The ferment which causes the cleavage of the 
bacterial proteins in the different infectious diseases is specific." 
Similar Vegetable and Animal Poisons.-Substances similar to those 
classed as bacterial endotoxins and as soluble toxins are formed by many 
varieties of cells other than bacteria. The i;icin and abrin poisons 
obtained from the seeds of the Ricinus communis and the Abrus pre- 
catorius have a number of properties similar to those of the diphtheria 
and tetanus poisons. The active poisons contained in ricin and abrin 
have not yet been isolated, but the impure substances are extremely 
poisonous. When injected into suitable animals anti-poisons are pro- 
duced and accumulate in the serum. These neutralize the poisons 
wherever they come in contact with them. 
They resemble the toxins in a general way in the manner in which 
they react to heat and chemicals. They are precipitated by alcohol. 
Through animal membranes they are less dialyzable than albumoses. 
Substances having these characteristics are called toxalbumins. 
Poisonous snakes secrete poisons which have many of the character- 
istics of the bacterial albumoses. The venom contains some substances 148 
PRINCIPLES OF MICROBIOLOGY 
similar to peptone and others similar to globulin. The former cause 
general nervous symptoms and paralysis of the respiratory centre, 
while the latter cause intense local reaction with hemorrhages around 
the point of injection. The injection of venins into animals is followed 
by the production of antivenins which neutralize the venins. When 
the serum containing abundant suitable antivenins is injected into 
an infected person it has considerable therapeutic value. 
The pyogenic action of their proteins is common to all microbes, 
this depending principally upon their being extraneous albuminous 
substances. Pyogenic effects may be produced in like manner by 
extraneous albumins of non-microbal origin. That every extraneous 
albuminous substance is harmful to the organism which seeks to 
resist its action is shown by those specific precipitating ferments, the 
precipitins, which are produced in the organisms after the introduction 
of every extraneous albumin. 
Ehrlich's Theories as to the Nature of Extracellular Toxins.- 
From a large number of most carefully conducted experiments with 
the toxin and antitoxin of diphtheria, Ehrlich has formulated a theory 
concerning the former. This theory has undergone several modifi- 
cations since it was first proposed, and it is difficult to give an exact 
statement of its present status. Generally speaking, however, in con- 
densed form its essential points are as follows: 
Toxins and antitoxins neutralize one another after the manner of 
chemical reagents. The chief reasons for this belief lie in the ob- 
served facts: (a) that neutralization takes place more rapidly in con- 
centrated than in dilute solutions, and (6) that warmth hastens and 
cold retards neutralization. From these observations Ehrlich con- 
cludes that toxins and antitoxins act as chemical reagents do in the 
formation of double salts. A molecule of the poison requires an exact 
and constant quantity of the antitoxin in order to produce a neutral 
or harmless substance. This implies that a specific atomic group 
in the toxin molecule combines with a certain atomic group in the anti- 
toxin molecule. 
The toxins, however, are not simple bodies, but easily split into 
other substances which differ from one another in the avidity with 
which they combine with antitoxin. 
These derivatives Ehrlich calls prototoxins, deuterotoxins, and 
tritotoxins. 
All forms of toxins are supposed to consist of two modifications, 
which combine in an equally energetic manner with antitoxin or with 
suitable substance in the cells, but differ in their resistance to heat and 
other destructive agents. 
The less resistant form passes readily into a substance called tox- 
oid, which has the same affinity for the antitoxin as the original toxin, 
but is not poisonous. The facts observed, Ehrlich thinks, are best 
explained on the supposition that the toxic molecule contains two 
independent groups of atoms, one of which may be designated as the 
haptophorous and the other as the toxophorous group. It is by the PTOMAINS 
149 
action of the former that toxin unites with antitoxin or cell molecule 
and allows the latter to exert its poisonous effect. 
The toxophorous group is unstable, but after its destruction the 
molecule still unites with the antitoxin or the sensitive molecule 
through its retained haptophorous group. 
Bordet has shown that toxin unites in different multiples with anti- 
toxin, so that the toxin molecule may have its affinity slightly, partly, 
or wholly satisfied by antitoxin. Slightly satisfied, it is still feebly 
toxic; combined with a larger amount of antitoxin, it is not toxic; but 
still may, when absorbed into the system, lead to the production of 
antitoxin. Fully saturated, it has no poisonous properties and no 
ability to stimulate the production of antitoxin. 
Ptomains.-Nencki, and later, Brieger, Vaughan, and others, suc- 
ceeded in isolating organic bases of a definite chemical composition 
out of putrefying fluids-meat, fish, old cheese, and milk-as well 
as from pure bacterial cultures. Some of these were found to exert 
a poisonous effect, while others were harmless. The poisons may be 
present in the decomposing cadaver (hence the name ptomain, from 
putrefaction), and, in consequence, have to be taken into 
consideration in questions of legal medicine. They may be formed 
also in the living human body, and, if not made harmless by oxida- 
tion, may come to act therein as self-poisons or leukomains. They 
possess the characteristics of alkaloid bodies and are different from 
the specific poisonous toxins. 
Many ptomains are known already and among them are some whose 
exact chemical constitution is established. Especially interesting is 
the substance cadaverin, which was separated by Brieger from portions 
of decomposing dead bodies and from cholera cultures, by reason of 
the fact that Ladenburg prepared it synthetically and showed it to 
be pentamethylenediamin [(NH2)2(CH2)5]. The cholin group is 
particularly interesting. Cholin itself (C5Hi5NO2) arises from the 
hydrolytic breaking-up of lecithin, the fat-like substance found in 
considerable amounts in the brain and other nervous tissue. By the 
oxidation of cholin there can be produced the highly toxic muscarin, 
found by Schmiedeberg in a poisonous toadstool and isolated by Brieger 
in certain decomposing substances: 
c6h15no2 
Cholin. 
+ o 
c6h16no3 
Muscarin. 
The ptomain tyrotoxicon was obtained from cheese, milk, and ice- 
cream by Vaughan. 
Pyocyanin (C14H14N2O), which produces the color of blue or blue- 
green pus, is a ptomainic pigment. Similar bodies of a basic nature 
may be found in the intestinal contents as the products of bacterial 
decomposition. Some of these are poisons and. can be absorbed into 
the body. Since the name ptomain was given to the poisonous products 
of bacterial growth before these products were chemically understood 150 
PRINCIPLES OF MICROBIOLOGY 
it is by many wrongly applied to all poisons found in food. Such 
poisoning may be due to true toxins or even living germs. 
The isolation of these substances can here be only briefly referred 
to. According to Brieger's method, which is the one now generally 
employed, the cultures having a slight acid reaction (HC1) are boiled 
down, filtered, and the filtrate concentrated to a syrupy consistency 
dissolved in 96 per cent, alcohol, purified and precipitated by means 
of an alcoholic solution of bichloride of mercury. CHAPTER VIII. 
IMMUNITY, ACTIVE AND PASSIVE. THE ANTAGONISM 
EXISTING BETWEEN THE LIVING BODY AND 
MICROORGANISMS. 
That certain races of animals and men, and certain individuals 
among these, are more refractory to disease than others is a fact 
which has long been known. Experience and observation have 
taught us, further, that the same individuals are at one time more 
resistant to disease than at another. This inborn or spontaneous 
refractory condition to an infectious disease is termed natural im- 
munity, in contradistinction to that acquired by recovery from 
infection. It is not always easy to separate these two forms of 
immunity. 
In regard to variations in susceptibility, certain known facts have 
been accumulated. Thus, cold-blooded animals are generally insus- 
ceptible to infection from those bacteria which produce disease in 
warm-blooded animals, and vice versa. This is partly explained by 
the inability of the bacteria which grow at the temperature of warm- 
blooded animals to thrive at the temperature commonly existing in 
cold-blooded animals. But differences are observed not only between 
warm-blooded and cold-blooded animals, but also between the several 
races of warm-blooded animals. The anthrax bacillus is very infec- 
tious for the mouse and guinea-pig, while the rat is not susceptible 
to it unless its body resistance is reduced by disease and the amount 
of infection is great. The inability of the microorganism to grow 
in the body of an animal does not usually indicate, however, an insus- 
ceptibility to its poison; thus, for instance, rabbits are less suscep- 
tible than dogs to the effects of the poison elaborated by the pneumo- 
cocci, but these bacteria develop much better in the former than in 
the latter. In animals, as a whole, it is noticed experimentally that 
the young ones are less resistant to infection than the older and larger 
ones, except in those still young enough to have immunizing sub- 
stances acquired from the mother before or shortly after birth. 
Decrease of Resistance.-The difficulty experienced by the majority 
of microbes in developing in the healthy body can be to a great extent 
removed by any cause which lowers the general or local vitality of 
the tissues. Among the causes which bring about such lessened 
resistance of the body are hunger and starvation, bad ventilation and 
heating, exhaustion from overexertion, exposure to cold, the delete- 
rious effects of poisons, bacterial or other, acute and chronic diseases, 152 
PRINCIPLES OF MICROBIOLOGY 
vicious habits, drunkenness, etc. Purely local injuries, such as 
wounds, contusions, etc., give a point of entrance for infection, and 
tissue of less resistance, where the bacteria may develop and through 
their poison produce adjacent injury and so predispose to further 
bacterial invasion in much the same way as the heat of the forest fire 
dries the green trees in front of it and so prepares them to ignite. 
Local affections, such as endocarditis, may also afford an area of 
lessened resistance. The presence of foreign bodies in the tissues in 
like manner predisposes them to bacterial invasion. Interference with 
free circulation of blood and retention in the body of poisonous sub- 
stances which should be eliminated also tend to lessen the vitality. 
In these and other similar ways animals which are otherwise refractory 
may acquire a susceptibility to disease. 
Increase of Resistance by Non-specific Means.-All conditions 
which are favorable to the health of the body increase its resistance, 
and thus aid in preventing and overcoming infection. The internal 
use of antiseptics against bacteria is so far unsuccessful, for the reason 
that an amount still too small to inhibit bacterial growth is found 
to be poisonous to the tissue cells. The efficacy of mercury in syphilis 
is, possibly, an exception to the rule. In dealing with animal para- 
sites, and with certain molds there are a number of notable exceptions 
to this rule; e. g., quinine in malaria, emetin in amebiasis, trypan red 
in trypanosomiasis, and iodides in sporotrichosis. Such substances as 
leukocytic extract, nuclein, and similar organic substances contained 
in blood-serum, when introduced into the body in considerable quan- 
tity, aid somewhat in inhibiting or preventing the growth of many 
microbes. Even bouillon, salt solution, and small amounts of urine 
have a slight inhibitory action. The hastening of elimination of the 
microbal poisons by free intestinal evacuation and encouragement of 
the functions of the skin and kidneys are also of some avail. The 
enzymes formed by certain bacteria have been found to exert a slight 
bactericidal action not only on the germs which have directly or indi- 
rectly produced them in the body, but also on other varieties. None of 
these enzymes are sufficiently protective to be of practical value, nor 
are they equal in power to the protective substances formed by the 
tissues from the bacterial products. 
Use of Local Treatment in Limiting Microbal Invasion.-The 
total extirpation of the infected area by surgical means, if thoroughly 
carried out, removes the microbes entirely; but, unfortunately, this 
procedure is rarely possible. When incomplete it is frequently help- 
ful; but it may be harmful, for by creating tissue injury and expos- 
ing fresh wounded surfaces to infection it may lead to the further 
development of the disease. In some cases, however, like anthrax 
and infection from bites of rabid animals, almost complete removal 
of the virus, either by the knife or thorough cauterization, will prevent 
a general infection or so lessen the number of organisms in the body as 
to allow the germicidal element of its fluids to exterminate them. So 
also in tetanus, the invasion being limited, surgical interference may be SPECIFIC IMMUNITY 
153 
of great use by removing not only the bacilli themselves, but also that 
portion of their poison which has not as yet been absorbed from the 
tissues. The beneficial effects of opening an abscess, or cleansing and 
draining the pleural, peritoneal or uterine cavities are well known. 
The retention of the poisonous products of the organisms leads to 
their absorption, and then through their combining with some of the 
tissue cells and with the protective substances of the adjacent fluids 
the tone of the tissues is lowered at the same time that germicidal 
substances have been neutralized. This enables the germs to penetrate 
into tissues which would otherwise resist them. The mechanical 
effect of pressure on the walls of an abscess by its contents also aids 
absorption of toxins and microbal progress. Local bleeding and the 
application of cold probably act by lessening absorption. The ap- 
plication of warmth increases the blood flow to the part, and so, when 
the general blood-supply is germicidal, as it often is, it acts favorably 
on the inflammation. A similar effect of operative interference is 
noticed in the frequently observed beneficial result of laparotomy 
in tuberculous peritonitis. 
Antiseptic solutions have the power of cleansing and rendering 
sterile the surfaces of a wound-that is, of lessening the introduction 
of infection. After infection has taken place, however, it is doubt- 
ful whether antiseptic washing has much more direct influence than 
simple cleansing, and it certainly can have no germicidal effect 
at any distance from the surface, either direct or indirect. Certain 
infectious diseases which are comparatively superficial are probably 
benefited by antiseptic solutions; such are gonorrhea, diphtheria, and 
other inflammations of the mucous membranes. Even here, how- 
ever, it is impossible to do more than disinfect superficially, and in 
some cases any irritation of the tissues is apt to do more harm than 
good. In the superficial lesions of syphilis, tuberculosis, and other 
chronic processes, the local use of antiseptics is sometimes of great 
value. In these diseases the irritant effects of the antiseptics which 
stimulate the tissues may also be beneficial. 
Specific Immunity, or a Condition of the Body which Prevents 
the Development in it of One Variety of Microorganisms or Renders 
it Unaffected by their Specific Poisons.-The'invasion of the body 
by almost every variety of microorganism is followed, if death does 
not quickly ensue, by conditions which for a variable period and to 
a variable degree are deleterious to the further growth of that variety. 
This more or less pronounced specific immunity may be created in 
various ways: 
1. Through recovery from disease naturally contracted or from 
infection artificially produced. According to the nature of the invading 
microorganism this immunity may be slight, as after recovery from 
erysipelas or pneumonia, marked for a limited period of time, as in 
diphtheria and typhoid fever, or prolonged, as after scarlet fever or 
syphilis. The extent of this protection also varies in different persons 
after the same infection. 154 
PRINCIPLES OF MICROBIOLOGY 
2. By inoculation with microorganisms attenuated by heat, chem- 
icals, or other means. In this case an infection of the animal is pro- 
duced, of moderate severity, as a rule, and the immunity is not quite as 
marked and lasting as after recovery from more serious attack; but 
it is, nevertheless, considerable. The inoculation of sheep with the 
attenuated anthrax bacillus and the use of vaccination with cow-pox 
in man are examples of this method. 
3. By the injection of the living organisms into tissues where develop- 
ment will not take place, as the injection of diphtheria bacilli, typhoid 
bacilli or cholera spirilla into the subcutaneous tissues. Here the de- 
struction of the bacteria with the absorption of their products causes 
mild chemical poisoning, with considerable resulting immunity. 
4. By the injection of the dead bodies of bacteria or of the chemical 
products which they elaborate and discharge into the surrounding 
culture media during their life. This produces a less marked immunity 
than when the living culture is used, but the method is a safer one. 
5. By the injection of the blood-serum of animals which have pre- 
viously passed through a specific disease or have been inoculated with 
the microbal products. The first, probably, to think of the possibility 
of effecting this was Raynaud, who in 1877 showed that the injection 
of large quantities of serum derived from a vaccinated calf into an 
animal prevented its successful vaccination. The results obtained by 
v. Behring and Kitasato upon diphtheria and tetanus, where the serum 
neutralized the poisons rather than the direct development of the bac- 
teria, gave a still greater impetus to these investigations. 
Suitable animals after repeated infections gradually accumulate 
in their blood considerable amounts .of these protective substances, 
so that very small amounts of serum inserted in another animal will 
inhibit the growth of the microbes or neutralize their products. Thus, 
0.1 c.c. of a serum from a horse frequently infected by the pneumococcus 
will prevent the development in the body of a rabbit of many thousand 
times the fatal dose of very virulent pneumococci, and a few times a 
fatal dose of less virulent ones, the actual number as well as the virulence 
of the bacteria affecting the protective value of the serum. 
These protective substances are found also in other fluids of the 
body than in the blood; they occur, indeed, in the substance of many 
cells to a greater or less extent. 
The immunity produced by these five methods affects the entire 
body, as is natural, since the blood and lymph which contain the pro- 
tective substances are distributed everywhere. These protective sub- 
stances pass from the blood through the walls of the capillaries and 
finally find their way to the lymph and back to the blood. When the 
immunity is but slight, infection may take place in the more sensitive 
regions or where a large number of organisms have gained access, and 
still be impossible in those tissues having more natural resistance or 
slighter infection. 
Passive as Contrasted with Active Immunity.-After the immune 
serum is injected into man the immunity is greatest at the time of its 
reception into the blood. This, of course, is instantaneous after an PRACTICAL THERAPEUTIC VALUE OF BACTERICIDAL SERA 
155 
intra venous injection, but only after thirty-six to forty-eight hours when 
given subcutaneously (Fig. 65), and then declines, being lost rather 
quickly (in several months or weeks, according as to whether or not 
the serum is injected into the same species of animal as the one from 
which it was drawn or into another species, when repeated injections 
are required to maintain the immunity). This passive immunity is 
distinctly in contrast to the active immunity acquired after the intro- 
duction of microbes or microbal products, where the tissues of the 
organism, in ways to us unknown, throw out, in response to the 
microbal stimulus, inhibitory or antitoxic substances. Here immunity 
is actually lessened for one or two days, and then is increased, and 
reaches its height a week or ten days after the injection, and then 
continues for a week or two, when it slowly declines again and is lost 
after several months or years. 
Fig. 65 
Units 
Average antitoxin content of one c.c. of the blood of goats receiving a subcutaneous 
injection of 10,000 units of antitoxin. 
Limitation of Curative Power of Serums which Act Directly Against 
Microorganisms.-As a rule, the serum has to be given before the 
bacteria introduced into the body have multiplied greatly. After that 
period has elapsed the serum usually fails to act. This is partly because 
the germicidal and antitoxic substances of the serum are insufficient 
in amount and partly because suitable antibodies develop for only a 
portion of the varied types of poison produced by microorganisms. 
Practical Therapeutic Value of Bactericidal Sera.-The use of 
serums having specific protective properties has been tried practically 
on a large scale in man as a preventive of infection. In susceptible 
animals injections of some of the very virulent bacteria, as pneumo- 156 
PRINCIPLES OF MICROBIOLOGY 
cocci, streptococci, meningococci, and typhoid bacilli, can be robbed 
of all danger if small doses of their respective serums are given before 
the bacteria have increased to any great extent in the body. If given 
later they are usually ineffective. For some bacteria, such as tubercle 
bacilli, no serum has been obtained of sufficient power surely to prevent 
infection. Through bactericidal serums, therefore, we can immunize 
against many infections, and even stop some just commencing; but 
as yet we cannot cure an infection which is already fully developed, 
though even here there is reason to believe that we may possibly pre- 
vent an invasion of the general system from a diseased organ, as by 
the pneumococcus from an infected lung in pneumonia. On the whole, 
the serums which simply inhibit the growth of microbes without neu- 
tralizing the toxins have not given conclusive evidence, as observed in 
practice, of great value in already developed disease, unless the serum 
can be brought in considerable concentration in contact with the 
infected tissues as is done by intraspinal injections in cerebrospinal 
meningitis. 
Relative Development of Antitoxins and Bactericidal Substances 
in the Different Infections.-Although the serum of animals which 
have been infected with any one of many varieties of bacteria is usually 
both antitoxic and bactericidal, still, one form of these protective sub- 
stances is usually present almost alone; thus antitoxic substances are 
present almost exclusively in animals injected with two species of 
bacteria which produce powerful specific poisons-viz., the bacilli 
of diphtheria and tetanus. When the toxins of either of these are 
injected in small amounts the animals after complete recovery are 
able to bear a larger dose without deleterious effects. To v. Behring and 
Kitasato we owe the discovery that this protecting substance accumu- 
lates to such an extent in the blood that very small amounts of serum 
are sufficient to protect other animals from the effects of the true extra- 
cellular toxins. 
Except the diphtheria and tetanus bacilli, a few only of the impor- 
tant parasitic bacteria attacking man produce extracellular toxins in 
any considerable amount, thus being capable of causing the production 
in the body of antitoxins, and even these bacteria do it to a far less 
extent than those of tetanus and diphtheria. Following them are the 
dysentery and plague bacilli, and then the cholera spirilla, the typhoid 
bacilli, the gonococci, meningococci, streptococci, etc. These latter 
bacteria when injected excite more of the substances which inhibit bac- 
terial growth than of those which neutralize their toxins. The bacillus 
of symptomatic anthrax and of botulismus and the vegetable poisons 
ricin, crotin, and abrin also produce specific antitoxins. So far, neither 
protozoa nor molds have been shown to produce specific antitoxin, 
though with a few species agglutinins and other antibodies have been 
demonstrated. 
Antitoxin a Preventive.-Antitoxin prevents the poisonous action 
of toxin. It does not restore the cells after they have been injured 
by the toxin; it is, therefore, like the bactericidal substances, a pre- METHOD OF ADMINISTRATION 
157 
ventive rather than a cure. We find, experimentally, that a very 
much smaller amount of antitoxin will neutralize a fatal dose of toxin 
in an animal, if given before or at the same time, than if given only 
shortly after it. An animal already fatally poisoned by the toxin is 
unaffected by any amount of antitoxin. 
Method of Administration.-Antitoxins and germicidal substances 
are absorbed by the gastro-intestinal tract to only a very slight extent 
-certainly less than 2 per cent. They must, therefore, be introduced 
subcutaneously, intraspinally, or intravenously to enter the body in 
appreciable amounts. CHAPTER IX. 
NATURE OF THE- PROTECTIVE DEFENCES OF THE BODY 
AND THEIR MANNER OF ACTION-EHRLICH'S "SIDE 
CHAIN" AND OTHER THEORIES. 
The fluids and tissues of the animal body under the normal con- 
ditions of life are, as we have seen, not only unsuitable for the growth 
of the great majority of the varieties of organisms, but even germicidal 
to the living organisms. 
In seeking to account for the germicidal property of the blood, 
which to a greater or less extent affects all microbes, we cannot find 
it either in the insufficient or excessive concentration of the nutritive 
substances, or in the temperature, or in the reaction. We are thus 
driven to the conclusion that the body fluids and cells contain substances 
which are deleterious to microorganisms. 
Germicidal Properties of the Blood.-The bactericidal effect upon 
most bacteria of the blood serum, noted by Nuttall in 1886, is now 
undisputed, and is readily shown by the fact that moderate numbers 
of bacteria when inoculated into freshly drawn blood usually die soon, 
and this destruction may be so rapid that in a few hours none of 
millions remains alive. Even when some of the bacteria survive there 
is for a time a decrease in the number living. Buchner in 1889 showed 
that serum heated to 55° lost its destructive power. He believed that 
in serum there was but a single bactericidal substance and called it 
alexin. 
In 1894-95 Pfeiffer showed that when cultures containing dead 
or living cholera spirilla or typhoid bacilli are injected subcutaneously 
into animals or man, specific protective substances are formed in 
the blood of the individuals thus treated. These substances confer 
a more or less complete immunity against the invasion of the living 
germs of the respective diseases. He also described the occurrence 
of a peculiar phenomenon when some fresh culture of the typhoid 
bacillus on agar is added to a small quantity of serum from an animal 
immunized against typhoid bacilli and the mixture injected into the 
peritoneal cavity of a non-immunized guinea-pig. After this procedure, 
if from time to time minute drops of the liquid be withdrawn in a capil- 
lary tube and examined microscopically, it is found that the bacteria 
previously motile and vigorous, and which remain so in control animals 
inoculated without the specific serum, rapidly lose their motility and 
die. They are first immobilized, then they become somewhat swollen 
and agglomerated into balls or clumps, which gradually become paler 
and paler, until finally they are dissolved in the peritoneal fluid. This GERMICIDAL PROPERTIES OF THE BLOOD 
159 
process usually takes place in about twenty minutes, provided a suf- 
ficient degree of immunity be present in the animals from which the 
serum was obtained. The animals injected with the mixture of the 
serum of immunized animals and typhoid cultures remain unaffected, 
while control animals treated with a fluid containing only the serum of 
non-immunized animals mixed with typhoid cultures die.' Pfeiffer 
claimed that the reaction of the serum thus employed is so distinctly 
specific that it could serve for the differential diagnosis of the cholera 
vibrio or typhoid bacillus from other vibrios or allied bacilli, such 
as Finkler's and Prior's or those of the colon group respectively. He 
thus showed that there was a great increase in the bactericidal power 
of a serum after immunization for the species of bacteria used in immu- 
nization. Metchnikoff then showed that the immunized serum added 
to peritoneal fluid in the test-tube would have the same effect on the 
spirilla. In March, 1896, Pfeiffer and Kolle published an article entitled 
"The Differential Diagnosis of Typhoid Fevers by Means of the 
Serum of Animals Immunized against Typhoid Infection," in which 
they claimed that by the presence or absence of this reaction in the 
serum of convalescents from suspected typhoid fever the nature of 
the disease could be determined. 
Bordet in 1895 reported that defibrinated blood filtered free of blood 
cells could be used instead of the peritoneal fluid and that if to a serum 
from an immunized animal, which had lost its bactericidal power through 
age, fresh serum from an untreated animal was added, the serum re- 
gained its destructive powers, i. e., it was activated, although the fresh 
serum by itself had almost no effect. These observations of Pfeiffer and 
Bordet indicated clearly that two types of substances were required 
to destroy cells. Both of these were present in fresh immune serum, 
one of which was stable and more or less specific, and the other un- 
stable and non-specific. The latter was proven to be present in all 
blood, while the former existed, except in minute amount, only in the 
blood of the immunized. The number of microbes introduced in a 
germicidal test is of great importance, for the serum with its contained 
substances is capable of destroying only a certain number, and after 
that it has lost its germicidal properties. 
Thus the following test illustrates this: 
No. of bacteria 
Amount of 
in 1 c.c. fluid 
serum added 
30,000 
0.1 c.c. 
100,000 
0.1 c.c.c 
1,000,000 
0.1 c.c. 
Approximate number alive after being kept at 37° C. 
One hour 
Two hours 
Four hours 
400 
2 
0 
5,000 
1,000 
2,000 
400,000 
1,000,000 
5,000,000 
Haas found that the circulating blood is not always bactericidal for 
any given variety of bacteria to the same extent the serum is. 
During the testing of the bactericidal power of the serum on dif- 
ferent bacteria it was discovered that numerous varieties were not 
destroyed by the serum alone, but only when exposed to both serum 
and leukocytes. 160 
PRINCIPLES OF MICROBIOLOGY 
During these earlier years Metchnikoff perceived that the infected 
host was too little considered, and he drew attention to the role of 
the leukocytes. He noted that in inflammation there is an active mi- 
gration of leukocytes through the walls of the vessels toward the infect- 
ing bacteria. If the bacteria are very virulent they continue to increase, 
destroying the leukocytes. If the bacteria are not sufficiently virulent 
to set up a progressive inflammation they are themselves disintegrated. 
Later it was discovered that bacteria were much more susceptible to 
the leukocytes if they had been acted upon by a serum from a body 
that had had an infection. (See chapter on Opsonins.) 
Buchner made many experiments on the nature of the process. He 
showed that bacteria absorbed these bactericidal substances. Later, 
Bordet, Ehrlich, and others established that the alexin of Buchner was 
really a mixture of two types of substances of which one, named 
"immune body," "sensitizer," or "opsonin," is developed as the re- 
sult of the injection of foreign-cell substance, and the other, named 
"complement" or "alexin," is present in the blood of normal animals, 
and is not increased by injection. Neither one of these types of sub- 
stances alone destroys bacteria, while together they destroy certain 
varieties. Other bacteria require the action of the complement-like 
ferment in the leukocytes also. 
The Nature of Bacteriolytic, Hemolytic, Cytolytic Sera.-Bordet, 
through his own researches and those of Gruber and Durham, was able 
to show that the same type of reaction took place in the animal body 
when cells of any kind were injected. He showed, for instance, that 
there was a close similarity between bacteria and the cells of the blood. 
By immunizing an animal, species A, with red-blood cells of animal, 
species B, he found that the blood of A became hemolytic for the cells 
of B, just as if immunized with cholera spirilla it would have been 
bacteriolytic for cholera spirilla. Since then truths obtained from 
investigation with any type of cells have been applied equally to all 
others. This made it possible for Ehrlich, Bordet, and others to study 
the nature of these processes upon blood cells instead of bacteria. 
Experiments Devised by Ehrlich to Show the Nature of Cytolytic (Bacterio- 
lytic, Hemolytic, etc.) Substances in the Blood.-Ehrlich asked himself two 
questions: (1) What relation does the hemolytic serum or its two active com- 
ponents, immune body and complement, bear to the cell to be dissolved? (2) 
On what does the specificity of this hemolytic process depend? He made his 
experiments with a hemolytic serum that had been derived from a goat treated 
with the red cells of a sheep. This serum, therefore, was hemolytic specifically 
for sheep-blood cells-i. e., it possessed increased solvent properties exclusively 
for sheep-blood cells. Ehrlich argued as follows: "If the hemolysin is able to 
exert a specific solvent action on sheep-blood cells, then either of its two factors, 
the immune body or the alexin (complement) of normal serum, must possess a 
specific affinity for these red cells." To show this he devised in conjunction 
with Morgenroth the following series of experiments: 
Experiment 1.-The serum that was specifically hemolytic for sheep-blood 
cells was made inactive by heating to 55° C., so that then it contained only the 
heat-resistant substance (immune body). To this was then added a sufficient 
quantity of sheep red-blood cells, and after a time the mixture was centrifuged. NAMES ATTACHED TO SUBSTANCES PRODUCING CYTOLYSIS 
161 
Ehrlich and Morgenroth were now able to show that the red cells had combined 
with all the heat-resistant substances, and that the supernatant clear liquid was 
free from the same. In order to prove that such was the case they proceeded 
thus: To some of the clear centrifuged fluid they added more sheep red cells; 
and, in order to reactivate the serum, a sufficient amount of alexin in the form of 
normal serum was also added. The red cells, however, did not dissolve-there 
was no sensitizing substance. The next point to prove was that immune body 
had actually combined with red cells. The red cells which had been separated 
by the centrifuge were mixed with a little normal salt solution after freeing them 
as much as possible from fluid. Then a little alexin in the form of normal serum 
was added. After remining thus for two hours at 37° C. these cells had all 
given up their hemoglobin to the surrounding fluid. 
In this experiment, therefore, the red cells had combined with all the sensitizing 
substance, entirely freeing the serum of the same. 
The second important question solved by these authors was this: What 
relation does the alexin bear to the red cells? They studied this by means of a 
series of experiments similar to the preceding. 
Experiment 2.-Sheep red-blood cells were mixed with normal-i. e., not 
hemolytic goat serum. After a time the mixture was centrifuged and the washed 
red cells tested with the addition of sensitizing substance to determine the pres- 
ence of alexin. It was found that in this case the red cells, in direct contrast to 
their behavior toward the sensitizing substance in the first experiment, did not 
combine with even the smallest portion of alexin, and remained unchanged. 
This experiment showed that the sensitizing substance first combined with the 
cell and then only could the alexin unite with the combined cell-immune body 
complex. 
Experiment 3.-The third series of experiments was undertaken to show 
what relations existed between the blood cells on the one hand and the sensitizing 
substance and the alexin on the other, when both were present at the same 
time, and not, as in the other experiments, when they were present separately. 
This investigation was complicated by the fact that the specific immune serum 
very rapidly dissolves the red cells for which it is specific, and that any pro- 
longed contact between the cells and the serum at ordinary temperatures, in 
order to effect union, is out of the question. Ehrlich and Morgenroth found 
that at 0° C. no solution of the red cells by the hemolytic serum takes place. 
They therefore mixed some of their specific hemolytic serum with sheep-blood 
cells, and kept this mixture at 0° to 3° C. for several hours. No solution took 
place. They now centrifuged and tested both the sedimented red cells and the 
clear supernatant serum. It was found that at the temperature 0° to 3° C. the 
red cells had combined with all of the sensitizing substance, but had left the 
alexin practically untouched. 
The addition of red cells in the experiments was always in the form of a 5 per 
cent, mixture or suspension in 0.85 per cent.-i. e., isotonic-salt solution. 
The significance of the last of the above-cited experiments is, according to 
Ehrlich, at once apparent. It is that the sensitizing substance possesses one 
combining group with an intense affinity (active even at 0° C.) for the red cell, 
and a second group possessing a weaker affinity (one requiring a higher tem- 
perature) for the alexin. 
Names Attached to Substances Producing Cytolysis.-Different 
investigators have applied to them different names. The one which 
is resistant to heat, which attaches itself directly to the cells, 
even at low temperatures, and is increased during immunization, is 
called sensitizing substance, interbody, amboceptor, or immune 
body. The other, which is sensitive to heat, is present in the 
healthy normal serum, is not increased during immunization, and 
• 11 162 
PRINCIPLES OF MICROBIOLOGY 
which unites with the cells' protoplasm only at temperatures above 
the freezing-point, is called alexin, or complement. 
The immune body attaches itself to the cellular substance, but does 
not appreciably harm the cells. The complement destroys the cells 
after the immune body has made the cell vulnerable. 
According to Ehrlich, the immune body first unites with the proto- 
plasm of the cell and this develops in the immune body an affinity for 
the complement and the two unite. (See Fig. 66.) He believes that 
it is through the immune body that the complement exerts its action 
on the cell. Very similar to the immune body is the substance called 
opsonin. This unites with the cell, but instead 
of making it sensitive to the complement it 
makes it sensitive to some ferment contained 
in the leukocytes. The destruction of organ- 
isms by the opsonins and leukocytes will be 
considered in detail in a special chapter. 
Bordet's Theory.-Bordet supposes that, 
instead of the tissue cell receptors which 
have combined with the toxin or foreign cell 
substance (antigen, haptine) producing an 
excess of similar receptors, the body of the 
animal that is immunized, instead of repro- 
ducing old receptors in large amount without 
changing them, builds up substances which 
in their character resemble but are not iden- 
tical with preexistent principles. These new 
substances have become endowed with a more 
marked affinity for the specific antigen in 
question. Bordet considers that Ehrlich, in 
offering explanations which seem definitive, 
has caused certain problems which have 
scarcely been touched upon to be regarded 
as settled. According to Bordet, Ehrlich is 
wrong in attributing such special properties 
to the immune body alone rather than to both antigen and immune body 
to an equal degree. He states that, "as a matter of fact, these pheno- 
mena should be related, not as regards antigen or antibody considered 
separately, but as regards the complexes which result from their union, 
and it is evident that the special properties of the antigen must affect 
markedly and perhaps to a preponderating degree the qualities of 
such complexes. Just as the union of agglutinins with microbes produces 
in them a remarkable sensitivity to the agglutinating effect of electrolytes 
by modifying their property of molecular adhesion, in a similar way 
sensitizers confer on their antigens a similar modified property of 
adhesion, namely, alexin absorption." In his opinion, antibodies, 
whatever their nature, act very much alike; but the effects which they 
produce differ with the antigen in question. 
Muir has shown that when cells are saturated with both immune body 
Fig. 66 
Graphic representation of 
amboceptor or receptors of the 
third order and of complement, 
showing on left the immune 
body uniting complement to 
foreign cell and on right the 
action of anticomplement, 
binding complement: A, com- 
plement ; B, intermediary body; 
C, receptor; D, cell; E, anti- 
complement. ORIGIN OF COMPLEMENT 
163 
and complement, the addition of fresh cells causes a splitting off of im- 
mune body, but not of complement. This throws further doubt upon 
the direct union of immune body and complement. 
There are exceptional normal sera, the complement of which may be fixed 
by certain cells without the presence of an immune serum. Malvoz showed 
that this is the case with dog serum mixed with B. anthracis. This serum acts, 
moreover, as if it contained a true sensitizer, because, in the presence of this 
organism it will cause the fixation of the complement of the sera of rabbits 
and guinea-pigs. 
Most of the experiments which have been made with the purpose 
of clearing up these difficult problems have been made upon red-blood 
cells. Here the absorption of the immune bodies at low temperatures 
and the lack of noticeable injury until the complement is added at a 
suitably high temperature is very striking. 
Multiplicity of Immune Bodies and Complements.-The immune 
bodies are very numerous and fairly specific in their action. The 
complement substance is much less specific and, although probably 
multiple, when chemically considered, each variety acts upon widely 
different bacteria and cells after they have united with the immune 
body. There is little reason to think that the complement of one 
animal is any more capable of attacking cells prepared by immune 
bodies developed in its blood than by immune bodies developed in 
some other species. 
Relation between Virulence and the Building of Immune Bodies. 
■-It is believed by most to take place the more rapidly the more viru- 
lent the infecting organisms. In our experiments this has not been 
evident. It must be remembered that increase of virulence for one 
species of animal does not mean increase for all animals; so that in 
order to draw conclusions, the animal upon which the virulence is 
tested must be the same variety as the one being immunized. 
Origin of Immune Bodies.-Their source must undoubtedly be at- 
tributed to the cells, but probably only certain cells produce them. 
The red-blood cells, for instance, seem rather to destroy than to 
increase them. Injections into the lung and into the subcutaneous 
tissues of toxins and microbal substances give rise to the formation of 
antibodies which are certainly formed partly, if not wholly, locally, and 
later find their way to the blood. The nuclein derived from the cells, 
although it has a general germicidal action, and may enter into the 
complement (alexin), has different properties,.and so cannot itself be 
one of these bodies. 
Origin of Complement (Alexin).-The cells which have abundant 
nuclear substance, such as the leukocytes and lymph cells, seem espe- 
cially to be a source, and Metchnikoff asserts their preeminent role 
as the producers of both complements and immune bodies. Buchner 
and others have found that after injection of bacterial filtrates the 
leukocytes were attracted in great numbers to the region of injection, 164 
PRINCIPLES OF MICROBIOLOGY 
and that the fluid here, which was rich in leukocytes, was more bac- 
tericidal than that of the blood-serum elsewhere. Some claim to 
have demonstrated that along with increased leukocytosis there is a 
general increase in the complement in the blood; still, it has not yet 
been positively established that the complement is derived solely from 
the leukocytes, nor from all leukocytes, and a mere increase in them does 
not always mean an increase in the complement. 
Multipartial or Polyvalent Sera.-Microbes are not homogeneous 
masses of protoplasm, but are made up of various molecules which 
differ biologically from one another. Conforming to this, the anti- 
substances, immune bodies (antitoxins, opsonins, etc.), which appear 
in a serum are made up of the sum of the antibodies which correspond 
to these partial elements in the microorganism. These separate groups 
are called "partial groups." An immune serum, therefore, consists 
of the partial groups which correspond to the separate partial elements 
of the microbal body. We are further able to show that these partial 
elements in one and the same microbal species are not the same for all 
individuals of that species. Thus one culture of streptococci or of 
Bacillus coli may have a few partial elements which differ from those 
of another culture. What is the consequence of this? The consequence 
will be that when we immunize with a culture a of such bacteria we 
shall obtain a serum which acts completely on this culture, for in this 
serum all the partial elements present in culture a are represented. 
If, however, we employ culture b, c, or d, which perhaps possesses other 
partial elements, we shall find that the serum does not completely affect 
these cultures. As already stated, such a condition of things is met 
with in inflammations due to streptococci and other bacteria, and is, 
therefore, of considerable practical importance. It is because of this 
fact that a serum from an animal immunized to one culture acts best 
only in a certain percentage of cases. In order to overcome this 
difficulty in persons infected with these bacterial species we have no 
choice but to make sera, not by means of one culture, but by means 
of a number of different strains of the same species. The result of 
this will be that, corresponding to the various partial elements in 
these different cultures, we shall obtain a serum containing a large 
number of the partial groups. Such a serum will then exert a specific 
action on a large number of different cultures, but not quite as great 
an influence on any one as if only that variety had been injected. 
In other words, the development and the closer analysis of the 
problem of immunity, especially during the past few years, have 
shown us that we must make use, more than heretofore, of so-called 
'polyvalent or multipartial sera. In the serum therapy of streptococcus 
infections, of dysentery, etc., the production of such multipartial sera 
is an advantage in practice. Owing to these partial groups also, a 
serum-e. g., antityphoid serum-can specifically affect to a very 
slight degree a closely allied species of bacterium, like Bacillus coli, 
for example. For it is known that closely related species of bacteria 
possess certain partial groups in common, and a serum is thus produced ANTITOXIN, NATURE. AND AFFINITY 
165 
which to a certain extent acts on such allied species. This constitutes 
what is known as the "group reaction." 
Antitoxin, Nature and Affinity.-During the investigations on the 
bactericidal power of the blood the discovery of the antitoxins which 
combine with the toxins, but leave untouched the bacteria, was made 
by v. Behring and Kitasato, and the nature of the union was investigated 
by Bordet, Ehrlich, and others. The peculiar facts developed by these 
extensive studies became the basis for Ehrlich's side-chain theory. 
During the earlier investigations on diphtheria toxin the filtered or 
sterilized bouillon, in which the diphtheria bacillus had grown and 
produced its "toxin," was supposed to require for its neutralization an 
amount of antitoxin directly proportional to its toxicity as tested in 
guinea-pigs. Thus, if from one bouillon culture ten fatal doses of 
"toxin" were required to neutralize a certain quantity of antitoxin, 
it was believed that ten fatal doses from every culture, without regard 
to the way in which it had been produced or preserved, would also 
neutralize the same amount of antitoxin. Upon this belief was 
founded the original v. Behring-Ehrlich definition of an antitoxin unit, 
viz., that it was ten times the amount of antitoxin which neutralized 
ten fatal doses of toxin. 
The results of tests by different experimenters with the same anti- 
toxic serum, but with different diphtheria toxins, proved this opinion 
to be incorrect. Ehrlich1 deserves the credit for first clearly perceiving 
this. He obtained from various sources twelve toxins and compared 
their neutralizing value upon antitoxin; these tests gave most interesting 
and important information. The results in four toxins, which are repre- 
sentative of the twelve, are as shown in the following table: 
Toxin 
specimen 
number of 
Ehrlich. 
Estimated 
"minimal" 
fatal dose 
for 250-gm. 
guinea-pigs. 
Smallest number 
of fatal doses of 
toxic bouillon re- 
quired to kill a 
250-gm. guinea- 
pig within 5 days, 
when mixed with 
one antitoxin 
Fatal doses required 
to "completely 
neutralize one anti- 
toxin unit" as de- 
termined by the 
health of the guinea- 
pig remaining unaf- 
fected "Lo" Ehrlich. 
L T - Lo 
= fatal 
doses. 
Data upon "toxin" 
specimen given 
by Ehrlich. 
unit, 
"L+ Ehr- 
lich." 
4 
0.009 
39.4 
33.4 
6 
Old, deteriorated 
from 0.003 to 
0.009. 
7 
0.0165 
76.3 
54.4 
22 
Fresh toxin, pre- 
served with tri- 
cresol. 
9 
0.039 
123.0 
108.0 
15 
A number of fresh 
cultures grown at 
37° C. 4 and 8 
days. 
12 
0.0025 
100.0 
50.0 
50 
Tested immediate- 
ly after its with- 
drawal. 
From the facts set forth in the table, Ehrlich believed that the 
diphtheria bacilli in their growth produce a toxin which, so long as 
1 Die Wertbemessung des Diphtherieheilserums und deren theoretische Grundlagen. 
Klinisches Jahrbuch, 1897. 166 
PRINCIPLES OF MICROBIOLOGY 
it remains chemically unaltered, has a definite poisonous strength 
with a definite value in neutralizing antitoxin. The toxin is, however, 
an unstable compound, and begins to change almost immediately into 
substances which are not, at least acutely, poisonous, but which retain 
their power to neutralize antitoxin. 
Fig. 67 
Graphic representation of receptors of the first and third orders and of complement 
as conceived by Ehrlich: A, complement; B, intermediary or immune body; C, cell receptor; 
D, part of cell; E, toxophorous group of toxin; F, haptophorous group. 
The results of some experiments of Atkinson and Park were fully in accord 
with those published by Ehrlich as to the varying neutralizing value of a 
minimal fatal dose of "toxin." Moreover they also indicate roughly a general 
law in accordance with which these changes occur. 
The neutralizing value of a fatal dose of toxin is at its lowest in the culture 
fluid when the first considerable amounts of toxin have been produced. After 
a short period, during which the quantity of toxin in the fluid is increasing, 
the neutralizing value of the fatal dose begins to increase, at first rapidly, then 
more slowly. 
While the culture is still in vigorous growth and new toxin is being pro- 
duced, the neutralizing value of the fatal dose fluctuates somewhat, but with 
a generally upward tendency. After the cessation of toxin production the 
neutralizing value of the fatal dose increases steadily until it becomes five to 
ten times its original amount. 
In our experiments the greatest value for L+ was 126, the least 27. As at 
six hours L-f- was only 72 and at twenty-eight hours only 91, we doubt whether 
L-f- ever reaches above 150.1 When we seek to analyze the above-described 
process we find certain facts which seem to explain it in part. 
In the fluid holding the living bacilli we have, after the first few hours of 
toxin formation, a double process going on-one of deterioration in the toxin 
already accumulated, which tends to increase the neutralizing value of the 
1 L+ = fatal doses of toxin required to kill a guinea-pig in four days after having 
been mixed with one unit of antitoxin. EHRLICH'S THEORIES UPON ANTITOXIN PRODUCTION 
167 
fatal dose; the other of new toxin formation, which probably tends to diminish 
the neutralizing value. The chemical changes produced by the growth of the 
bacilli in the bouillon tend to aid one or the other of these processes, and so to 
make, from hour to hour, slight changes in the value of the fatal dose. Later, 
with the period, of cessation of toxin production, the gradual deterioration of 
the toxicity alone continues, and the fatal dose gradually and steadily increases 
in its neutralizing value. We believed that two or more types of - toxin were 
produced by the bacilli. 
With greater information Ehrlich has had to modify greatly the details of 
his earliest explanation of the reason of the variation in the ratio between tox- 
icity and neutralizing value of toxin. He now accepts the fact that diphtheria 
culture fluid contains at least two toxins which differ in their char- 
acteristics. 
To summarize Ehrlich's present views as to the nature of diphtheria toxin: 
The diphtheria bacillus secretes two toxic substances, one of which, the toxin, 
cause the acute phenomena of diphtheria intoxication, while the other, the toxon, 
causes cachexia and paralysis after a rather long period of incubation. The 
non-toxic toxin, or toxoid, appears as the result of the degeneration of the toxo- 
phore group of the toxin, the haptophore group remaining intact. The toxin 
may be separated into three divisions, which vary in their affinity for anti- 
toxin-prototoxin, deuterotoxin, and tritotoxin. On the same basis there are 
three toxoids-prototoxoids, syntoxoids, and epitoxoid (the toxon)-the first 
having the greatest affinity for antitoxin, while the epitoxoid has the least. 
The toxins are divided into an alpha and a beta portion, depending on the ease 
with which they are changed into toxoids. All of these substances unite with 
tissue cells and with antitoxin through the agency of a haptophore group, while 
the toxicity depends on the presence of a toxophore group in the toxin or 
toxon molecule. 
Bordet and others refuse to accept these conceptions of Ehrlich and the whole 
matter is at the present time under active discussion. Thus the existence or 
non-existence of toxons has excited a great deal of discussion among investi- 
gators. The Swedish chemist, Arrhenius, has quite recently given much atten- 
tion to toxons and is applying the principles of physical chemistry to the study 
of toxins and antitoxins. It is a well-known fact that some chemical substances 
when in solution have the power of breaking up into their constituent parts; 
thus sodium chloride breaks up in part into sodium and chlorine, as sodium or 
chlorine ions or electrolytes. . The dissociated sodium and chlorine may then 
enter into combination with any other suitable substance which may be present. 
Arrhenius holds that this is the case with the toxin-antitoxin molecule, that it 
may to a certain extent again break up into separate toxin and antitoxin. He 
believes that this dissociated toxin is the substance which Ehrlich has been 
calling toxon. Madsen, who formerly had done much work with toxons, has 
now joined with Arrhenius in support of the dissociation theory. In spite of 
their reasoning Ehrlich and his followers continue to uphold the toxon as an 
independent toxic substance. Recent investigations throw doubt on both 
explanations as being at all final. 
Ehrlich's Theories upon Antitoxin Production.-Ehrlich began by 
observing that of the many poisonous substances known to us only 
a comparatively small number existed against which we could truly 
immunize--i. e., obtain specific antibodies in the blood serum of the 
immunized organism. Let us look at two poisons which are very similar 
in their physiologic action, for example, strychnine and tetanus poison, 
both of which excite spasms through the central nervous system. One, 
strychnine, produces no antibody whatever in the serum, while the 
injection of the other, the tetanus poison, causes the formation of the 168 
PRINCIPLES OF MICROBIOLOGY 
specific tetanus antitoxin. Ehrlich says that this is because these sub- 
stances enter into entirely different relations with the cells of the living 
organism. The one substance, strychnine, merely enters into a loose 
combination with the cells of the central nervous system, so that it 
can again be abstracted from these cells by all kinds of solvents-e. g., 
by shaking with ether or chloroform. The combination, therefore, 
is a kind of solid solution, such as has been shown in the staining with 
aniline dyes. The tetanus poison, on the contrary, Ehrlich says, is firmly 
bound to the cell; it enters the cell itself, becoming a chemical part of the 
same, so that it cannot again be abstracted from the cell by solvent 
agents. Ehrlich says that the first requirement for every substance 
against which we can obtain a specific serum must be its power to enter 
into such a combination with one or more types of cells in the living 
animal. The substance must possess a definite chemical affinity for 
certain parts of the organism. Hence, in each substance against which 
we can specifically immunize, Ehrlich assumes a group of atoms which 
effects the specific binding to certain cells, the haptophore group (Fig. 
56, F). Corresponding to this is a group in the cell of the living 
organism C, the receptor group, with which the haptophore group 
combines. The haptophore group is distinct from that part of the 
substance which exerts the physiologic or pathologic effect in toxins, 
for example, from the group which is the carrier of the poisonous ac- 
tion, the so-called toxophore group E, or in ferments, from the group 
which exerts the ferment action, the zymophore group. Both groups, 
haptophore and functional, are independent of each other, and their 
separate presence can easily be demonstrated because the functional 
group-e. g., in poisonous toxins the toxophore group-is more readily 
destroyed by heat than the haptophore group. Thus by heating a toxin 
for some time to from 60° to 65° C. a product will be obtained which 
is much less poisonous, but which still possesses largely its power to 
bind antitoxins. In the case of toxins such substances are called 
toxoids. Ehrlich conceived the finer mechanism of the formation of 
specific substances to be somewhat as follows: The haptophore group 
is bound to the receptor of the living organism owing to a specific 
affinity. As a result of this the receptor is lost to the living organism, 
disposed of, and a biologic law formulated by Weigert now comes 
into action, the law of supercompensation; that is, the organism seeks 
to replace this defect, but in doing so, not merely replaces the receptors 
in question, but, according to Weigert, produces more of them than 
were previously present. The conditions are somewhat like those seen 
in the callus after a fracture, in which the organism likewise produces 
a greater amount of bone than was previously present. 
In this way, Ehrlich states, such a large number of one type of 
receptors are produced by certain cells, that these become excessive; 
they are then thrust off into the blood, and these free receptors cir- 
culating in the blood constitute the specific antibodies. Ehrlich 
therefore believes that the specific antibodies in the serum are nothing EHRLICH'S THEORIES UPON ANTITOXIN PRODUCTION 
169 
else than receptors for which the substance employed in immunization 
possesses specific affinity. Hence, the same substance which, so long 
as it remains a vital part of the cell, attracts the toxin and enables it 
to exert its poisonous action on the cell, now, when it is cast off circu- 
lates free in the blood or tissue fluids, acts as a protection by combining 
with and neutralizing the poison while still free in the blood, and thus 
prevents the poison molecule from reaching the cell itself. 
Fig. 68 
Graphic representation of Ehrlich's theory of the production of antitoxin and the neu- 
tralization of toxin: a, diphtheria toxin molecule; x, toxophore atom group; y, haptophore 
or combining group; b, cell receptors with affinity for diphtheria toxin; c, other cell receptors 
1. Cell with its receptors. Outside of cell, free toxin molecules. 
2. Toxin molecules combined with the cell receptors having affinity for diphtheria 
toxin. 
3. After three days, showing multiplication of cell receptors similar to those combined 
with toxin. 
4. After four days, excess of receptors cast off in the blood. 
5. Toxin molecules largely neutralized by combining with free receptors in blood of 
immunized animal or in animal into which blood with free receptors had been transferred. 
In the formation of the specific antibodies we must therefore dis- 
tinguish three stages (Fig. 68): 
The binding of the haptophore group to the receptor (2). 
The increased production of the receptors following this bind- 
ing (3). 
The thrusting off of these increased receptors into the blood (4). 170 
PRINCIPLES OF MICROBIOLOGY 
The idea of Weigert, that the cells are biologically altered so as 
to continue to make receptors (antitoxin) after the cessation of the 
injections, and that they increase in capacity to produce antitoxin 
as they become accustomed to forming it through the stimulus of re- 
peated injections, is not in accord with the observations made by us. 
The first point is opposed by the fact that there is uniformly a great drop 
in antitoxin ten days or two weeks after the cessation of the stiumlus 
of the injection. The second point is, we believe, rendered improbable 
by the fact that by partially neutralizing toxin before injecting it into 
animals, we have found it possible to excite the cells to produce as much 
antitoxin from the first as from any later injections. Twelve years 
ago we showed that an injection into a previously untreated horse of 
one liter of strong toxin which had been neutralized just sufficiently not 
to poison a guinea-pig was followed by the development of antitoxin 
during the following seven days so that each c.c. of serum contained 60 
units of antitoxin. 
It is true that by the ordinary methods of immunizing the first 
injections of toxin produce a very small response in antitoxin, but 
this is because it is possible to give only minute amounts of toxin 
without causing the death of the animal. Very few cells are thus 
brought in contact with the toxin. When horses are used for a long 
period they cease to produce as much antitoxin as at the beginning in 
spite of the fact that they appear to be in good health. 
The properties of antitoxins have been most accurately studied in the 
case of diphtheria antitoxin. Tetanus and all other antitoxins seem 
to be very similar in their characteristics. 
Nature of Diphtheria and Tetanus Antitoxin.-Experiments have 
seemed to show that it was either closely bound to the serum 
globulins or was itself a substance of proteid nature closely allied to 
serum globulin. A fact developed by Atkinson is that the globulins 
tend to increase markedly in the serum of horses as the antitoxic 
strength increases. It seems possible from the above that diphtheria 
antitoxin has the characteristics of the serum globulins. Antitoxin is 
but little injured by prolonged moderate heat (56° C.), but is destroyed 
by short exposure to higher temperatures (95° to 100° C.). It is less 
sensitive than diphtheria toxin. Atkinson, when research chemist 
in our laboratory, found that in the case of antitoxic serum the globulin 
precipitate carries with it all of the antitoxic power of the serum, 
leaving the filtrate without any neutralizing power against the diph- 
theria toxin. Independently of Atkinson, Pick obtained similar results. 
These experiments were continued later by Gibson and Banzhaf, who 
proved that the globulins which were insoluble in saturated sodium 
chloride solution carried with them no antitoxin. The soluble globulins 
which become insoluble on heating also contain no antitoxin. With 
this knowledge a practical method of eliminating much of the non- 
antitoxic portion of the serum was perfected. 
Production of Diphtheria and Tetanus Antitoxin for Therapeutic 
Purposes.-As a result of the work of years in the laboratories of the PRODUCTION OF DIPHTHERIA AND TETANUS ANTITOXIN 
171 
Health Department of New York City, the following may be laid down 
as a practical method: 
A strong diphtheria or tetanus toxin should be obtained by taking a 
very virulent culture and growing it in broth under the conditions 
already described. The followiny directions apply to the production of 
diphtheria antitoxin: 
The horses used should be young, vigorous, of fair size, and abso- 
lutely healthy. The horses are severally injected with 10,000 units of 
antitoxin so as to allow giving a much larger dose of toxin1 than would 
otherwise be safe and thus gain several weeks in time. The following 
figures give the actual injections in a horse which produced an unusually 
high grade of serum. 
Injections of toxin were given at first every two days and then later every 
three days in the following amounts: 
First day, 12 c.c. toxin c.c. fatal dose), the 10,000 units of diphtheria 
antitoxin having been given the previous day. 
Second and later injections of toxin without antitoxin at three-day intervals 
as follows: 15 c.c., 20 c.c., 30 c.c., 40 c.c., 50 c.c., 60 c.c., 80 c.c., 100 c.c., 125 
c.c., 150 c.c., 170 c.c., 205 c.c., 250 c.c., 300 c.c. (fortieth day). The injections 
were gradually increased until, on the sixtieth day, 675 c.c. were given. The 
whole injection should not be in one place, but divided into six or eight portions. 
The antitoxic strength of the serum was on the twenty-eighth day, 
225 units; on the fortieth day, 850 units; and on the sixtieth day, 
1000 units. Regular bleedings were made weekly for the next four 
months, when the serum had fallen to 600 units in spite of weekly, 
gradually increasing doses of toxin. 
If the antitoxin is not given we begin with 10 fatal doses of toxin 
diluted to 10 c.c. and increase the amount each time about 25 per cent. 
There is absolutely no way of judging which horses will produce the highest 
grades of antitoxin. Very roughly, those horses which are extremely sensitive 
and those which react hardly at all are the poorest, but even here there are 
exceptions. The only way, therefore, is to bleed the horses and test their serum 
at the end of six weeks or two months. If only high-grade serum is wanted 
all horses that give less than 150 units per c.c. are discarded. The retained 
horses receive steadily increasing doses, the rapidity of the increase and the 
interval of time between the doses (two days to one week) depending somewhat 
on the reaction following the injection, an elevation of temperature of more 
than 3 ° F. being undesirable. At the end of three months the antitoxin serum 
of all the horses should contain over 300 units, and in about 10 per cent, as much 
as 800 units in each cubic centimeter. Not more than 1 per cent, give above 
1000 units, and none so far has given as much as 2000 units per c.c. The very 
best horses if pushed to their limit continue to furnish blood containing the 
maximum amount of antitoxin for several months, and then, in spite of increas- 
ing injections of toxin, begin to furnish blood of gradually decreasing strength 
1 The culture, after a week's growth, is removed, and having been tested for purity 
by microscopic and culture tests is rendered sterile by the addition of 10 per cent, of a 
5 per cent, solution of carbolic acid. After forty-eight hours the dead bacilli have set- 
tled on the bottom of the jar and the clear fluid above is siphoned off, filtered, and 
stored in full bottles in a cold place until needed. Its strength is then tested by giving 
a series of guinea-pigs carefully measured amounts. Less than 0.005 c.c., when injected 
hypodermically, should kill a 250-gram guinea-pig. 172 
PRINCIPLES OF MICROBIOLOGY 
If an interval of three months' freedom from inoculations is given every nine 
months, the best horses furnish high-grade serum during their periods of treat- 
ment for from two to four years. 
Tetanus Antitoxin Production.-This is carried on exactly as in the 
case of diphtheria antitoxin except that one proceeds more slowly. 
Good horses yield a serum containing 200 or 300 units per c.c. For 
the collection and preservation of the antitoxin the blood is withdrawn 
from the jugular vein by means of a sharp-pointed cannula, which is 
plunged through the vein wall, a slit having been made in the skin. 
The blood is carried by a sterile rubber tube into large Erlenmeyer 
flasks, held slanted, or into cylindrical jars, and allowed to clot. The 
serum is drawn off after four days by means of sterile glass and rubber 
tubing, and is stored in large flasks. When the globulins are to be 
separated the blood may be added directly to one-tenth of its volume 
of a 10 per cent, solution of sodium citrate. This prevents clotting 
of the blood. Small phials are filled with the serum or globulin solu- 
tion. The phials and their stoppers, as indeed all the utensils used for 
holding the serum, must be absolutely sterile, and every possible pre- 
caution must be taken to avoid contamination. An antiseptic may be 
added to the serum as a preservative, but it is not necessary except 
when the serum is to be sent to great distances, where it cannot be kept 
under supervision. 
Kept from access of air and light and in a cold place it is fairly 
stable, deteriorating not more than 30 per cent., and often much less 
within a year. Diphtheria antitoxin, when stored in phials and kept 
under the above conditions, contains within 10 per cent, of its original 
strength for at least two months; after that it can be used by allowing 
for a maximum deterioration of 2 per cent, for each month. The 
antitoxin in old serum is just as effective as in that freshly bottled, 
only there is less of it. The serum itself is less apt to produce rashes. 
All producers put more units in the phials than the label calls for, so 
as to allow for gradual loss of strength. 
Standardizing of Antitoxin Testing.-Ehrlich, with the knowledge 
gained by his investigations, called attention to the necessity of all 
laboratories using the same toxin so that a unit would become a definite 
amount. The German Government undertook to supply this need by 
testing all sera produced in Germany and by supplying a carefully tested 
serum to be used by the producers as a standard to test their toxins 
and antitoxins by. In this way smaller testing stations can make their 
results correspond with those of the central station. The United States 
Marine Hospital Laboratory has also distributed to laboratories in the 
United States an equally carefully standardized serum for diphtheria 
and a toxin for tetanus. 
It is seen that the old definition of v. Behring and Ehrlich, that a diph- 
theria antitoxin unit contains the amount of antitoxin which will protect 
the life of a guinea-pig from one hundred fatal doses of toxin, is true 
only in a general sense and for freshly prepared toxins. The important 
point to remember is that for fourteen years the German, United States, THE SEPARATION OF ANTITOXIN FROM SERUM 
173 
and other governments have kept carefully preserved a serum which is 
supplied to all testing stations, so that the unit measure is the same in 
all countries and from year to year. 
The actual test to estimate the number of units in one c.c. of an 
antitoxin serum is, therefore, carried out as follows: Six guinea-pigs 
are injected with mixtures of tested toxin and the serum to be tested. 
In each of the mixtures there is the amount of toxin sufficient to just 
neutralize 1 unit of antitoxin. This has been standardized by testing 
with the serum from Washington. In each of the mixtures the amount 
of the unknown serum varies; for instance, No. 1 would contain 0.002 c.c. 
serum; No. 2, 0.003 c.c.; No. 3, 0.004 c.c.; No. 4, 0.005 c.c., etc. If at 
the end of four days, Nos. 1, 2, and 3 were dead and Nos. 4, 5, and 6 
were alive we would consider the serum to contain over 200 units per c.c. 
and less than 250 units because 0.004 (off c.c.) c.c. of the serum did 
not protect the guinea-pig for a full four days, while c.c. protected 
the guinea-pig for longer than four days. We would then test again 
between these limits. When we test for experimental purposes sera with 
very little antitoxin, we often use only one-tenth the above amount of 
toxin. 
In this case the resistance of the guinea-pig must be considered so 
that the guinea-pig must not only not die, but must remain well. The 
mixed toxin and antitoxin must remain together for fifteen minutes before 
injecting, so that complete union may occur. Doerr showed that very 
small amounts of the mixture could be injected intracutaneously. 
Tetanus antitoxin is tested in the same way except that the serum is 
tested against 1000 fatal doses of standard tetanus toxin instead of 100 
of diphtheria toxin. 
The Separation of Antitoxin from Serum.-There have already 
been many attempts to accomplish this in the case of the antitoxins. 
Those interested in the chemical side of these investigations are re- 
ferred to the recent article by Banzhaf, already mentioned. In 1900, 
Atkinson, working in the Research Laboratory of the Health Depart- 
ment, eliminated all but the globulin from the antitoxic serum, and we 
tried this partially refined serum in 36 cases. The results were so 
nearly identical with an equal number of cases treated with the whole 
serum from the same horse that it did not seem "to be worth while to go 
to the expense of preparing such an antitoxic solution. The idea that 
a practical separation of the antitoxin from much of the protein non- 
antitoxic portion of the serum was possible was not given up. In 
August of 1905 we began trials with an antitoxic preparation which 
offered grounds for hoping for better success. Dr. R. B. Gibson, 
chemist in the Research Laboratory, placed the ammonium sulphate 
precipitate from the antitoxic serum in saturated sodium chloride so- 
lution and found that the portion of the globulin soluble in this con- 
tained all the antitoxin. In this way the nucleoproteins and the 
insoluble globulins present in the Atkinson preparation were elimi- 
nated, as the following summary shows: 
Ordinary antitoxic serum contains serum globulins (antitoxic), 174 
PRINCIPLES OF MICROBIOLOGY 
serum globulins (non-anti toxic), serum albumins (non-antitoxic), 
serum nucleoproteins (non-antitoxic), cholesterin, lecithin, traces of 
bile-coloring matter, traces of bile salts and acids, traces of inorganic 
blood-salts and other non-protein compounds. Refined serum con- 
tains serum globulins (antitoxic), traces of serum globulins (non- 
antitoxic), dissolved in dilute saline solution. Later, Dr. E. J. Banzhaf, 
who had succeeded Gibson, discovered that if the antitoxic serum or 
plasma was heated to 57° for 18 hours there was a change of a con- 
siderable portion of the soluble globulins into insoluble globulins. The 
antitoxin remained unchanged. This permitted a greater elimination 
of the non-antitoxic proteins. 
Method of Concentration.-The material we use is blood-plasma 
instead of blood-serum. This is obtained by allowing the blood to 
flow directly from the jugular vein of the immunized horse into 10 
per cent, sodium citrate solution, which prevents it from clotting and 
allows the red corpuscles to settle out. This plasma is used, in place 
of serum, merely as a matter of convenience and economy. 
Isolating the Antitoxin Globulins.-From year to year Banzhaf 
has succeeded in discovering methods for further purifying the anti- 
toxin. These methods have greatly lessened the early rashes and subse- 
quent "serum sickness." Briefly his latest method is as follows: The 
citrated plasma is diluted with half its volume of water and saturated 
ammonium sulphate solution is added up to 30 per cent, saturated 
solution. This mixture is heated up to 60° centigrade and held there 
for one hour. Then filtered while hot. The. precipitate contains the 
native non-antitoxic proteins and a large amount of non-antitoxic pro- 
teins newly formed by the above method of heating. This precipitate 
is discarded. The filtrate is brought up to 50 per cent, saturated 
ammonium sulphate solution. The resulting precipitate contains only 
pseudoglobulin and antitoxin. The albumin remains in solutions and 
is discarded. The pseudoglobulins and antitoxin precipitate is pressed 
to remove excess of fluid and then dialyzed until free from salts. After 
dialysis is completed 0.8 per cent, sodium chloride is added for iso- 
tonicity and 0.3 per cent, trikresol for preservation. It is then filtered 
through paper pulp to remove extraneous matter, then through a 
Berkefeld clay filter to remove bacteria, tested for sterility and potency 
and filled in sterile syringes. The above method gives a concentration 
of about six times the original potency. 
Aggressins.-A further contribution has recently been made to 
the problems of virulence and immunity in the form of the "aggres- 
sin theory" of Bail. Apparently it grew out of an attempt to explain 
the so-called "phenomenon of Koch"-an observation made years 
ago by Koch-to the effect that tuberculous animals when inoculated 
intraperitoneally with a fresh culture of tubercle bacilli succumb 
quickly to an acute attack of the disease, the resulting exudate contain- 
ing almost exclusively lymphocytes. Bail found that if tubercle bacilli, 
together with sterilized tuberculous exudate, were injected into healthy 
guinea-pigs, the animal died very suddenly-i. e., in twenty-four hours AGGRESSIN S 
175 
or thereabouts. The exudate alone had no appreciable effect on the 
animal, while inoculation with tubercle bacilli alone produced death 
in a number of weeks. He therefore concludes that there is something 
in the exudate that allows the bacilli to become more aggressive, and 
hence has called this hypothetical substance "aggressin." He thinks 
it is an endotoxin liberated from the bacteria as a result of bacteriolysis 
and that it acts by paralyzing the polynuclear leukocyte, thereby 
preventing phagocytosis. Heating the exudate to 60° C. increases 
rather than diminishes its aggressive properties apd small doses act 
relatively more strongly than larger ones. These facts he explains 
by assuming the presence of two properties, one that prevents rapid 
death, is thermolabile and acts feebly in small doses, and one that 
favors rapid death and is thermostabile. He assumes that in a tuber- 
culous animal the tissues are saturated with the aggressin and when 
fluid collects in the body cavities, as it does on injection of tubercle 
bacilli, it contains large quantities of aggressin, which prevents migra- 
tion of the polynuclear leukocytes, but not of the lymphocytes, and 
hence allows the bacilli to develop freely, producing acute symptoms. 
In the peritoneal cavity of the normal animal injected with tubercle 
bacilli, on the other hand, are large numbers of polynuclear leukocytes 
which engulf the bacilli, thus inhibiting their rapid development, there 
being here no aggressin to prevent phagocytosis. 
This theory has been applied to a number of infections, including 
typhoid, cholera, dysentery, chicken cholera, pneumonia, and staphylo- 
coccus infections. In all, similar results have been obtained as with 
tubercle bacilli. When exudates, produced by virulent cultures of 
these various organisms and properly sterilized, are injected with fresh 
cultures into an animal, death occurs in much shorter time than when 
the organisms alone are injected. 
Moreover, it has been possible to immunize animals against these 
various infections by repeated injections of the aggressin in the form 
of exudates. This results in the formation of an "antiaggressin," 
which opposes the action of the aggressin, thereby enabling the leuko- 
cytes to take up the bacteria and thus protect the animal. This 
has been done in staphylococcus, dysentery, typhoid, cholera, pneu- 
mococcus, and chicken-cholera infection in animals. In addition, a 
very marked agglutinative property of the blood is acquired for the 
bacteria in the animals so immunized. The present view is that the body 
fluids contain both the split products and the unchanged protoplasm 
of the bacteria. This accounts both for their aiding the virulence 
of the bacteria and for the production of antibodies from their 
injection. 
References. 
Bail. Wiener klin. Woch., 1905, No. 9. Ibid., 1905, Nos. 14, 16, 17. Berliner klin. 
Woch., 1905, No. 15. Zeit. f. Hyg., 1905, vol. i, No. 3. Arch. f. Hyg., vol. lii, pp. 272 
and 411. 
Banzhaf. Collected studies, Department of Health, City of New York, 1912, vol. vii, 
p. 114. 176 
PRINCIPLES OF MICROBIOLOGY 
Bordet Resume of Immunity from 1898 to 1909, trans, by Gay. 
Buchner. Cent. f. Bakt., 1889. 
Ehrlich and Morgenroth. Berl. klin. woch., 1899, 1900. 
Ehrlich and Sachs. Collected Studies on Immunity, trans, by Bolduan. 
Malvoz. Annales de 1'Institut Pasteur, Aug., 1902. 
Metchnikoff. Ann. de 1'Inst. Past., 1889. 
Nuttall. Zeit. f. Hyg., 1886. 
Park and Atkinson. Journal of Experimental Medicine, vol. iii, No. 4. 
Pfeiffer u. Isaeff. Ztschr. f. Hyg., 1894. 
Vaughan and Vaughan. Protein Split Products, etc., 1913, Phila. and New York. 
Vaughan. Herter Lectures, 1913-14, New York. CHAPTER X. 
COMPLEMENT-FIXATION: THE TECHNIQUE OF THE 
TEST AND ITS PRACTICAL APPLICATIONS. 
Bordet1 and Gengou showed that the existence of a sensitizer or 
specific immune body in an antimicrobal serum, by uniting with its 
specific antigen (bacterium or other cell or proteid material), absorbs 
alexin (complement). 
This experiment is usually spoken of as the "Bordet-Gengou phe- 
nomenon." 
Wassermann and others applied this method in measuring the am- 
boceptor content of specific sera, the first important practical applica- 
tion of its use being in the diagnosis of syphilis. 
In order to demonstrate fixation of complement the phenomenon of 
hemolysis is brought into use. 
Hemolysis.-Hemolysis consists of the breaking up of the red-blood 
cell with the consequent passing of hemoglobin into the outer medium 
(serum or physiologic salt solution), the originally opaque fluid 
becoming transparent. 
If an animal (A) be inoculated with the erythrocytes (red-blood cells) of an 
animal (B) of a different species, the blood-serum of A acquires the property of 
hemolyzing the red-blood cells of B. The immune serum of A, through heating 
at 55° to 56° C. for 30 minutes or through standing for over 24 hours, loses its 
hemolyzing power, but the latter may be restored by the addition of fresh (not 
over 24 hours old) serum. 
The hemolyzing power of an immune serum depends on the pres- 
ence in the serum of two components, one of which is thermostabile 
and specific, the other thermolabile and non-specific. The thermostabile 
component is, according to Ehrlich's side-chain theory, an antibody 
of the third order, the amboceptor type, and is known as hemolysin, 
hemolytic immune body, hemolytic amboceptor, substance sensibilatrice 
(Bordet), or sensitizer. Erythrocytes to which sufficient hemolysin 
has been added to prepare them for complete dissolution by comple- 
ment are called sensitized erythrocytes. The thermolabile activating 
complement of serum is a normal constituent of all fresh serum and 
is known as complement, alexin (Buchner and Bordet), or cytase 
(Metchnikoff). The hemolytic system consists of three substances: 
erythrocyte suspension, homologous immune serum, and complement; 
and the presence of all three in definite proportions is necessary for 
hemolysis. 
1 Anuales de 1'Institut Pasteur, 1901, xv, 290. 
12 178 
PRINCIPLES OF MICROBIOLOGY 
Complement Fixation Defined.-If an animal be immunized, naturally 
or artificially, against a species (C) of bacterial or other protein the 
serum (D) of that animal acquires, through the development of certain 
antibodies of amboceptor type, the property of fixing or absorbing 
complement when mixed in proper proportion with the antigen (C). 
As this phenomenon, termed complement fixation, complement binding, 
or complement deviation, is invisible, sensitized erythrocytes are used 
as an indicator of the reaction. If complement is bound by the inter- 
action of antigen1 (C) and amboceptor (D) it cannot join sensitized 
erythrocytes to form the hemolytic system; hence the latter is incomplete 
and hemolysis does not occur (Fig. 69). If antigen (C) is mixed with 
Fig. 69 
DIAGRAM OF POSITIVE COMPLEMENT FIXATION TEST 
BACTERIAL ANTIGEN 
PLUS 
HOMOLOGOUS IMMUNE 
SERUM 
ERYTHROCYTES 
PLUS 
HEMOLYSIN 
COMPLEMENT 
BACTERIAL ANTIGEN 
PLUS 
ERYTHROCYTES 
PLUS 
HEMOLYSIN 
HETEROLOGOUS IMMUNE 
SERUM 
DIAGRAM OF NEGATIVE COMPLEMENT FIXATION TEST 
an immune serum (E) that is heterologous instead of homologous, i. e., 
the serum from an animal immunized with an antigen unrelated to C, 
then complement instead of being bound is still free and sensitized 
erythrocytes are hemolyzed (Fig. 69). The complement-fixation test 
has been developed from the following classical experiment of Bordet 
and Gengou (1901): 
Typhoid bacteria (antigen), inactivated typhoid immune serum (amboceptor), 
and normal serum (complement) were mixed in a test-tube; after an interval 
washed erythrocytes and inactivated homologous immune serum (hemolysin) 
1 In complement-fixation the term antigen is confined to substances capable of pro- 
ducing, after one or more administration to a suitable animal, antibodies of the ambo- 
ceptor type. TECHNIQUE 
179 
were added. No hemolysis occurred, indicating that the complement had been 
fixed by the interaction of antigen and amboceptor, whereby the hemolytic 
system was left incomplete. 
The Bordet-Gengou phenomenon may be applied to the identification 
of an unknown organism or other proteid material that is tested as 
antigen against known immune serum, or it may be applied to the 
identification of unknown amboceptor in an immune serum that is 
tested against known antigen, inhibition of hemolysis showing that 
the antigen and immune serum are homologous. Complement-fixation 
is used in the diagnosis of various infectious diseases, e. g., syphilis, 
gonococcus infections, and glanders, in the differentiation of proteids, 
in the standardization of some immune sera (for example, antigono- 
coccus and antistreptococcus horse sera), and in establishing the 
etiology of infectious diseases. 
Technique.-Preparation of Reagents.-The reaction of glassware 
and reagents has been found to have an important bearing on the 
accuracy of the test, acidity or alkalinity giving rise to false reactions, 
positive or negative according to the degree of acidity or alkalinity. 
All new glassware should be neutralized by standing overnight in 1 
per cent, hydrochloric acid. Then, like previously neutralized glass- 
ware, it is washed in tap-water, thoroughly rinsed in hot distilled water, 
and sterilized. The distilled water and salt solution used in the prepara- 
tion of reagents and in the performance of the test are tested for neu- 
trality to phenolphthalein. Physiologic salt solution, 0.85 per cent, to 
0.9 per cent., is used in blood-washing, in diluting other reagents, and 
in the performance of the test. 
Immune Serum.-The method of withdrawing immune serum from 
an animal depends on the animal and the purpose of bleeding. In 
the case of a horse, the bleeding is made from the jugular vein. Rabbits 
are bled from the marginal ear-vein if only a few cubic centimeters 
(less than 5 c.c.) of serum are required. If a large amount is required, 
the rabbit is etherized, tied on a board made for that purpose, and under 
aseptic precautions bled to death from the carotid artery. A cannula 
may be inserted in the artery and the blood allowed to flow into one 
or more tubes. To obtain the maximum amount of serum, the tube 
of blood should be slanted at room temperature until coagulation has 
taken place. In obtaining blood for complement-fixation tests on human 
beings venous puncture is the most satisfactory method. The drawing 
of sufficient blood from finger or ear-lobe is a tedious process and the 
blood-cells are apt to be somewhat broken, so that the serum is tinged 
with hemoglobin and rendered unfit for testing. 
Blood is most easily obtained from the median basilic vein of the elbow. 
The arm should be rendered aseptic by the usual method of scrubbing with 
soap and water and the application of a 1 to 2000 bichloride of mercury 
pack for ten minutes. The site of puncture should then be rubbed with 
alcohol and ether. A ligature is placed above the elbow sufficiently tight to 
fill the vein, but not tight enough to impede the arterial circulation. A sterile 180 
PRINCIPLES OF MICROBIOLOGY 
needle is then introduced and 5 to 10 c.c. of blood allowed to flow into a sterile 
test-tube, which is corked and left slanted at room temperature until the blood 
is firmly clotted. The tube should then be placed in the ice-box until serum has 
separated. If it is necessary to mail a blood specimen, serum only should be 
sent, as lysis of the blood-cells would be caused by the heat and shaking to which 
the specimen would be exposed in transit. The serum should be mailed in a 
sealed,ampoule or tightly corked tube. 
All immune serum is separated from the red-blood cells and fibrin of 
the blood by centrifugalization before coagulation has taken place, or, 
better, by pipetting or pouring from the clot after coagulation. Serum 
thus removed may be entirely freed from erythrocytes by centrifugaliza- 
tion. Serum should be removed from the clot before spontaneous lysis 
of the blood-cells occurs, as hemoglobin has the power of fixing com- 
plement in itself and a hemolyzed specimen of serum, one containing 
hemoglobin, is anticomplementary, i. e., it inhibits hemolysis without the 
presence of a specific antigen, hence cannot be tested for a specific ambo- 
ceptor. Serum, both before and after removal from the clot, should be 
kept in a cool place, at a temperature not higher than 0° C., as the 
antibody content weakens more rapidly at a high temperature than at a 
low. For practical purposes serum is best preserved by freezing.1 The addi- 
tion of a preservative is not advisable, as the accuracy of the test may 
thereby be invalidated. Contamination of an immune serum should be 
avoided, as it may result in an anticomplementary and non-specific 
action of the serum. All immune serum before use in tests should be 
inactivated, i. e., heated for one-half hour at 56° C. to destroy complement 
and serum components that might give rise to a non-specific fixation. 
Antigen.-The method of preparing antigen depends on the nature 
of the test to be made. Each antigen is described in detail below. A 
bacterial antigen may be prepared as in the original method of Bordet- 
Gengou, by suspending in physiologic salt solution a 24-hour agar 
growth of the bacteria, making a rather concentrated emulsion. 
Bacterial extracts give more specific results than emulsions. The best 
method of extraction depends on the organism. 
Complement.-Not all fresh serum has the power of reactivating the 
serum of an alien species, but that of the guinea-pig has unusual power 
in this respect; hence guinea-pig serum is generally used for complement 
in hemolytic work. There is a wide variation in the activating power 
of guinea-pig serum, and also in its power of combining with antigen 
and immune serum in the complement-fixation reaction. It is advisable 
to use the pooled serum of at least three pigs, in order to obtain comple- 
ment of average activating and combining properties. 
Guinea-pigs that have been used for antitoxin tests and other purposes may 
be of use in complement-fixation work after a rest of three months, but their 
serum is less apt than that of unused pigs to be of normal activating power. 
In the complement-fixation test for glanders pigs that have been inoculated 
1 Serum is perfectly preserved by evaporating to dryness in a vacuum desiccator, but 
the procedure is complicated. ANTIGEN 
181 
with horse serum must never be used, as substances are formed that cause a 
reaction with the horse serum that is being tested for glanders and the test is 
unsatisfactory; serum controls in themselves inhibit hemolysin and no reading 
can be made of a specific reaction. Gravid pigs should not be used for comple- 
ment, as their serum is apt to be weak in activating power. 
To obtain complement, guinea-pigs may be bled from the heart 
by aspiration and used again after several weeks' rest, or they may 
be bled to death. In this laboratory the pigs, after being stunned by a 
blow at the base of the skull, are bled from the throat1 into Petri dishes, 
which are left at room temperature until the serum begins to separate 
and then set in the ice-box for several hours. The serum is drawn off with 
a capillary pipette and centrifuged if not free from blood-cells. Before 
being pooled, the serum from each pig should be tested separately for 
natural hemolysin, which is occasionally present, and for activating 
power. Serum containing natural hemolysin or serum of weak 
activating power should be discarded. Complement deteriorates 
rapidly if exposed to sunlight or to a warm temperature (over 70° F.); 
if kept in the ice-box (at a temperature below 17° C.) it is good for at 
least twenty-four hours. Complement may be preserved for several 
weeks by freezing. Its activating power is retained, but its capacity 
for being bound may weaken, so that it is not considered reliable. In 
our experience complement frozen for one week is as good as perfectly 
fresh complement. 
Complement is used in a 10 per cent, dilution made with physiologic 
salt solution. 
Erythrocytes from sheep, goat, man, ox, or other animal may be used, 
and they must be washed free from serum, with physiologic salt 
solution. In our laboratory sheep's cells, washed six times, are used, 
in a 5 per cent, suspension. A sheep is bled from the jugular vein into 
a sterile bottle or flask containing glass beads, and before the blood 
coagulates it is thoroughly shaken for the purpose of defibrination. 
Blood is washed in the following manner: 
Iii washing a small amount of blood it is convenient to put 2 to 4 c.c. in a 15- 
c.c. graduated centrifuge tube. The tube is filled with physiologic salt solu- 
tion and a mixture of blood and saline is effected by means of a pipette. The 
volume of saline should be three times that of the blood, otherwise the washing 
is not thorough. The blood is rapidly centrifugalized long enough for the ery- 
throcytes to fall to the bottom of the tube. The supernatant fluid is decanted 
or drawn off with a pipette attached to a pressure pump or fitted with a rubber 
bulb. Physiologic salt solution is again mixed with the cells and the tube 
centrifugalized. This process is repeated until the blood-cells are entirely 
free from serum. In most laboratories three washings are considered sufficient, 
but we have found that after even four washings the serum is not always com- 
pletely removed. Hence we make it a rule to wash six times. After the last 
washing the level of the erythrocytes is marked on the tube, before the cells 
have been disturbed by the removal of the supernatant fluid. The amount 
1 Care must be taken not to cut the esophagus, as the stomach contents might render 
the serum unfit for use in complement-fixation work. 182 
PRINCIPLES OF MICROBIOLOGY 
of blood per centrifuge tube, the speed and duration of centrifugalization should 
always be the same, at least for the last washing, in order that the packing of 
the cells be uniform day by day. 
Blood that has been drawn for over two days in summer or three days 
in winter is unsuitable for hemolytic work, as the resistance of the 
erythrocytes weakens on standing and hemolysis occurs rapidly, so that 
perfect balance of the hemolytic system cannot be obtained. In places 
where it is inconvenient to obtain sheep's cells, human erythrocytes 
are frequently used. An advantage in the human system arises from 
the fact that by its use one error in the test is avoided. Human serum 
frequently contains natural antisheep amboceptor and the excess 
of hemolysin introduced into the hemolytic system when such a serum 
is being tested may, according to some investigators, cause a weakening 
or loss of a positive reaction. In our experience this error rarely or 
never occurs in the Wassermann reaction, owing to our practice of 
reading the reaction as soon as controls are completely hemolyzed; 
hence the use of a hemolytic system other than sheep or the absorption 
of the natural hemolysin from the serum is not necessary in Wassermann 
work. In tests with bacterial antigens where the fixation of com- 
plement is less firm than in the Wassermann reaction the native anti- 
sheep amboceptor in human serum may be a source of error. 
Hemolysin is obtained by successive inoculations of a rabbit (or 
other experimental animal) with the type of erythrocytes to be used 
in tests. Inoculations may be made intraperitoneally or intravenously. 
Owing to the frequency of abscesses after intraperitoneal inoculations 
the intravenous method is preferable. In this laboratory, rabbits are 
inoculated intravenously at intervals of 2 days with fresh, thoroughly 
washed sheep's cells in a 50 per cent, suspension in physiologic salt 
solution, the doses being 2 c.c., 4 c.c., and 6 c.c. On the ninth day after 
the last inoculation the rabbit is bled to death from the carotid. The 
serum is put up in small bottles or ampoules and heated for one-half 
hour at 56° C. on three successive days, to destroy the complement 
and to insure sterilization. 
Standardization of Reagents.-An accurate daily standardization of 
the hemolytic system is very important for reliable results, and this 
may be accomplished by means of a hemolysin or a complement titra- 
tion. In the former, varying amounts of hemolysin are incubated with 
a constant amount of complement and erythrocyte suspension. In 
a complement titration the amounts of complement are varied and 
hemolysin is constant. In most laboratories it is considered preferable 
to follow a hemolysin titration. In this laboratory preliminary titra- 
tions of new hemolytic immune serum are made in dilutions of 1 to 
100, 1 to 1000, etc., to determine the dilution of hemolysin in which 
0.05 c.c. is one unit. By a unit of hemolysin is meant the smallest 
amount that gives complete hemolysis of 0.1 c.c. of a 5 per cent, sheep's 
cell suspension in the presence of an excess of complement after one hours 
incubation in a 37° C. water bath. STANDARDIZATION OF REAGENTS 
183 
Once standardized the hemolysin is used in the same dilution every 
day. The technique of the titration is given below. The volume, like 
that of all our complement-fixation tests, is one-tenth the volume of 
the classical Wassermann-i.e., 0.5 c.c. instead of 5 c.c. After thorough 
shaking the titration is incubated one hour in the water-bath at 37° C. and 
examined frequently to determine the rapidity of the reaction, according 
to which from two to three units are used in the test. As hemolysin 
is stable and fresh, and erythrocytes from a healthy sheep vary little 
in resistance, such a titration indicates sufficiently for practical work 
the value of the complement. If the complement is normally active 
and free from hemolysin, the erythrocytes fresh and thoroughly washed, 
the saline solution isotonic and all reagents accurately diluted, hemolysis 
at the end of the hour's incubation is complete (Plate V, e) in the first 
six tubes, nearly complete (Plate V, d) in the seventh, partial (Plate V, c) 
in the eighth, slight in the ninth, and lacking (Plate V, a and 6) in the 
last four tubes. Then since 0.05 c.c. is the unit of hemolysis, 0.1 c.c. 
is, in general, used to sensitize 0.1 c.c. of erythrocytes. The interpreta- 
tion of a hemolysin titration can be learned by experience only. 
Hemolysin Titration 
Number of 
tube. 
Hemolysin in 
standard 
dilution. 
C.c. 
10 per cent, 
complement. 
C.c. 
5 per cent, 
erythrocyte 
suspension. 
C.c. 
0.85 per cent, 
saline. 
C.c. 
1 
0.1 
0.1 
0. 1 
0.0 
2 
0.09 
0.1 
0.1 
0.21 
3 
0.08 
0.1 
0.1 
0.22 
4 
< 0.07 
0.1 
0.1 
0.23 
5 
0.06 
0.1 
0.1 
0.24 
6 
0.05 
0.1 
0.1 
0.25 
7 
0.04 
0.1 
0.1 
0.26 
8 
0.03 
0.1 
0.1 
0.27 
9 
0.02 
0.1 
0.1 
0.28 
10 
0.01 
0.1 
0.1 
0.29 
11 
0.1 
0.0 
0.1 
0.3 
12 
0.0 
0.1 
0.1 
0.3 
13 
. 0.0 
0.0 
0.1 
0.4 
Incubated for one hour in the 
water bath at 37° C. 
Complement Titration 
Number of 
tube. 
10 per cent, 
complement. 
C.c. 
Hemolysin in 
standard 
dilution. 
C.c. 
5 per cent, 
erythrocyte 
suspension. 
C.c. 
0.85 per cent, 
saline. 
C.c. 
1 
0.1 
0.05 
0.1 
0.25 
2 
0.09 
0.05 
0.1 
0.26 
3 
0.08 
0.05 
0.1 
0.27 
4 
0.07 
0.05 
6.1 
0.28 
5 
0.06 
0.05 
0.1 
0.29 
6 
0.05 
0.05 
0.1 
0.3 
7 
0.04 
0.05 
0.1 
0.31 
8 
0.03 
0.05 
0.1 
0.32 
9 
0.02 
0.05 
0.1 
0.33 
10 
0.01 
0.05 
0.1 
0.34 
11 
0.1 
0.0 
0.1 
0.3 
12 
0.0 
0.05 
0.1 
0.35 
13 
0.0 
0.0 
0.1 
0.4 
Incubated for one hour in the 
water bath at 37° C. 184 
PRINCIPLES OF MICROBIOLOGY 
Number of 
tube. 
Immune serum. 
C.c. 
Antigen. 
C.c. 
10 per cent, 
complement. 
C.c. 
0.85 per cent, 
saline. 
C.c. 
Sensitized 
erythrocyte 
suspension. 
C.c. 
1 
0.01 
0.25' 
0.1 
0.0 
s 
0.2 
2 
0.01 
0.2 
0.1 
0.0 
0.2 
3 
. 0.01 
0.15 
0.1 
0.05 
o 
0.2 
4_ 
5 
0.01 
0.01 
0.1 
0.05 
0.1 
0.1 
0.1 
0.15 
co 
0.2 
0.2 
6 
0.01 
0.025 
0.1 
0.2 
0.2 
7 
0.0 
0.4 
0.1 
0.0 
o 
0.2 
8 
0.0 
0.3 
0.1 
0.0 
0.2 
9 
0.0 
0.2 
0.1 
0.05 
. 0.2 
10 
0.0 
0.1 
0.1 
0.1 
S £ 
0.2 
11 
0.0 
0.05 
0.1 
0.15 
g -a 
0.2 
12 
0.02 
0.0 
0.1 
0.2 
0.2 
Antigen Titration. 
Incubated one hour in the 
water bath at 37° C. 
If an exact determination of complement value is desired the hemoly- 
sin titration may be followed by a complement titration as given above. 
The unit of complement is the smallest amount that completely hemolyzes 
0.1 c.c. of 5 per cent, erythrocyte suspension sensitized by one unit of 
hemolysin. If the balance of the hemolytic system is to be obtained 
by varying complement instead of hemolysin, two units of hemolysin 
instead of one unit should be used in titrating complement. In our 
opinion more uniform and accurate results are secured by varying 
hemolysin than by varying complement. 
Antigen.-There are two requirements for a good antigen, a long 
range and specificity.1 The range of an antigen is determined by 
mixing varying amounts of the antigen with a constant amount of a 
previously tested homologous immune serum, a constant amount of 
complement and a constant amount of sensitized erythrocyte suspen- 
sion. The specificity of an antigen is determined by using heterologous 
instead of homologous immune serum in the titration. The technique 
employed in this laboratory in the titration of an antigen is given 
above. Readings are made after the erythrocytes have settled, and 
meantime the titration should be kept in the ice-box to prevent a con- 
tinuation of hemolysis. An immediate reading may be made by centrifu- 
galizing the tubes. If fixation is complete through 0.025 c.c. (tube 6) 
a 10 per cent, dilution of the antigen should be titrated in the same 
manner to determine the unit of antigen, which is the smallest amount 
that with two units of homologous immune serum (or 0.01 c.c. of a 
human serum which has given a + + + + reaction) gives complete fixation 
of complement. The anticomplementary dose is the samllest amount 
of antigen that is in itself inhibitory. The longer the range of the antigen 
the greater is the probability of success in diagnosis. An amount of 
antigen that fixes complement completely with a serum of high anti- 
body content may give incomplete or no fixation with a serum of low 
1 The range of an antigen is the difference between the anticomplementary dose (the 
smallest amount of antigen that is in itself inhibitory) and the minimum fixing dose, 
the antigen unit. PLATE V 
a. Complete absence of hemolysis before settling of cells. 
b. Complete absence of hemolysis after settling of cells. 
c. Partial hemolysis. 
d. Nearly complete hemolysis. 
e. Complete hemolysis. STANDARDIZATION OF REAGENTS 
185 
antibody content. Hence in making diagnostic tests, where the detec- 
tion of even a small amount of antibody is desired, it is advisable to 
use much more than one unit of antigen. As the maximum amount of 
antigen that may be used with safety is one-fourth the anticomplementary dose 
an antigen of long range is necessary. If in table inhibition is complete 
(Plate V, a and b) in the first five or six tubes, hemolysis is complete 
(Plate V, e) in tubes 8 to 12, and there is very slight inhibition (Plate 
V, d) in tube 7 only, then 0.1 c.c. is the amount of antigen to be used in 
diagnostic tests. Antigen should always be so diluted that 0.1 c.c. may be 
used. If one-fourth the anticomplementary dose gives complete fixation 
with a heterologous immune serum the antigen is non-specific and unsuit- 
able for tests. Occasionally an antigen is lytic for erythrocytes. In such 
a case, tubes containing the largest amount of antigen show more hemo- 
lysis than those containing less. The fixation curve instead of dropping 
(Fig. 70, a) first rises and then drops again (Fig. 70, bf If a lytic antigen 
is also anticomplementary and has a long fixation range it may be used, 
otherwise it should be discarded. 
Complete fixation 
Fig. 70 
- Incomplete fixation 
hemolysis 
Complete fixation 
Incomplete fixation 
- hemolysis 
hemolysis 
Test 
Antigen in ' 
Sensitized 
Number of 
tube. 
1 
Patient's 
serum. 
C.c. 
0.02 
standard 
dilution. 
C.c. 
0.1 
10 per cent, 
complement. 
C.c. 
0.1 
0.85 per cent, 
saline. 
C.c. 
0.1 
erythrocyte 
suspension. 
C.c. 
0,2 
2 
0.01 
0.1 
0.1 
0.1 
ri 
.o 
0.2 
3 
0.04 
0.0 
0.1 
0.2 
o 
0.2 
4 
5 
0.02 
0.0 
0.0 
0.2 
0.1 
0.1 
0.2 
0.0 
.2 
fl 
X 
cc 
0.2 
0.2 
rt 
fl 
o 
6 
0.0 
0.1 
0.1 
0.1 
Ah 
0.2 
fl 
>-< 
Incubated 
until hemo- 
lysis is com- 
plete in tubes 
3 to 6. 
A test for diagnosis is set up as given above. Fixation of complement 
may be allowed to take place for one-half hour in a water bath at 
37° C., for one hour in the incubator at 37° C., at room temperature for 186 
PRINCIPLES OF MICROBIOLOGY 
four hours or more1 or in the ice-box for from four to eighteen hours. 
Whichever method is employed an antigen titration fixed by the same 
method should be followed, as the antigenic range varies with the 
time and temperature allowed for fixation. Positive and negative control 
sera should always be included in the test. Citron's standard for the 
strength of a complement-fixation reaction is used by this laboratory 
in the reading of all tests (Plate V): 
Complete absence of hemolysis in tubes 1 and 2 + + + +, very 
strong positive. 
Complete absence of hemolysis in tube 1, faint hemolysis in tube 2 
+ + +, strong positive. 
Complete absence of hemolysis in tube 1, complete or nearly complete 
hemolysis in tube 2 ++, positive. 
Partial hemolysis in tube 1, complete or nearly complete hemolysis 
in tube 2 +, weak positive or doubtful. 
Nearly complete hemolysis in tube 1, complete hemolysis in tube 2 
doubtful. 
Complete hemolysis in bath tubes -, negative. 
The distinction between + + and + is very important, as the latter 
reaction, we have found, is frequently non-specific. We therefore make 
a positive diagnosis on only ++, + + +, or + + + + reactions.2 
An antibody content titration is for the purpose of measuring more 
accurately than in the diagnostic test just described the amboceptor con- 
tent of an immune serum. Our technique is given in table below. The 
serum is first titrated in a 10 per cent, dilution; if fixation is complete 
through 0.01 c.c. a similar titration of a 1 per cent, dilution is made. 
The amount of antigen used is double the antigen unit; for example, 
if 0.05 c.c. of a 10 per cent, dilution of antigen is the smallest amount 
giving complete fixation with a homologous immune serum 0.1 c.c. of 
that dilution is used in the antibody content titration. The reading is 
made as in an antigen titration, after the erythrocytes have settled. The 
antigen and serum controls (tubes 11 and 12) should, of course, be hemo- 
lyzed. An antibody unit is the smallest amount of serum that with two units 
of a homologous antigen gives complete fixation of complement. The number 
of antibody units per cubic centimeter may be calculated by dividing 
1 c.c. by the minimum fixing dose; that is, if 0.05 c.c. of a 1 in 10 dilu- 
tion is the antibody unit, the number of antibodies per cubic centi- 
meter equals = 200. An antibody content titration is used for the 
standardization of antimicrobic sera, such as the antigonococcus and 
antistreptococcus horse sera. The power of a serum to fix complement 
is not, however, always parallel with its protective or therapeutic 
power. Besredka found that antistreptococcus serum of high protec- 
tive power had little complement fixing power, and vice versa. 
1 In our experience, room temperature is not desirable for fixation, as complement may 
deteriorate and the hemolysis of control tubes cannot be relied upon even though the 
hemolytic system when standardized is active. 
2 A + Wassermann reaction following the treatment of a case that has given a ++, 
+ + +, or + + + + reaction is considered positive and indicates the need of further 
treatment. THE WASSERMANN REACTION 
187 
Antibody Content Titration 
Immune 
serum. 
Antigen in 
dilution 
Sensitized 
Number 
tube. 
1 
of Diluted 1 
to 10 (etc.). 
C.c. 
0.1 
determined 
by titration. 
C.c. 
0.1 
10 per cent, 
complement. 
C.c. 
0.1 
0.85 per cent, 
saline. 
C.c. 
0.0 
.s 
erythrocyte 
suspension. 
C.c. 
0.2 
2 
0.09 
0.1 
0.1 
0.01 
6 
o 
0.2 
3 
0.08 
0.1 
0.1 
0.02 
o 
0.2 
4 
0.07 
0. 1 
0. 1 
0.03 
co 
0.2 
5 
0.06 
0.1 
0.1 
0.04 
"5 
0.2 
6 
0.05 
0.1 
0. 1 
0.05 
0.2 
7 
0.04 
0.1 
0.1 
0.06 
1 
0.2 
8 
0.03 
0.1 
0.1 
0.07 
75 
0.2 
9 
0.02 
0.1 
0.1 
0.08 
O 
0.2 
10 
0.01 
0.1 
0.1 
0.09 
g3 
£ 
0.2 
11 
0.0 
0.2 
0.1 
0.0 
0.2 
12 
0.2 
0.0 
0.1 
0.0 
a 
»-< 
0.2 
Incubated one hour in the 
water bath at 37° C. 
The Wassermann Reaction.-Wassermann, Neisser, and Bruck were 
first to apply the Bordet-Gengou phenomenon to the diagnosis of 
syphilis. The antigen originally employed and still preferred by some 
workers consists of a saline extract of liver from a syphilitic fetus. 
The finely divided tissues are mixed in the proportion of one to four 
with normal salt solution, to which 0.5 per cent, carbolic has been added, 
agitated at room temperature for twenty-four hours, centrifuged, and 
the supernatant fluid drawn off into sterile vessels and kept in the ice- 
box until needed. Since the cultivation of the Treponema pallidum 
has been made possible, antigens have been made from the organisms 
in pure culture instead of from luetic tissue; but the results have, on 
the whole, been less satisfactory, i. e., fewer cases of syphilis give a 
positive Wassermann reaction with a Treponema pallidum antigen 
than with a luetic tissue antigen, or even with a lipoid antigen from 
normal tissues. The exact nature of the antigen that produces the 
antibodies (called by Citron Luesreagine} taking part in the Wasser- 
mann reaction is unknown. It appears to be neither the pure spiro- 
chetes, nor a pure lipoid substance. Although a pure lipoid cannot 
stimulate the production of antibodies when inoculated into an experi- 
mental animal, it reacts in vitro with the Luesreagine in the blood of 
the syphilitic. The Wassermann reaction is a lipotropic or lipoidophilic 
reaction and not due to the interaction of specific antigen and anti- 
body. An extract of heart, liver, or kidney in 96 per cent, alcohol may 
be used. Some serologists recommend the addition of cholesterin to 
a crude alcoholic extract; such an antigen gives a higher percentage of 
positive reactions than most other antigens, but its reliability has not 
yet been established. 
In our experience a cholesterinized antigen is of a special value in determining 
the effectiveness of antiluetic treatment, as old cases of syphilis that are or 
have recently been under treatment are more apt to react with this antigen 
than with a crude alcoholic antigen. In making a fresh diagnosis it is not wise 
to depend on a cholesterinized antigen alone, since cases of scarlet fever, leprosy, 
and other non-syphilitic conditions have been found to react strongly with this 188 
PRINCIPLES OF MICROBIOLOGY 
antigen. These false positive reactions do not occur with a crude alcoholic 
antigen. A positive reaction is, however, presumptive of syphilis and a negative 
reaction has more value in excluding a diagnosis of syphilis than Jias a negative 
reaction witli the crude alcoholic antigen. 
A safe and stable antigen is Noguchi's acetone insoluble fraction of 
beef heart, liver, or kidney, prepared by extracting macerated tissue 
with ten times the amount of absolute aclohol at room temperature 
for several days, filtering, evaporating the filtrate to dryness, taking 
up the residue with ether, treating the ethereal solution with five times 
its volume of acetone, and making a saturated solution of the precipi- 
tate in absolute methyl alcohol. 
In our laboratory the most satisfactory results have been obtained with a crude 
alcoholic extract of guinea-pig hearts prepared as follows: hearts (from pigs 
bled to death for complement) are minced and washed in tap-water until free 
from blood and macerated in C. P. 96 per cent, alcohol in the proportion of 
one gram of heart to 5 c.c. of alcohol. Extraction is allowed to take place in the 
ice-box for at least three months and then in the incubator for a week. The 
extract is filtered through paper and kept in the ice-box, where it remains stable 
for at least a year. The titer of different antigens varies from one in twenty to 
one in a hundred. In diluting the antigen the first 10 c.c. of saline are added 
drop by drop and the remainder slowly, the mixture being effected by gently 
rotating the receptacle, not by shaking. 
The table on page 189 gives the classieal Wassermann technique. The 
readings are made according to Citron's standard. Our technique differs 
in three respects from Wassermann's: in the size of the test, in the 
amount of antigen used, and in the method of fixation. In our experience 
the results obtained through using all the reagents in one-tenth the 
amounts used by Wassermann are absolutely reliable, provided the 
test is accurately performed, and the saving in material is considerable. 
Another advantage in the small size is that a very small amount (about 
0.2 c.c.) of the patient's serum is sufficient for a test. Concerning the 
amount of antigen to be used in the test, we have found the use of a 
constant amount to give the same results as the use of a varying 
amount. For fixation of complement a four-hour period in the ice- 
box is allowed instead of incubation, as it has been found that about 
10 per cent, more positive reactions in cases of syphilis are obtained 
by this method, and without any apparent danger of obtaining false 
positive reactions. Though biologically non-specific the Wassermann 
reaction is clinically specific except, perhaps, in cases of leprosy, yaws, 
sleeping sickness, and scarlet fever.1 With these exceptions a positive 
reaction indicates the presence of luetic infection, either active or 
latent. A positive reaction may be given in any stage of syphilis but 
is most apt to occur in the secondary stage. A negative reaction at 
any stage of the disease does not exclude the possibility of syphilis. 
Antiluetic treatment, especially mercury, frequently results in the 
reaction becoming negative, though the disease may still be active. 
1 With the use of the crude alcoholic antigens, in one-quarter the anticomplementary 
dose, we have never obtained false reactions in such cases even by ice-box fixation. TEST FOR GONOCOCCUS INFECT JON 
189 
Classical Wassermann Test 
Luetic 
liver 
10 per 
0.85 per 
Sensitized 
Number 
extract 
Patient's 
cent, com- 
cent. 
erythrocyte 
of tube. 
diluted. 
serum. 
plement. 
saline. 
suspension. 
C.c. 
C.c. 
C.c. 
C.c. 
C.c. 
1 
1.0 
0.2 
1.0 
0.8 
J. » 
2.0 
2 
0.5 
0.1 
1.0 
1.4 
a! C .' 
.O O ~ 
2.0 
3 
2.0 
0.2 
1.0 
1.8 
2 7} O 
2.0 
4 
2.0 
0.0 
1.0 
0.0 
c o -0 
1-4 
2.0 
Incubat- 
ed until 
control 
tubes 
are com- 
pletely 
hemo- 
lyzed. 
If the Wassermann reaction is persistently negative for two years 
after cessation of treatment, tests being made at intervals of three 
months, it is fairly certain that a cure has been effected. The inges- 
tion of alcohol or the administration of an anesthetic within twenty- 
four hours of collection of the blood-specimen interferes with the 
accuracy of the test, alcohol weakening the reaction and an anesthetic 
giving rise to false positive reactions. 
The Complement-fixation Test for Gonococcus Infection.-This 
method was first applied to the study of gonococcus infection by Muller 
and Oppenheim (1906). The technique developed by Schwartz and 
McNeil has been widely followed and the value of the test in the 
diagnosis of secondary gonococcus infections, especially in conditions 
in which a bacteriologic diagnosis is difficult, as in arthritis, has been 
thoroughly established. A polyvalent antigen, one made from as many 
different strains of the gonococcus as possible, is essential.1 A twenty- 
four-hour growth on salt-free veal2 agar neutral to phenolphthalein 
is washed off with sterile neutral distilled water, autolyzed in a 56° C. 
water bath for one hour, and in an 80° C. water bath for one hour (to 
destroy a. ferment that would render the antigen unstable), filtered 
through paper pulp and a Berkefeld filter,3 and heated on three successive 
days at 56° C. for one-half hour for sterilization. This antigen is stable 
for at least six months and is highly specific.4 . 
Immediately before use the antigen must be made isotonic by the 
addition of one part of 9 per cent, saline solution to nine parts of the 
antigen. The test is performed in the usual way. The optimum period 
for fixation has been found to be six hours in the ice-box. Readings 
must be made with great care. A positive diagnosis should not be 
made unless 0.02 c.c. of serum gives complete inhibition of hemolysis, 
1 In this laboratory ten strains of gonococci isolated by Torrey are used in the prepa- 
ration of the antigen. 
2 Medium from bob-veal is most desirable. 
3 New Berkefeld filters are very alkaline, and before use for the filtration of bacterial 
antigens they should be boiled in distilled water at least three times for five minutes each 
time, and scrubbed thoroughly with a small brush in fresh water, after each boiling. 
After the filter is set up, hot, neutral, distilled water should stand in it for about five 
minuses. Then hot, neutral distilled water should run through under gentle pressure 
until the fluid is clear and neutral to phenolphthalein, when the filter is ready for use. 
Under high pressure a-filter still alkaline might test neutral. After use, the filter should 
be boiled in distilled water, scrubbed, and dried in the air. A filter used for gonococcus 
antigen should never be used for any other bacterial antigen, unless it is first boiled in 
1 per cent, sodium hydroxide solution and reneutralized. 
4 The specificity of a gonococcus antigen may be best determined by an antigen titra- 
tion with an antimeningococcus serum of high antibody content. 190 
PRINCIPLES OF MICROBIOLOGY 
as serum containing heterologous amboceptors, for example, strepto- 
coccus, may give a + reaction with gonococcus antigen. Only gonococcus 
amboceptors give a + + , + + +, or + + + + reaction. Cases of 
anterior gonorrheal urethritis and acute vulvovaginitis rarely give a 
positive complement-fixation test. A positive reaction is indicative 
of the presence or recent activity in the body of a focus of living 
gonococci. A positive reaction may persist for from six to eight weeks 
after a cure has been effected. Persistently negative results obtained 
through a considerable period of time indicate the probability of a 
cure. 
The Complement-fixation Test for Glanders.-Complement fixation 
has proved to be a valuable aid in the diagnosis of glanders. It is 
generally considered specific and more reliable than the agglutination 
test. In this laboratory the antigen is prepared from a twenty-four- 
hour growth of B. mallei on salt-free veal-agar 1.6 per cent. acid. The 
growth is washed off with sterile distilled water, autolyzed for six to 
eight hours at 80° C., filtered through paper pulp and a Berkefeld, and 
the filtrate sterilized at 56° C. on three successive days for a half-hour. 
This antigen, like the gonococcus and all other aqueous extracts, must 
be made isotonic before use. The test is performed like the others, 
but the optimum period for fixation has been found to be 6 to 18 
hours in the ice-box. The New York Health Department condemns 
all horses that give a + + + + complement-fixation reaction when it 
is confirmed by the eye mallein test, though + + and + + + are con- 
sidered certain indications of glanders infection.1 
Streptococcus Infections.-The value of complement fixation in the 
diagnosis of streptococcus infections is still uncertain. In this labora- 
tory fairly satisfactory results are obtained with a saline antigen pre- 
pared as follows: A twenty-four-hour growth on salt veal-agar neutral 
to phenolphthalein is washed off with 0.85 per cent, saline solution. 
The emulsion is heated at 60° C. one hour, left in the ice-box twenty-four 
hours, and centrifugalized. The supernatant fluid is used for tests, 
either water bath or incubator being used for fixation. The test seems 
to be specific, but is far from perfected. 
Pertussis.-Complement fixation has been used for the determina- 
tion of the etiological cause of whooping cough and for the diagnosis 
of the disease. The results of many investigators, including ourselves 
confirm the findings of Bordet, that the Bordet-Gengou bacillus is the 
etiologic cause of whooping cough. As to the diagnostic value of the 
test, reports vary. The figures given by. the workers who use active 
serum are undoubtedly too high, as investigations in this laboratory 
have proved that active serum may give non-specific fixation with even 
a highly specific antigen. We have found the most satisfactory antigen, 
that is, the strongest and most specific, to be obtained by shaking and 
then autolyzing at 56° C. A twenty-four-hour culture on the Bordet- 
1 Macroscopic lesions are always shown by horses giving a + + + + complement- 
fixation test, rarely by those giving a + + or a + + + reaction. COMPLEMENT-FIXATION TEST FOR TUBERCULOSIS 
191 
Gengou potato-blood-agar medium is scraped off with a platinum 
spud and put in sterile distilled water. The emulsion is shaken for 
about two hours, left in a thermostat at 56° C. for about eighteen hours, 
and centrifugalized. The supernatant fluid is used for tests, being 
made isotonic immediately before using. Water bath or incubator is 
used for fixation; room temperature, at which some workers allow 
fixation to take place, gives such variable results as to be unsuitable 
for routine work. We obtain a positive reaction in about 50 per 
cent, of whooping-cough cases in the paroxysmal stage. The ad- 
ministration of whooping-cough vaccine may increase the strength 
of the complement-fixation reaction, but experiments with normal 
individuals have proved that vaccine in itself, in the absence of whoop- 
ing cough, does not bring about a positive reaction. Immune serum 
of high antibody content may be produced by the intraperitoneal 
inoculation of rabbits once a week with a live culture of the Bordet- 
Gengou bacillus, beginning with a dose of one-eighth of a slant and 
increasing to three slants, the dosage depending on the condition of 
the rabbit. The height of immunity is usually reached through five 
or six inoculations; if other inoculations are given, the antibody content 
decreases. The rabbits are bled nine days after the last inoculation. 
Complement-fixation Test for Tuberculosis.-The complement- 
fixation test for tuberculosis is of uncertain value. Koch's old and 
new tuberculins have been used as antigens, but may give non-specific 
fixation. Besredka's antigen, a thirty-day culture from egg-bouillon 
medium sterilized at 115° C., is said to give a high percentage (over 90 
per cent.) of positive reactions in cases of active tuberculosis and to 
be of value in the diagnosis of the disease. It has been reported that 
syphilitics may give a positive tuberculosis reaction and that the test 
is therefore not specific. As an individual may suffer from both diseases 
at the same time the occurrence of both reactions is not a proof of non- 
specificity. The subsequent development of tuberculosis in cases that 
at the time of the complement-fixation test showed no clinical symp- 
toms suggests that the statistics of non-specificity that have been 
published may be inaccurate. Bronfenbrenner's work1 has been par- 
ticularly careful and thorough and maintains the specificity of the 
tuberculosis reaction with the use of. the Besredka antigen. Our own 
findings confirm his insofar as specificity is concerned. Our percentage 
of positive reactions among cases of active tuberculosis is much lower 
than Bronfenbrenner's. 
Meningitis.-The complement-fixation method has been successfully 
applied in epidemic meningitis by Briick, but the diagnosis can more 
readily be made by the bacteriologic examination of cerebrospinal 
fluid. Complement fixation is a valuable means of differentiating strains 
of meningococci. The antigen used here for this purpose is prepared 
in the same manner as gonococcus antigen. Water-bath fixation is 
used for all titrations. 
1 Personal communication of preliminary work. 192 
PRINCIPLES OF MICROBIOLOGY 
Typhoid.-Complement fixation is a valuable method of corroborating 
the Widal. Its exact clinical value and specificity have not yet been 
established. The use of a highly polyvalent antigen is essential. Gar- 
bat's method of preparing the antigen is to wash off with sterile distilled 
water a twenty-four-hour growth on agar. Heat the emulsion at 60° 
to 70° C. for twenty-four hours, shake with glass beads for twenty- 
four hours, and centrifugalize until the supernatant fluid is absolutely 
clear. A positive reaction usually appears only after bacteria have 
disappeared from the circulation; it becomes stronger during con- 
valescence and persists for several months afterward. CHAPTER XI. 
THE NATURE OF THE SUBSTANCES CONCERNED 
IN AGGLUTINATION. 
By the phenomenon of agglutination is meant the aggregation into 
clumps of uniformly disposed microorganisms in a fluid because of the 
action of a hemologous immune serum. If the organisms are motile 
they become immobile. 
This phenomenon had probably been noted by several observers 
(Charrin and Roger in 1889) but it was first extensively studied by 
Gruber and Durham in 1896, who determined that the serum of those 
passing through many infections contained a specific substance (agglu- 
tinin) which caused the infecting organisms to clump. Several months 
later Widal reported that in typhoid fever the development of agglutinins 
could be used for diagnostic purposes. It was thus demonstrated 
by these studies and those of Griinbaum, Bordet and others that through 
agglutinins a new means was available for the identification of bacteria. 
As to the nature of these phenomena a 
number of theories have been advanced. 
As in the case of the immune body, there 
is positive proof that the agglutinin com- 
bines directly with agglutinable sub- 
stances in the bacterial body, the two 
bodies affecting a loose combination. But 
since a certain amount of salt is necessary 
(Bordet) for the reaction it must be 
classed as a physico-chemical reaction. 
Bordet believes that all the antibodies 
act very much alike in their method 
of union and that the effects produced 
vary with the antigen in question and 
the characteristics which, on account 
of its own nature, it can produce as 
soon as it unites with the appropriate 
antibody. He does not believe in diff- 
erent families of antibodies, but in an 
infinite variety of antigens. Ehrlich, on the other hand, considers that 
the agglutinin consists of a haptophore or combining atom group which 
is stabile and of a ferment group which is labile (receptors of the 
second order). The latter causes the phenomenon of agglutination. 
In some types of infection there is a great accumulation of agglu- 
tinins in the blood. Thus in typhoid patients and convalescents distinct 
13 
Fig. 71 
Receptors of the second order are 
pictured in c. Here e represents 
the haptophore group, and d the 
zymophore group of the receptor, 
f being the food molecule with 
which this receptor combines. 
Such receptors are possessed by 
agglutinins and precipitins. It 
is to be noted that the zymophore 
group is an integral part of the 
receptor. 194 
PRINCIPLES OF MICROBIOLOGY 
agglutination has been observed in dilutions of 1 to 5000, and this action 
persisted for months, though not, of course, in the same degree. Even 
normal blood-serum, when undiluted, often produces agglutination 
through group agglutinins. But the specific agglutinins, which are 
formed only in consequence of an infection, are characterized by this, 
that they produce agglutination even when the serum is highly diluted, 
and, furthermore, that after this dilution the action is generally specific 
-i. e., the high dilutions of cholera-immune serum agglutinate only 
cholera bacilli, of typhoid-immune serum only typhoid bacilli, etc. 
This specificity, however, for some bacteria such as the colon and 
dysentery group, is not always absolute, as the group agglutinins 
previously present may be in unusual amount. 
It was formerly assumed that agglutination was a prerequisite for 
bacteriolysis. This, however, is not so, for both in cholera and in 
typhoid immunity bacteriolytic substances have been observed with- 
out agglutinins, and agglutinating substances without bacteriolysins. 
Characteristics of Agglutinins.-The union of agglutinin with re- 
ceptors in bacteria is a physicochemical reaction, and is quantitative. 
By chemical means it is possible again to separate a portion of the 
agglutinin from bacteria saturated with it and use it to agglutinate 
bacteria anew. Before agglutination occurs sodium chloride or a 
similarly acting compound must be present. The amount of bacteria 
in the emulsion used to test the amount of agglutinin must, there- 
fore, be known. An emulsion one hundred times as dense as another 
would require one hundred times as much agglutinin to give an equally 
complete reaction. Agglutinin acts upon dead bacteria. 
Heated at 55° C. agglutinins are not destroyed although bactericidal 
action is annulled. 
Agglutinins changed by heat, acids, and other influences become 
"agglutinoids," which are comparable to toxoids, complementoids, etc., 
e. g., agglutinating sera heated to a certain temperature loses its power 
to agglutinate but act upon bacteria so that they are unable to be 
agglutinated by active serum 
Heat diminishes the agglutinability of bacteria when above 60° C. 
Dreyer found that if a twenty-four-hour bouillon culture of Bacillus 
coll required 1 part of agglutinin to agglutinate it, then if heated to 
60° C. it required 2.3 parts; if to 80° C., 18 parts; if to 100° C., 24.6 
parts. He found the surprising fact that long heating of the culture 
restored to some extent its ability to be agglutinated by smaller amounts 
of agglutinins. Heated thirteen hours to 100° C., the culture was 
agglutinated by four parts. Heating the serum above 60° C. injures 
the agglutinin slightly, above 70° C. greatly, and above 75° C. 
destroys it. 
Weak and strong acids agglutinate bacteria, while medium acidity 
does not. Alkalies inhibit agglutination. 
Agglutinin does not dialyze through animal membranes. In diluted 
solution agglutinin slowly deteriorated. Dried, it lasts longer. It is 
precipitated with the globulins by ammonium sulphate. When a GROUP AGGLUTINATION 
195 
solution containing agglutinin is passed through a stone filter the first 
few cubic centimetres contain no agglutinin. The next contain a 
moderate amount and the remainder the same as the solution. 
It is important to remember that in low dilutions of a serum agglu- 
tination may fail, while in higher dilutions agglutination may take place 
readily. 
The Development of Agglutinin.-Experimental or natural infection 
of animals and men is followed in seven to ten days by an appreci- 
able development of agglutinin. This development is much greater 
for some bacteria than for others. 
Group Agglutination.-Many varieties of organisms have, among the 
different protein substances composing their bodies, some that are 
common to other microbes which are more or less allied to them (Fig. 
72). If these substances are of the type that excite agglutinins, we 
have from an animal immunized by any one of them a serum acting on 
other organisms somewhat in proportion to the amount of agglutinin 
producing protoplasm which they have in common with the infecting 
organism. These agglutinins, acting on common substances in other 
microorganisms which are generally allied varieties are called, there- 
fore, group agglutinins. Thus, in a case, the infecting paratyphoid 
bacilli type B were agglutinated 1 to 5700; typhoid bacilli, however, 
only 1 to 120, while paratyphoid bacilli type A were agglutinated only 
1 to 10. In a case of typhoid fever an agglutination of paratyphoid 
type B occurred with a dilution 1 to 40, while typhoid bacilli were 
agglutinated with 1 to 300. 
Fig. 72 
Typhoid Bacillus 
Colon Bacillus 
Specific and common agglutinins producing protoplasm. 
Dysentery Bacillus 
The bacteria which are agglutinated by one and the same serum need 
not at all be related in their morphologic or other biologic charac- 
teristics, as at first assumed. Conversely, microorganisms which, 
because of the characteristics mentioned, are regarded as entirely 
identical are sometimes sharply differentiated by means of their ag- 
glutination. Tn other words, the "groups" arrived at by means of a 
common agglutination have no necessary relation to species as the 
term is usually employed, but only of chemical similarity. This is 
indicated by the diagrams in Fig. 72. The letters indicate chemical 
substances capable of stimulating the production of agglutinin and of 
combining with it when made. Thus both the typhoid and colon will 196 
PRINCIPLES OF MICROBIOLOGY 
stimulate and react to B agglutinins, but the typhoid bacilli will only 
react to the type A and the colon to the D type. Because of this lack 
of absolute specificity of the agglutinated reaction the diagnosis of the 
type of infection or the absolute identification of bacteria through the 
agglutination or bacteriolytic tests can only be determined in those 
cases where the group agglutinins are not abundantly present. This 
suffices for some infections such as those caused by the typhoid bacillus 
and the cholera spirillum, but not for others such as those due to the 
colon group of bacilli. 
The Relative Development of Specific and Group Agglutinins.- 
The study of a large number of series of agglutination tests obtained 
from young goats and rabbits injected chiefly with typoid, dysentery, 
paradysentery, paracolon, colon, and hog-cholera cultures has shown 
that there is considerable uniformity in the development of the specific 
and group agglutinins. The specific agglutinins develop a larger 
amount of their total in the early days, being in the second week usually 
from five to one hundred times as abundant as the-group agglutinins. 
Later the total amount of the group agglutinins tends to approach more 
nearly to that of the specific, and may reach as high as 20 to 50 per cent. 
In a number of tests carried out by us we found that many group 
agglutinins supplement specific ones in their action, causing by their 
addition an increased agglutinating strength. In our experience the 
variety of microorganism used for inoculation is, if equally sensitive, 
agglutinated in a higher dilution by the combined specific and group ag- 
glutinins produced through its stimulus than any microorganism affected 
merely by the group agglutinins. It is true that bacteria not injected 
were at times agglutinated in higher dilutions than the variety injected; 
this, if not due to greater sensitiveness, was caused by normal group 
agglutinins present in the animal before immunization. For this 
reason untreated horse serum is a very dangerous substance to use 
in differentiating the intestinal bacteria. The great height to which 
the group agglutinins may rise is seen in the following table: 
Agglutinin in the Serum of a Horse Injected with Paradysentery Bacillus, Culture 
Type Manila. 
Culture. 
After 18 injections. 
After 21 injections. 
1:3000 1:5000 1:10,000 
1:3000 
1:5000 
1:10,000 
Paradysentery type Manila . 
+ + - - 
+ + 
+ + 
+ + 
Colon B. X 
+ + ++ - 
+ + 
+ + 
+ + 
The great amount of agglutinins acting upon the colon bacillus X. 
is remarkable. A serum is here seen to be acting in dilutions as high 
as 1 to 10,000 upon a culture possessing different characteristics from 
the one used in the injections. 
Although a considerable proportion of the group agglutinins act- 
ing on colon bacillus X. was undoubtedly due to the stimulus of the 
injections of the paradysentery culture, still a portion of them was THE USE OF ABSORPTION METHODS 
197 
probably due to the agglutinins developed by the stimulus of the 
absorbed intestinal bacteria. In the table given below is seen the 
marked accumulation of agglutinins which may occur in a normal 
horse before injections are begun: 
Culture. 
A young horse before inoculation. 
1:100 
1:500 
1:1000 
1:5000 
Dysentery B., Japan 
+ 
- 
- 
- 
Paradysentery, Mt. Desert . 
+ 
- 
- 
- 
Paradysentery, Manila . 
+ + 
+ + 
+ + 
- 
Colon B. X 
4" 4" 
+ 
- 
- 
The Relative Accumulation of the Group and Specific Agglutinins for 
the Organism Injected and for Allied Varieties.-A test was carried 
out with different types of dysentery bacilli. For the Manila culture 
of Flexner, which is nearest to the colon in its characteristics, the specific 
agglutinins were in the serum of an animal which had received injec- 
tions of the Manila cultures at the end of the fourth month five times 
as abundant as the group agglutinin acting on the Mt. Desert culture 
of Park, which represents a type lying between the Flexner and Shiga 
cultures. For the dysentery bacillus (Shiga) the development of agglu- 
tinins was the least (Fig. 73). 
Another point of interest is that the proportional amounts of agglu- 
tinins from the different cultures varied at different times. If on tests 
made of a single bleeding we had attempted to draw conclusions as to 
the relative development of specific and group agglutinins between the 
cultures, we would have had an imperfect view. Many conflicting 
statements in literature are undoubtedly due to this lack of appreciation 
of the variability in the relative amount of these two types of agglutinins 
during a long process of immunization (Fig. 74). 
The Use of Absorption Methods for Differentiation between 
Specific and Group Agglutinins due to Mixed Infection and to a 
Single Infection.-It is now well established that if an infection is due 
to one microorganism there will be specific agglutinins for that organ- 
ism and group agglutinins for that and other more or less allied organ- 
isms. If infection is due to two or more varieties of bacteria, there 
will be specific agglutinins for each of the microorganisms and group 
agglutinins produced because of each of them. 
The following experiments will illustrate these points: 
A rabbit immunized to B. typhi agglutinated B. typhi 1 to 5000, B. coli (31) 
1 to 600. After saturation with B. typhi all agglutinins were removed for'both 
microorganisms. A rabbit immunized to both B. typhi and B. coli (31) agglu- 
tinated B. typhi 1 to 4000, B. coli (31) 1 to 1000. (After saturation with B. 
typhi the serum did not agglutinate B. typhi, but agglutinated B. coli (31) 1 to 
900.) After saturation with B. coli (31) it failed to agglutinate B. coli (31), but 
still agglutinated B. typhi 1 to 3500. Some other strains of B. coli still agglu- 
tinated in 1 to 20 or more because many strains included in this group act as 
differently toward each other in respect to agglutinins as they do to the typhoid 
baccili. 198 
.PRINCIPLES OF MICROBIOLOGY 
Fig. 73 
The rise and fall of common and specific agglutinins during seven months in a rabbit 
injected with the Manila culture. 
Colon bacillus X. 
- - Paradysentery type (Mt. Desert). 
Paradysentery type (Manila). 
Dysentery type (Japan). 
• Test dates for all four sera 
* Injections stopped. 
Fig. 74 
Similar conditions to those noted in previous chart, except that a young goat has been 
used for the injections of the colon bacillus X. The great accumulation of common agglu- 
tinins for the paradysentery bacillus in the third month of the injections of the bacillus 
X is very striking. 
• Tests made. 
The following tables give the outcome of several experiments: 
Absorption by the Typhoid Bacillus of Group Agglutinins Acting upon a Number 
of Varieties of B. coli which were Produced by Another Variety of B. coli. 
Agglutination by Serum of Rabbit Immunized to Colon Bacillus X. 
Before addition of 
typhoid bacilli. 
After attempt at absorption 
with typhoid bacilli at 22° C. 
Colon bacillus X 
6000 
5000 
Colon bacillus 1 .... 
500 
20 
Colon bacillus 2 .... 
500 
30 
Colon bacillus 3 .... 
250 
30 
Colon bacillus 4 .... 
250 
10 
Colon bacillus 5 .... 
10 
less than 10 
Colon bacillus 6-1S 
less than 10 
less than 10 
Typhoid bacillus .... 
less than 10 
less than 10 LOSS OF CAPACITY IN BACTERIA 
199 
The absorption tests were carried out by adding the bacilli from 
recent agar cultures to a 10 per cent, solution of the serum in a twenty- 
four-hour bouillon culture. The mixture was allowed to stand for 
twenty-four hours at about 22° C. It was found that the agglutinin 
in a simple dilution of serum when left at 37° C. rapidly deteriorated. 
Thus, in an extreme instance a serum positive at 1 to 1500, when diluted 
with bouillon or salt solution 1:25 and left at 37° C. for twenty-four 
hours, lost 30 to 40 per cent, of its strength; at 22° C. it lost at times 
15 to 20 per cent. Left for three hours, the loss only was 5 to 10 
per cent. 
Fig. 75 
Showing the effect of saturating with bacilli of types of Shiga, Manila, and Mt. Desert, a 
serum from a horse which had received combined injections of dysentery bacilli of the three 
types. Note that the Manila type removed almost all the specific and group agglutinins 
acting upon its own type and the group agglutinins acting upon the Coney Island and 
normal types, leaving the specific agglutinins for types Shiga and Mt. Desert. The same 
is true for types Shiga and Mt. Desert when they were used. 
- Manila paradysentery. 
Japan dysentery. 
Mt. Desert paradysentery. 
and-- Atypical paradysentery. 
The great number of varieties of the colon group of bacilli that are in the normal 
intestine and which are absorbed slightly in health and more markedly in intes- 
tinal diseases make the use of absorption test for diagnostic purposes too com- 
plicated except for peculiarly important cases. 
Loss of Capacity in Bacteria to be Agglutinated or to Absorb 
Agglutinins Because of Growth in Immune Sera-The loss of these 
characteristics by growth in sera has been demonstrated by Marshall 
and Knox. The experiments of Collins and ourselves are recorded 
because they were undertaken in a slightly different way and also 
because a certain number of confirmatory observations are of value. 
The maltose fermenting paradysentery bacillus of Flexner was grown on each 
of eleven consecutive days in fresh bouillon solutions of the serum from a horse 
immunized through oft-repeated injections of the bacillus. The solutions used 
were 1.5, 4, and 15 per cent. The serum agglutinated the culture before its 200 
PRINCIPLES OF MICROBIOLOGY 
treatment in dilutions up to 1 to 800, and was strongly bactericidal in animals. 
After the eleven transfers the culture grown in the 15 per cent, solution ceased to 
be agglutinated by the serum and ceased to absorb its specific agglutinins. 
The cultures grown in the 1.5 and 4 per cent, solutions agglutinated well in 
dilutions up to 1 to 60 and 1 to 100 and continued to absorb agglutinins. The 
recovery of the capacity to be agglutinated was very slow when the culture was 
transplanted from time to time on nutrient agar. After growth for sixteen 
weeks, during which it was transplanted forty-three times, it agglutinated in dilu- 
tions of 1 to 200. The culture grown in 4 per cent, agglutinated 1 to 500, and the 
one in 1.5 per cent. 1 to 800. 
This diminution and final cessation of development of agglutinable 
substance in bacteria grown in a serum rich in agglutinin and immune 
bodies is interesting both as showing the variation of the bacteria and 
as one means of adapting themselves to resist destruction, since the 
bacteria which ceased to produce agglutinable substance probably also 
produced less substance with affinity for other antibodies. This inhibi- 
tion of the production of agglutinable substance was also very note- 
worthy in the case of pneumococci grown in serum media. 
Non-agglutinable Strains.-Sometimes strains of microorganisms 
are obtained which possess all the other characteristics of a type and 
yet do not agglutinate. When grown on artificial media for some 
time they may gain the property. Organisms freshly obtained from 
the blood and tissues frequently agglutinate to a much less degree than 
after several transfers on media. 
Fig. 76 
1 and 21 3 and 4 5 and 6 7 and 8 8 and 9 9 and 10 11 and 12 13 and 14 15 and 16 
Relation of agglutinative to bactericidal power. Horse injected with culture of 
dysentery bacilli over a period of sixteen months. 
Agglutination index. 
Bactericidal index. 
• Test dates. 
Relation between Agglutinating and Bactericidal Power.-In spite 
of proof to the contrary good observers hold to the belief that there 
is some relation between the agglutinating and the bactericidal strength 
of a serum. The tests we carried out on the serum of a number of 
horses showed no such relation. In Fig. 76 are recorded a number 
1 Months. MACROSCOPIC METHOD OF ESTIMATING AGGLUTININS 
201 
of comparative tests during a period of sixteen months. While such 
an experiment shows no definite relation between agglutinin and 
immune bodies it must be remembered that these antibodies are the 
products of processes which are governed by similar laws, and so they 
have many points in common. 
Variation in the Agglutinating Strength of a Serum.-There is 
usually a continued increase in the amount of agglutinin in the blood 
of an infected person from its first appearance at the end of from 
four to twenty-one days until convalescence and then a decrease. At 
times, however, there is a marked variation from day to day, so that 
it may be abundantly present one day and very slight in amount the 
next. 
Mode of Obtaining Serum from Blood or Blisters for Examination. 
-Fluid blood-serum can easily be obtained in two ways: First, the 
serum may be obtained directly from the blood, thus: The tip of the 
finger or ear is pricked with a lancet-shaped needle, and the blood 
as it issues is allowed to fill by gravity a capillary tube having a central 
bulb. The ends of the tube are than sealed by heat or with melted 
wax, or candle-grease, and as the blood clots a few drops of serum 
separate. To obtain larger amounts of serum for a microscopic 
examination the blood is milked out from the puncture into a small 
homeopathic vial or test-tube. One cubic centimetre of blood can 
easily be collected in this way. The vial is then corked and placed 
on the ice to allow the serum to separate. As a rule, one or two drops 
of serum are obtainable at the end of three or four hours. Second, 
the serum may be obtained from blisters. This gives more serum, 
but causes more or less delay. The method is as follows: A section 
of cantharides plaster, the size of a 5-cent piece, is appplied to the skin 
at some spot on the chest or abdomen. A blister forms in from six 
to eighteen hours. This should be protected from injury by a vaccine 
shield or bunion plaster. The serum from the blister is collected in 
a capillary tube, the ends of which are then sealed. Several drops of 
the serum can easily be obtained from a blister so small that it is prac- 
tically painless and harmless. The serum obtained is clear and 
admirably suited for the test. A piece of blotting paper soaked in 
strong ammonia when placed on the skin and covered by a watch- 
glass or strips of adhesive plaster will quickly raise a blister. A little 
vaselin should be smeared on the skin surrounding the blotting paper. 
The whole blood may be used. It is dropped on glass or stiff paper, 
dried, and when it reaches the laboratory it is brought into solution. 
The Reaction. -The reaction can be observed either macroscopically 
by sedimentation of the agglutinated clumps in a test-tube or micro- 
scopically in a hanging drop. 
The Macroscopic Method of Estimating the Amount of Agglutinins. 
-The tests are carried out with sets of test-tubes in racks as in the 
complement-fixation tests (see Chapter X). Some prefer tapering tubes 
so that the sediment can be more easily estimated. As in complement- 
fixation tests, great care must be taken to have the test-tubes clean and 202 
PRINCIPLES OF MICROBIOLOGY 
free from chemicals. The bacterial suspension used, as in the micro- 
scopic test, must be standardized. The addition of the bacilli on a 
24 hours' slanted agar tube to 150 c.c. of normal saline solution gives 
a good suspension. There must be a uniform cloudiness, not too heavy, 
with no signs of antiagglutination. 
A series of dilutions are made iir which the suspensions of bacteria 
are of similar strength. 
Salt 
Suspension of 
Final 
Tube. 
Serum. 
solution. 
bacteria. 
solution. 
C.c. 
C.c. 
C.c. 
C.c. 
1 
0.4 
1.5 
0.1 
1 to 5 
2 
0.2 
1.7 
0.1 
1 to 10 
3 
0.1 
1.8 
0.1 
1 to 20 
4 
0.05 
1.85 
0.1 
1 to 40 
5 
0.0 
1.9 
0.1 
control 
After thorough mixing, the tubes are allowed to stand in the incu- 
bator for one hour. They are then observed and the amount of flocculent 
precipitate noted. They are then placed in the ice-box at 10° or under, 
examined in twelve to twenty-four hours, and readings again made. 
The tubes holding the greatest dilution of serum in which the fluid 
has cleared by the complete precipitation of all the bacteria shows 
the measure of the agglutinins. The control tube must be closely 
examined to note any sedimentation. 
Comparison of Microscopic and Macroscopic Methods.-The 
reaction is the same in both and one is as reliable as the other. The 
ice-box readings are apt to be higher than those obtained by micro- 
scopic examination. Dead cultures are more frequently used in the 
macroscopic method because the motility is of no importance. 
The growth of bacteria in fresh blood containing agglutinins inhibits 
the development of agglutinable substance in bacteria or causes 
them to produce substances which prevent the union of agglutinin 
with them. Bacteria should therefore not be grown on serum media 
when they are to be used in agglutination tests. Even the addition of 
ascitic fluid to broth has some effect. 
Microscopic Reaction.-If the reaction is observed through the micro- 
scope in a hanging drop, a formation of clumps is seen which, if it takes 
place rapidly, reveals the reaction almost completed at the first glance, 
that is, most of the bacilli are in loose clumps and nearly or altogether 
motionless (Figs. 77 and 80). Between the clumps are clear spaces con- 
taining few or no isolated bacilli. If the reaction is a little less complete 
a few bacilli may be found moving slowly between the clumps in an aim- 
less way, while others attached to the clumps by one end are apparently 
trying to pull away, much as a fly caught in fly-paper struggles for free- 
dom. If the agglutinating substances are present, but still less abundant, 
the reaction may be watched through the whole course of its develop- 
ment. Immediately after mixing the blood and the culture together 
it will be noticed that the bacilli move more slowly than before the 
addition of serum. Some of these soon cease all progressive movement, PSEUDOREACTIONS 
203 
and it will be seen that they are gathering together in small groups of 
two or more, the individual bacilli being still somewhat separated from 
each other. Gradually they close up the spaces between them, and 
clumps are formed. According to the completeness of the reaction, 
either all of the bacilli may finally become clumped and immobilized 
or only a small portion of them, the rest remaining freely motile, and 
those clumped may appear to be struggling for freedom. With blood 
containing a large amount of agglutinating substances all the grada- 
tions in the intensity of the reaction may be observed, from those shown 
in a marked and immediate reaction to those appearing in a late and 
indefinite one, by simply varying the proportion of blood added to the 
culture fluid. The reaction takes place more quickly at a higher 
temperature (36° C). 
Fig. 77 
Gruber-Widal reaction. Bacilli gathered into one large and two small clumps, the few 
isolated bacteria being motionless or almost so. 
Pseudoreactions.-If too concentrated a solution of dry blood from 
a healthy person is employed a picture is often obtained which may 
be mistaken for a reaction. Dissolved blood always shows a vary- 
ing amount of detritus, partly in the form of fibrinous clumps, and 
prolonged microscopic examination' of the mixture of dissolved 
blood with a culture fluid shows that the bacilli, inhibited by sub- 
stances in the blood, often become more or less entangled in these 
clumps, and in the course of one-half to one hour very few isolated 
motile bacteria are seen. The fibrinous clumps alone, especially if 
examined with a poor light by a beginner, may be easily mistaken 
for clumps of bacilli. Again, the bacilli may become fixed after 
remaining for one-half to two hours, by slight drying of the drop 
or the effect of substances on the cover-glass. The reaction in 
disease is chiefly due to specific substances, but clumping and inhi- 
bition of movement similar in character may be caused by group 
agglutinins such as exist in normal horse and other serums. This is 
a very important fact to keep in mind. 204 
PRINCIPLES OF MICROBIOLOGY 
In order to help the student thoroughly to understand what com- 
prises a reaction, Wilson prepared a set of drawings, which are here 
reproduced. The culture to be tested should be of about twenty 
Fig. 78 
Fig. 79 
Microscopic field, showing the top of a hanging 
drop in a normal typhoid culture. 
Microscopic field, showing a cross- 
section of the drop in Fig. 78. 
hours' growth, either in bouillon or on agar. If on the latter a sus- 
pension is made in broth or normal salt solution. A loopful of the 
fluid containing the bacteria is placed on the cover-glass, and to it an 
equal quantity of the desired serum dilution is added. 
Fig. 80 
Fig. 81 
Microscopic field, showing the top of a 
drop with the typhoid reaction. 
Microscopic field, showing a cross-section of 
the drop in Fig. 80. 
In making the hanging drop to be examined it is necessary to have 
it of such a depth that it will show at least three focal planes, other- 
wise the examination will be incomplete and unsatisfactory. The PSEUDOREACTIONS 
205 
moist chamber must be well sealed by vaselin so as to prevent drying, 
and kept at a temperature of at least 20° and not over 35° C. 
Fig. 78 shows a microscopic field of the top of a hanging drop of a 
normal bouillon culture of typhoid bacilli. The culture is twenty 
hours old and the organisms are freely motile. This represents the 
control drop used for camparison with the drop of the same culture to 
which has been added a little of the blood of a person suspected to have 
typhoid. Note these points in Fig. 78; the organisms are evenly 
distributed throughout the field, except at the edge of the drop, where 
they are gathered in great numbers; they show great activity here, 
seemingly trying to crowd to the very edge. This attraction is probably 
due to the action exerted on the organisms by the oxygen in the air, 
which naturally exerts positive chemotaxis on all aerobic organisms. 
Fig. 79 shows a cross-section of the drop represented in Fig. 78, and 
it will be noticed that the bacilli are evenly distributed throughout the 
drop, except at one place in the focal plane a, and again in the focal 
plane c. 
It sometimes happens that there is a substance adhering to a sup- 
posedly clean cover-glass which attracts the bacilli to that point, where 
they appear as fairly well defined clumps, more or less like the true 
clumps due to the agglutinating substance in typhoid blood. The 
increase in organisms at the bottom of the drop in the focal plane c 
is easily accounted for by the fact that gravity naturally carries the 
dead and non-motile organisms to the bottom, these frequently assum- 
ing the character of clumps. 
If a field can be found in any focal plane of the hanging drop free 
from clumps, one can be quite sure that any clumping present is not 
due to any agglutinating substance which necessarily will affect organ- 
isms in every focal plane. 
Fig. 80 shows the microscopic appearance of the top of a drop 
where the reaction is present. Notice first that the organisms have 
been drawn together in groups and that the individuals of each group 
appear to be loosely held to gether. Viewed under the microscope 
these clumps are practically quiescent, there being very little move- 
ment either of the individual organisms or of the clump as a whole. 
The edge of the drop is practically free from organisms, showing that 
the air no longer exerts any influence on them. 
Fig. 81 shows a cross-section of the hanging drop shown in Fig. 80. 
The clumps are evenly distributed throughout the drop, with perhaps 
some increase in the numbers and compactness of the clumps at the 
bottom. 
Fig. 82 shows the microscopic appearance of the top of a hanging 
drop of a bouillon culture to which has been added some blood of a 
patient suffering from a felx-ile condition not caused by typhoid 
infection, but which exerts a slight non-specific influence on the 
typhoid organisms. It will be seen that the reaction is incomplete 
and that there are many organisms at the edge of the drop. The air 
exerts the same influence on the bacilli that it did before the addition 206 
PRINCIPLES OF MICROBIOLOGY 
of the blood. Note the character of the clumps, generally small and 
compact at the centre, with the bacilli at the edge of the clump, usually 
attached by one end only. 
Very frequently these clumps have the appearance of being built up 
around a piece of detritus present in the clump. All the organisms 
comprising the clump seem to have retained part, at least, of their 
motility, those on the edges being particularly motile, so far as their 
free ends are concerned. 
When motility is very much inhibited these clumps have a peculiar 
trembling movement, which is like the molecular movement described 
by Brown. 
Fig. 83 shows a cross-section of the drop represented in Fig. 82. 
Note the same character of the clumps in every focal plane: the large 
number of motile bacilli and the number attracted at the edge of the 
drop by the air. 
Fig. 82 
Fig. 83 
Microscopic field, showing the top of a drop 
of culture with reaction not due to typhoid. 
Microscopic field, showing a cross- 
section of Fig. 82. 
Precipitin.-A substance similar to and possibly identical with 
agglutinin is precipitin. This substance was discovered by Kraus 
in 1897. He found that when a little immune serum was added to 
the bacteria-free filtrate of a culture of the organism used to produce 
the immunization there occurred a precipitate. This same reaction 
took place between the serum of an animal and various proteid sub- 
stances, such as white of egg, blood-serum, or milk, with which the 
animal had been injected. That part of the proteid-producing pre- 
cipitin is called precipitinogen (antigen). Precipitins in their develop- 
ment, their resistance to heat and chemicals, and in their specific and 
non-specific forms are similar to agglutinins. The specificity of pre- 
cipitins is, like that of the agglutinins, not absolute. Group precipitins 
act upon similar chemical substances derived from cells having very PRODUCTION OF PRECIPITATING SERA 
207 
different characteristics. The precipitin test is mostly employed in 
blood-identification and in testing sera and tissue extracts rather than 
bacterial filtrates. As the reaction depends on the formation of a 
precipitate it is important that the solution of serum and antigen be 
absolutely clear before being placed together. 
The test is carried out by placing a constant amount of the precipitinogen 
in a row of test-tubes and to each of these adding different amounts of immune 
serum (precipitin). A series of control tubes of each serum above should be 
observed. The tubes are placed in the incubator for twenty-four hours. To 
prevent contaminating growths 0.5 per cent, carbolic acid is first added to each 
tube. 
As the action of bacterial precipitins seems to be parallel with the 
action of the agglutinins, it is possible that where tube reactions 
are depended upon, some confusion may occur as to which substance 
is really affected by certain processes or agents, especially those having 
a solvent action upon the bodies of the bacteria. 
Production of Precipitating Sera.-Animals, usually rabbits, are in- 
jected with broth cultures or with emulsions of agar culture, or with a 
foreign proteid, just as in the production of agglutinins. CHAPTER XII. 
EXTRACT OF LEUKOCYTES. 
The original theory of Metchnikoff, that the leukocytes were the 
only actual protective bodies which warded off disease, and that they 
did this by attacking the bacteria, was founded on the fact that certain 
of the white cells possess the power of taking up into themselves patho- 
genic bacteria, which are there destroyed. It was later observed that 
these cells have the property of taking from, the blood many lifeless 
foreign elements. 
The question thereby arose as to whether these cells engulfed and 
then killed the bacteria, or whether perhaps other substances previously 
prepared them before the cells took them up. The latter supposition 
was found to be the fact. Neufeld called these substances bacterio- 
tropins and Wright opsonins. The leukocytes and the chemical 
substances of the blood thus both play an important part. The death 
of the bacteria also liberates positive chemotactic substances, and the 
disintegration of the white-blood cells gives rise to bactericidal bodies. 
We find that phagocytosis is most marked when the disease is on the 
decline or the infection mild, but is usually absent in rapidly increasing 
infection. This would seem to indicate that the course of the infection 
is often already determined before the leukocytes become massed at 
the point of its entrance. The first determining influence is given by 
the condition of the tissues and the amount of bactericidal substances 
contained in them, and then, later, in cases where the bacteria have 
been checked, comes the additional help of the leukocytes. If the 
tissues are wholly free of bactericidal and sensitizing substances, neither 
they nor the leukocytes, nor both combined, can prevent the bacterial 
increase. The simple engulfing by the cells of bacteria is not neces- 
sarily a destructive process. Metchnikoff believes that the polymor- 
phonuclear leukocytes are especially antibacterial in relation to acute 
infections. The large phagocytes are conceived to deal chiefly with the 
resorption of animal cells and with immunity to certain chronic diseases, 
such as tuberculosis. 
The present great interest in the subject of the opsonins is largely 
due to the investigations and influence of Wright.1 We should, how- 
ever, recognize the important earlier work of others. Denys and 
Leclef had previously shown that in the case of rabbits immunized 
against streptococci, the increased phagocytosis was due to an altera- 
OPSONINS. 
1 Greek "opsono"-I cater for. OPSONINS 
209 
tion in the serum and not to changes induced in the leukocytes. They 
demonstrated that the leukocytes of the immunized animal when 
placed in normal serum showed no greater phagocytic activity than 
normal leukocytes did, and that therefore the substances in the serum 
favoring phagocytosis united with the bacteria. Wright and Douglass 
showed definitely that phagocytosis of bacteria in normal serum depends 
upon a special substance in the serum which becomes fixed to the bac- 
teria and prepares them for phagocytes. They called this substance 
opsonin. Neufeld and Rimpau discovered the same point independ- 
ently. Wright dealt mostly with normal serum, -while Neufeld used 
serum from immunized animals. 
Wright originated the idea of estimating the changes in the opsonic 
power of the blood for the purpose of guiding the use of vaccines in the 
treatment of bacterial infections. Thus he states: 
Fig. 84 
Opsonic curve showing the slight immediate rise and the later negative and positive phases 
following inoculation. The changes here are more regular than generally occurs. 
"I have found that there exists in the serum of the successfully inoculated 
patient an increase of opsonin. This is a substance which lends itself to very 
accurate measurement by a modification of Leishman's method. By the aid 
of this method the patient's progress or regress can be very accurately followed." 
Where vaccines are injected, Wright states, there "supervenes a negative 
phase where there is a diminished content in protective substances. This is 
succeeded by a positive phase. This inflowing wave of protective substances 
rapidly flows out again, but leaves behind in the blood a more or less perma- 
nently increased content of protective substances. When a small dose of vaccine 
is given the negative phase may hardly appear, but the positive phase may 
be correspondingly diminished. Where an unduly large dose of vaccine is 
inoculated the negative phase is prolonged and much attenuated. The posi- 
tive phase may in such a case make default. 
" It will be obvious that, if we, in the case of a patient who is already the 
subject of a bacterial invasion, produce by the injection of an excessive dose 
of vaccine a prolonged and well-marked negative phase, we may, instead of 
benefiting the patient, bring about conditions which will enable the bacteria 
to run riot in his system. 
. 14 210 
PRINCIPLES OF MICROBIOLOGY 
"Now, consideration will show that we may obtain, according as we choose 
our time and our dose wisely or unwisely, either a cumulative effect in the 
Fig. 85 
Opsonic outfit. THE OPSONIC INDEX 
211 
direction of a positive phase or a cumulative effect in the direction of a nega- 
tive phase. We may, in other cases, by the agency of two or more successive 
inoculations, raise the patient by successive steps to a higher level of immu- 
nity, or, as the case may be, bring down by successive steps to a lower level. 
We can select the appropriate time and dose with certainty only by examining 
the blood and measuring its content in protective substances in each case before 
reinoculating." 
An immense amount of investigation has revealed the fact that the 
index cannot be obtained accurately enough in single tests to be a safe 
guide to be used in diagnosis or treatment unless the variation from the 
normal is exceptionally great and that the opsonic content is not a 
safe guide for the measure of the total antibodies in the blood. 
THE OPSONIC INDEX. 
Technique.-Wright's technique of measuring the opsonic power is 
a slight modification of the Leishman1 method and is as follows: An 
emulsion of fresh human leukocytes is made by dropping twenty drops 
of blood from a finger prick into 20 c.c. normal salt solution containing 
1 per cent, sodium citrate. The mixture is centrifuged, the super- 
natant clear fluid removed, and the upper layers of the sedimented 
blood cells transferred by means of a fine pipette to 10 c.c. normal 
salt solution. After centrifuging this second mixture the supernatant 
fluid is pipetted off and the remaining suspension used for the opsonic 
tests. Such a "leukocyte emulsion," of course, contains a mixture of 
leukocytes and of red-blood cells; the proportion of leukocytes, how- 
ever, is much greater than in the original blood. The bacterial emul- 
sion is prepared by gently rubbing a little of the culture to be tested 
in salt solution (0.85 to 1.2 per cent.). When thoroughly mixed the 
fluid is centrifuged for a few minutes so as to remove any clumps. The 
emulsion should be so thick that in a trial test the leukocytes take up 
about five apiece on the average. 
One volume of the leukocytes is mixed with one volume of the bac- 
terial suspension to be tested and with one volume of the serum. This 
is best accomplished by means of a pipette whose end has been drawn 
out into a capillary tube several inches- in length. With a mark made 
about three-quarters of an inch from the end it is easy to suck up one 
such volume of each of the fluids, allowing a small air-bubble to inter- 
vene between each volume. All three are now expelled on a slide and 
thoroughly mixed by drawing back and forth into the pipette. Then 
the mixture is sucked into the pipette, the end sealed, and the whole 
put into the incubator at 37° C. The identical test is made using a nor- 
mal serum in place of the serum to be tested. Both tubes are allowed 
to incubate fifteen minutes and then the end of the tube is broken off, a 
large drop mounted on a clean slide the surface of which was previously 
roughened by emery-paper, and a spread made with a second slide as in 
ordinary blood-work, only a little thicker and using no force whatever. 
1 Leishman, British Medical Journal, January, 1902. 212 
PRINCIPLES OF MICROBIOLOGY 
After drying in the air the smears are stained without previous fixation 
either with a 1 per cent, aqueous solution of methylene blue or some 
other suitable stain. The degree of phagocytosis is then determined in 
each by counting a consecutive series of fifty or one hundred leukocytes 
and finding the average number of bacteria ingested per leukocyte. 
This number for the serum to be tested is divided by the number ob- 
tained with the normal serum and the result regarded as the opsonic 
index of the serum in question. Thus, if the tubercle bacilli, sensitized 
by a patient's blood, are taken up by the leukocytes to the average 
number of three per leukocytes, and bacilli from the same emulsion 
sensitized by normal blood are taken up by leukocytes to the average 
of five, then the index will be three-fifths of one, or 0.6. In this case 
the index would indicate a deficiency in opsonins. The presence of 
a high opsonic index Wright regards as indicative of increased resist- 
ance. He further states that the fluctuation of the- opsonic index in 
normal healthy individuals is not more than from 0.8 to 1.2, and 
that an index below 0.8 is, therefore, almost diagnostic of the presence 
of an infection with the organism tested. 
A number of workers use the washed whole blood. This saves in 
original labor, but makes the search for a suitable number of leukocytes 
more difficult. 
Simon's Method.-Simon has suggested a modification of Wright's 
method. He estimates the percentage of phagocyting cells in the 
mixture containing the serum to be tested and compares this with 
the mixtures containing normal serum. He also suggests that dilu- 
tions of blood be tested. 
The Dilution or Extinction Method recommended by Dean and by 
Klein. The degree of dilution of the serum necessary for the extinc- 
tion of its opsonic index is determined; that is, the serum to be tested 
is diluted until a dilution is found which shows the same small amount 
of phagocytosis shown in preparations in which no serum is used, 
namely, an index below 0.5. Klein claims that results by this method 
are more accurate than by the method of Wright. The method is 
too tedious for practical use in routine work. 
Most workers are now agreed that the use of the opsonic index is 
limited to experimental investigations. 
The Accuracy with Which the Opsonic Power of the Blood 
Can Be Determined by Wright's Method.-An examination of 
any slide will show that the different leukocytes vary in their size 
and in their content of bacteria. This is due partly to variation in 
phagocytic activity, and partly to the interference of the red-blood 
cells, which are present in great numbers in the emulsion and separate 
the bacteria in different degrees from the white cells. These and 
other reasons bring it about that the different leukocytes vary greatly 
in the number of bacteria they take up and in their distribution on 
the slide. Partly to overcome this, large numbers of leukocytes are 
counted. Beyond one hundred, or at most one hundred and fifty, 
the increase of accuracy hardly compensates for the extra labor. THE OPSONIC INDEX 
213 
The following table shows the difference between counting larger or 
smaller numbers of cells in five opsonic tests as determined by counting 
different numbers of cells in one .specimen: 
Opsonic Index Estimations in Five Blood-specimens. 
Cells counted. 
Average number of bacteria in each leukocyte. 
50 ... . 
1.18 
1.88 
1.34 
1.42 
1.90 
100 .... 
1.22 
1.78 
1.24 
1.42 
1.59 
150 .... 
1.18 
1.62 
1.22 
1.44 
1.50 
200 .... 
1.18 
1.51 
1.22 
1.46 
1.37 
600 .... 
1.28 
1.62 
1.23 
1.36 
1.36 
1,200 .... 
1.34 
1.44 
1.25 
1.30 
1.42 
It is noticed that the variation between the average cell count 
obtained from fifty cells and larger numbers is much greater than 
between that obtained at from one hundred or one hundred and fifty. 
It is necessary to have the counts that are compared all counted by 
the same person, as each individual has a somewhat different method 
and will average higher or lower for all counts than any other person. 
When two specimens of blood are tested not only the inaccuracy of 
counting due to the different arrangement of the unequally filled cells 
on the slides to be counted is met, but the fact that in making the test 
the conditions are not similar, for in different mixtures slightly dif- 
ferent proportions of leukocytes, bacteria, and red cells will always be 
mixed together. If smears from a series of tubes of the same blood 
are compared with a series of smears from one of the tubes, the former 
will always show the greater variation. 
This variation is much greater than most examiners believe. North 
has collected a series of tests carried out in nearly all the important 
laboratories in the Eastern United States that are working upon 
opsonins. The results recorded prove absolutely that while an average 
counting error of only about 10 per cent, is present, there may be an 
exceptional error of at least 100 per cent., and one of at least 20 per 
cent, may be expected once in about every ten determinations. 
The following is a fair average of the correctness of routine tests 
by experienced workers. 
Absolute Count or Bacteria in One Hundred Leukocytes. 
Blood-specimen A. 
Blood-specimen B. 
Blood-specimen C. 
Tube 
1 . . 
. . 156 
Tube 1 ... 
142 
Tube 1 ... 
89 
Tube 
2 . . 
. . 168 
Tube 2 ... 
182 
Tube 2 ... 
102 
Tube 
3 . . 
. . 172 
Tube 3 ... 
188 
Tube 3 ... 
121 
This error, which occurs because of the technique, applies not only 
to the examination of the specimen of blood, but also to the measure 
we employ to estimate the amount of opsonins. As these are not 
stable, we cannot have a standardized solution, as we do with anti- 
toxins. We must, therefore, determine our measure afresh in each 
test, taking for this purpose a supposedly normal blood. Wright, 
from a great many tests, has determined that the opsonic power of 214 
PRINCIPLES OF MICROBIOLOGY 
the blood in non-infected persons for tubercle bacilli does not vary, 
as a rule, more than 10 per cent, above or below the average power 
of healthy blood. For staphylococci there is more variation. It is 
found also that many things besides infection decrease the amount of 
opsonins in the blood. Hemorrhage, fatigue, starvation, and other 
influences which lower the resistance of the body have this effect. 
Wright gets this measure as uniform as possible by determining 
the average opsonic strength of five supposedly healthy persons at 
the time of each test. If any one of these five is considerably below 
or above others it is omitted for that day. The measure so obtained 
will probably vary about 5 per cent, from day to day, though seldom 
getting far away from what we might call the absolute normal. The 
following results were obtained by us from examining at one test a 
number of supposedly normal persons against staphylococci. 
Opsonic Counts in Test of Twenty-one Normal Sera with Stock 
Staphylococcus Culture. 
1 
4.13 
8 . . . . 
3.82 
15 . . . 
. . 9.09 
2 
2.93 
9 . . . . 
3.95 
16 . . . 
. . 5.17 
3 
2.78 
10 ... . 
3.98 
17 . . . 
. . 4.04 
4 
4.37 
11 ... . 
. 4.27 
18 . . . 
. . 3.82 
5 
3.58 
12 ... . 
. 3.69 
.19 . . . 
. . 4.00 
6 
2.90 
13 ... . 
. 3.80 
20 . . . 
. . 3.79 
7 
3.56 
14 ... . 
. 3.59 
21 . . . 
. . 3.44 
■ The Influence upon the Opsonic Test of the Specific Differ- 
ences between Strains of a Single Species.-The general practice 
in laboratories is to use stock cultures of tubercle bacilli, staphylococci, 
and other bacteria for the opsonic tests. To obtain a culture from a 
case may be at first impossible and, if successful, causes a delay of at least 
one or two days. The culture when obtained may also, as is frequently 
the case with pneumococci and streptococci, fail to opsonize readily. 
These and other reasons tend to establish the use of laboratory 
stock cultures, and yet we must acknowledge that when we test the 
amount of opsonins by both the stock and fresh cultures a marked 
difference sometimes develops. 
The Leukocytes to be Employed.-To many it seems a matter 
of indifference whether one person's leukocytes or another's are used, 
but our experience agrees with that of others that the leukocytes from 
different persons not only vary in their activity, but also in their selective 
action, and that the index is not the same when obtained with one 
person's leukocytes as with another's. 
The Influence of the Strength of the Bacterial Emulsion. 
-The more abundant the bacteria the greater will be the number 
taken up by the leukocytes. It is very important therefore that the 
tests be made with the same strength of emulsion. 
The Opsonic Variation during Treatment by Inoculations.- 
Wright lays stress on the considerable uniformity of the degree and 
persistence of development of opsonins after inoculation. We have THE OPSONIC INDEX 
215 
found in a small percentage of cases typical increases and decreases, 
as seen in Fig. 84, but in the majority of those inoculated there has 
been great irregularity. Frequently the negative phase does not occur 
or at least it is not detected. The following chart for three staphylo- 
coccus cases illustrates this (Fig. 86): 
Fig. 86 
x = injection of vaccine. 3 types of lines = 3 cases 
Fig. 87 
Dotted and crossed lines, normal persons. Continuous lines, tuberculosis. 
The Variation in the Amount of Opsonins in Supposedly 
Healthy Persons.-It has already been noted that in getting our 
measure we test a number of persons and exclude the blood of those 
which varies greatly from the average. We are so in the habit of 216 
PRINCIPLES OP MICROBIOLOGY 
seeing the index of normal blood placed at unity because it is each 
day the measure of comparison that even investigators are apt to think 
of the indices of normal persons as being unchanged from day to day. 
This is not the fact. A glance at the next chart (Fig. 87), in which 
four cases of tuberculosis are charted together with two normal 
persons, shows that the variation is only slightly greater in infected 
than in normal cases. If one normal person is charted against another 
for several weeks, marked differences will usually appear. The 
indices of the twenty-one normal cases tested against staphylococci 
(page 214) illustrate this variation in the amount of opsonins in normal 
blood. 
The Opsonic Index Cannot be Known at the Time the Treat- 
ment is Given.-Most of those who have not carried out inoculations 
under the guide of the opsonic tests think that the vaccinator is guided 
at the moment of injection by his knowledge of the opsonic power of 
the blood at the time. A moment's thought reveals that this is an 
absolute impossibility. In fact, except under very unusual conditions, 
it is impossible to have the test of the opsonic power reported within 
twenty-four hours, and in the treatment of the poor in out-patient 
practice longer intervals usually elapse, so that the treatment is given 
on a test made either the'day before or, more often, on from three to 
seven days before. As can be seen by the three curves in Fig. 86, 
which are quite as uniform as the average, it is impossible to judge 
what the index is at any moment by looking at the indices of blood 
taken from one to seven days previously. 
Other methods have been devised to get more accurate information 
upon the opsonic contents of the blood. The dilution method and 
that combined with determining the percentage of phagocytes ab- 
sorbing bacteria are the most valuable1 (p. 212). For experimental 
work they have advantages, but for practical use in governing the dos- 
age of vaccines they have most of the drawbacks of Wright's method. 
The Diagnostic Value of Opsonins.--The presence of a great 
excess or deficiency of opsonins for a microorganism, or of marked 
variation in the index after massage or exercise, has been thought by 
some to indicate the type of infection. Extreme caution should be 
used in making such an application of the index determinations. 
The Nature of Opsonins.-Wright and Neufeld, in their orig- 
inal experiments, differed as to the effect of heat on opsonins. Further 
investigation has shown that opsonins in those not immunized are 
largely thermo-labile, while opsonins developed after immunization 
are resistant. Muir and Martin believe from their experiments that 
the thermo-labile opsonin of normal serum and the thermo-stable 
opsonin are two entirely distinct classes of substances. The thermo- 
stable substance is of the nature of a true antibody and possesses the 
comparatively specific qualities of antibodies in general. Powerful 
complement absorbers have no effect on the thermo-stable opsonin, 
1 Simon, Jour. Exp. Medicine, vol. ix, No. 5, 1907, p. 487. LEUKOCYTE EXTRACT IN INFECTIONS 
217 
but do remove almost completely the thermo-labile opsonin. Neufeld 
agrees with them. 
Emulsions of other than the organisms used in immunization do 
not absorb a large percentage of the immune opsonin, but do of the 
complement opsonin. 
We have carried out absorption experiments with staphylococci, 
colon, and tubercle bacilli. Our results were similar to those of Muir 
and Martin. 
Opsonin Deficient in Cerebrospinal Fluid and in Exudates. 
-Opie1 has shown that exudates produced by injecting microorgan- 
isms usually have little or no opsonin for the variety injected or for 
other varieties. Hektoen has showed that opsonins, like other anti- 
bodies, are almost absent in the spinal fluid. McKenzie and Martin2 
showed that in a case of cerebrospinal meningitis the spinal fluid 
showed no immune bodies, while the blood contained them in abun- 
dance. 
Comparison between Opsonins and Bactericidal Substance 
in the Serum.-We have made comparative tests between the op- 
sonic and bactericidal power of the cell-free serum in typhoid infection 
and found that they did not run parallel. The frequent rapid increase 
in opsonic power within twelve hours of an injection of bacteria is 
striking and very different from the development of bactericidal 
strength. 
LEUKOCYTE EXTRACT IN INFECTIONS. 
Hiss3 recommends the treatment of certain diseases with a leukocyte 
extract. He makes this extract as follows: 
The leukocytes are obtained by double pleural inoculations with 
aleuronat into the animal (rabbit, dog). The amount of leukocyte- 
filled fluid obtained after twenty-four hours from rabbits has usually 
been from 30 to 60 c.c. This is immediately centrifugalized, the serum 
poured off, and the extracting fluid (distilled water) added in amounts 
about equal to the fluid poured off. The cells are then thoroughly 
emulsified in the distilled water, allow.ed to stand for eight hours at 
37.5° C., and then at ice-box temperature until used. Varying amounts 
of the entire fluid (after shaking) are inoculated. Hiss's animal ex- 
periments were made on rabbits and guinea-pigs infected with staphyl- 
ococcus, streptococcus, pneumococcus, typhoid bacillus, or menin- 
gococcus. Hiss stated that animals suffering from severe septicemias 
and poisonings following intravenous injection of any one of the above 
organisms have shown the beneficial effect of treatments with extracts 
of leukocytes, and have, in many instances, survived infections fatal to 
the control animals in thirty-six hours, even when treatment has been 
1 Opie, Jour. Exp. Med., vol. ix, No. 5, p. 515. 
2 Jour, of Path, and Bact., vol. xii, p. 539. 
3 Jour. Med. Research, xiv, No. 3. 218 
PRINCIPLES OF MICROBIOLOGY 
delayed as late as twenty-four hours. Zinsser1 has carefully studied 
the nature of the substances extracted from leukocytes. He finds 
they contain no complement, are not destroyed by heating to 56° C., 
and are no more abundant in cells derived from immunized than from 
normal rabbits. 
The Hiss leukocytic extract has now been used by a number of ob- 
servers for some six years. Cases of pneumonia, erysipelas, septicemia, 
and some other infections have been treated. It is difficult to deter- 
mine just what the value of the treatment is. No harmful results have 
been noticed. In a certain number of cases the temperature and 
symptoms have bettered, in a way which seemed clearly to indicate 
that the extract had done good. In other cases no results whatever 
were apparent. In our personal experience we have not seen definite 
results. It is usually given subcutaneously in 10-c.c. doses every four 
to six hours. As high as 500 c.c. have been given in some cases. 
1 Jour. Med. Research, xxii, No. 3. CHAPTER XIII. 
ACTIVE IMMUNIZATION. VACCINES. 
The success of vaccine virus in immunization against smallpox led 
Pasteur to his successful attempts to immunize sheep against anthrax 
by the use of attenuated living cultures of the anthrax bacillus, and to 
inoculate human beings against rabies by the inoculation of attenuated 
rabies virus. Later experimenters learned, as already stated, that the 
parenteral injection of the whole bodies of microorganisms or of extracts 
of them produced more or less immunity. This could be determined 
both by the resistance of the animals to infection or by the estimation 
of the various antibodies in the serum. This success led to the use of 
injection of measured doses of living or attenuated organisms or their 
extracts into persons already diseased, with the hope that healthy 
tissues at a distance from the diseased area might make antibodies to 
add to the insufficient amount produced in the infected area. Experi- 
mental investigations showed that the cells constantly poisoned were 
not in as good condition as normal cells to make antibodies and that 
the bacterial substances entering the blood were neutralized by anti- 
bodies in the serum as soon as they had accumulated so that the 
antigen acted less strongly to produce additional antibodies. 
Koch attempted this in tuberculosis by means of graded doses of the 
various tuberculins and Wright and others in various infections. 
A general consideration of the subject only is given here as the use 
of vaccines which have been successful will be considered under the 
individual pathogenic microorganisms. 
There are two points to be kept in constant view, namely, that the 
giving of bacterial vaccines during infection is adding more or less 
poisonous proteins and that unless the body tissues respond to produce 
antibodies, harm rather than good is obtained. Experience has taught 
us that those who become infected are*less able to produce antibodies. 
In such diseases as pneumonia, typhoid fever, septicemia, and other 
general infections due to different microorganisms, the treatment by 
bacterial vaccines has not met with sufficient success to be recognized 
by most careful observers. Here, an overdosage of the foreign more or 
less toxic protein should be carefully avoided. The vaccine treatment 
of these diseases must still be considered in the experimental stage and 
the exaggerated claims of certain manufacturers as wholly unwarranted. 
Localized Infections.-The results here in a considerable variety of in- 
fections have been good. The clinical observations are also borne out 
by the increase of the various immune bodies in the cases treated. 
The Use of Single and Multiple Varieties of Bacteria.-The classification 
of bacteria upon any grounds other than their production of antibodies 220 
PRINCIPLES OF MICROBIOLOGY 
is useless in vaccine therapy. Two otherwise characteristic strains 
of colon bacilli may be as widely separated from the antibody standpoint 
as a colon and a typhoid bacillus. This adds very much to the difficulty 
of treating many infections and leads frequently to the necessity of 
using cultures made from the infected persons (autogenous cultures) 
to prepare the vaccines. 
In mixed infections the patient should theoretically be immunized 
against each of the varieties causing the different infections. The 
organism of the patient may not find the task of responding to a series 
of different vaccines (always supposing that each of these is adminis- 
tered in appropriate and properly interspaced doses) much more difficult 
than the task of responding to one variety of vaccine only. 
General Opinion of Results.--Although during the past few years 
many thousands of cases of infection have been treated by vaccines, there 
is at present considerable difference of opinion as to their value. The 
majority of observers agree that it is in subacute and chronic infections 
that vaccines give the best results. Thus a case of acute streptococcus 
septicemia, which after a week or ten days shows a tendency to abate 
with localization in a joint or in a valve of the heart, offers a much 
better chance for vaccine treatment than such a case during the early 
more acute stage. Pneumonia, which persists after partial recovery, a 
gonorrheal joint, a persistent pus sinus, a localized inflammation due 
to colon bacilli, are all considered suitable for treatment. The use of 
vaccines in cases of acute inflammation of the mucous membranes of 
the intestines, bladder, throat, etc., have in most hands given rather 
negative results. Most believe that the inflammation is lessened, but 
that, as a rule, the bacterial infection, though it becomes latent, still 
remains and that a relapse may occur later. 
The different bacteria seem to differ in the successes with which 
they are used as vaccines. Nearly all observers report the greatest 
success with staphylococcus infections. These not only when they are 
severe as an osteomyelitis or an extensive carbuncle, but also when 
they are mild, such as ordinary furuncles or acne, seem to be aided 
by the vaccine treatment. Gonorrheal joints respond well, while acute 
gonorrhea of the mucous membranes responds little if at all. 
The streptococcus infections seem on the whole to be more resistant, 
but considerable success has been met with in treating subacute and 
chronic joint affections following local points of infection, as in a tonsil 
or tooth. The pneumococcus also seems resistant, perhaps even more 
so than the streptococcus. Numerous cases of pneumonia have been 
treated with somewhat doubtful results. Some observers treat these 
pneumococcus and streptococcus inflammations with daily doses of 
10,000,000 instead of the large semiweekly inoculations. Various sinus 
infections of the head have been treated, but with doubtful results. 
Streptococcus and other inflammations of the gums and teeth have 
yielded apparently good results. The treatment of tuberculosis with 
the different tuberculins has given perhaps the most extended use of 
vaccines. The majority of observers are convinced that in cases of PREPARATION OF BACTERIAL VACCINES 
221 
tuberculosis, in which the symptoms have become quiescent, whether 
they are incipient or fairly advanced, tuberculin injections do good. 
Some believe in giving very minute doses, and only slightly increas- 
ing their size. Others believe that different cases will receive with 
benefit different amounts, and so try to detect which will stand larger 
amounts. Nearly all physicians try to prevent definite reactions. The 
treatment of tuberculosis is considered in detail under Tuberculosis. 
The results of vaccine treatment, so far as we have observed them, 
have not come very quickly or been striking. There may be a slight 
betterment after the second or third injection, but at other times this 
improvement is delayed until ten or twelve have been given. We are 
always skeptical of the influence of vaccines when sudden improvement 
ensues. We remember, several years ago, two cases of septicemia, one 
of which received vaccine and the other did not. Both made a striking 
recovery. If circumstances had not prevented giving the vacccine to 
the second case, it would have seemed as almost certain that the vaccine 
had produced the cures. 
We believe that a final decision as to the value of vaccines would 
be arrived at more quickly if it were possible frequently to control 
treated cases with similar ones not treated, so that one might obtain 
more readily a correct impression of the effect of the injections. Even 
then it will undoubtedly be a number of years before we will clearly 
appreciate the class of cases which vaccines will benefit and those 
which they will not. Probably only the observations made by especially 
trained observers will ever give us this correct impression. 
Preparation of Bacterial Vaccines.-Cultures are preferably grown on 
agar, although broth may be used. The growth after twenty-four to 
forty-eight hours, depending on the rapidity of growth of the organism, 
is washed off in a small amount of saline solution. This suspension 
should be well shaken to give an even distribution of bacteria and is 
then standardized. The Wright method is most commonly used. 
A capillary pipette (see p. 210) is marked about one inch from the tip. The 
finger is pricked and blood drawn up to this mark; a bubble of air is 
then allowed to enter the tube and the bacterial supension then drawn up 
to the mark. The contents are then mixed on a slide and thin smears (as for 
blood) made. On the under lens of the eye-piece a one-quarter inch square is 
marked out with blue pencil, and using an oil-immersion objective the number of 
bacteria and red cells appearing in this square are counted. About fifty squares 
are counted and the average per square obtained. The number of red cells 
per c.c. being known, the number of bacteria are obtained according to the 
following proportion: 
Number of red cells • Number of bacteria • • , .1V x , .... . 
per square • per square • • 5000 t™11™) : X (millions) 
Other methods are employed as direct counting either by the Prescott 
method, in a counting chamber as used for red cells, centrifuging and deter- 
mining the column of bacteria, or by determining the weight of the bacteria 
after evaporation and drying. 
Methods of Killing the Bacteria.-Heated above 58° the bacteria 
show marked changes in their chemical composition, and do not yield 222 
PRINCIPLES OF MICROBIOLOGY 
the same amount of antibodies as those not so heated. Even lower 
degrees, such as 56°, are preferable to 58°. It has lately been suggested 
that instead of killing by heat the vaccines be killed by adding, when 
made, 0.5 per cent, carbolic acid, or 0.25 per cent, lysol. Vaccines can 
be kept for a period of three months with only slight loss of activity. 
The vaccines may be diluted to the required dosage when used. 
Amount of Vaccine Injected.-The intervals and size of dosage 
are still under active investigation. Some advise daily small doses, 
others larger doses every 2 to 7 days. We prefer giving small injections 
every other day. Certainly the interval should not be longer than 4 
days. The dose should be gradually increased at each injection. Differ- 
ent observers advise that for each organism the following number of 
millions be given: 
Minimum. 
Maximum. 
Average. 
Staphylococcus . 
50.0 m. 
2000.0 m. 
250.0 m. 
Streptococcus 
5.0 m. 
500.0 m. 
25.0 m. 
Pneumococcus . 
5.0 m. 
500.0 m. 
25.0 m. 
Gonococcus .... 
5.0 m. 
500.0 m. 
30.0 m. 
B. coli group 
25.0 m. 
1000.0 m. 
100.0 m. 
B. pyocyaneus . 
25.0 m. 
1000.0 m. 
100.0 m. 
Sensitized Vaccines.-Besredka1 has suggested a plan of injecting 
virus which has been immersed in specific serum; in other words, of 
using virus which has been sensitized. Heated vaccines alone some- 
times give severe symptoms when inoculated, e. g., typhoid, cholera; 
but after exposure to serum, inoculations of the sensitized vaccines are 
found to give only slight local and general reactions. Immunity, 
moreover, is produced rapidly by this procedure. Such sensitized 
vaccines can be given in doses thirtyfold those of pure virus. Marie2 
has used the same method in rabies treatment. 
The method is as follows: For bacteria: A twenty-four-hour agar 
culture of bacteria is mixed with specific antiserum at room temperature 
and allowed to stand for three hours. The bacteria are then washed 
free from unattached serum by repeated centrifugalizations with sterile 
normal salt solution. 
Typhoid and cholera bacteria are killed at this stage of the procedure 
by heating at 56° C. for one hour. Plague bacilli are killed before the 
addition of the serum. By this method the endotoxins are neutralized 
and the symptoms of intoxication are less severe. 
Besredka and those working with him have used • sensitized living 
typhoid bacilli for treatment and immunization. We have had no 
experience with these. It probably is incorrect to consider that many 
of the sensitized bacilli are alive after a few days' storage. 
Autogenous and Stock Vaccines.-The usual practice is to use a stock 
vaccine for the first inoculation and autogenous vaccines later. Some- 
times it is impossible to obtain a culture from the patient. The staphy- 
1 Review of previous work. Bull. Inst. Pasteur, 1910, viii, 241. 
2 See Rabies, Sec. II. VACCINES AS IMMUNIZING AGENTS 
223 
lococci, tubercle, and typhoid bacilli from different cases seem much 
alike, so that it is less important with these organisms to get autogenous 
vaccines than when streptococci, pneumococci, or colon bacilli must 
be used. A stock vaccine of certain organisms like the gonococcus 
require a stock vaccine composed of the different varieties in the group. 
Vaccines as Immunizing Agents.-The injection of vaccines in healthy 
subjects for the prevention of disease has been made so extensively 
that .no one doubts the advisability of their use. Typhoid vaccines are 
used extensively in the army and among persons going into special 
danger. Cholera vaccines and vaccines against bubonic plague also 
have been widely used. (See Individual Organisms, Part II.) CHAPTER XIV. 
PROTEIN HYPERSENSITIVENESS OR ANAPHYLAXIS. 
Perhaps the most important recent addition to our scientific 
knowledge has been the development of our conception of the fact 
and meaning of protein hypersensitiveness. Magendie is probably the 
first to record observations on this subject. He noted that rabbits 
which were injected intravenously for the first time with egg albumen 
showed no untoward effects, but that if after a lapse of time a second 
injection was given, serious and perhaps fatal results followed. 
Richet, in 1902, noted that when the blood of rabbits, which had 
received a previous protein injection was transferred to other rabbits, 
it rendered them sensitive to a second injection. The fact that this 
transfer of serum rendered them susceptible to protein poisons instead 
of protecting them caused Richet to give the term anaphylaxis to this 
condition. Arthus showed that in animals sensitized to horse serum 
a later injection given subcutaneously produced striking cutaneous 
reactions ranging from infiltration to gangrene. Theobald Smith 
studied and reported the reaction in guinea-pigs and Rosenau and 
Anderson noted the fact that the young of anaphylactic mothers 
inherited the same protein sensitiveness. Vaughan and others explain 
the poisoning of the second injection as follows: The introduction of 
a foreign protein into the tissues or blood of an animal develops in that 
animal a proteolytic ferment which is specific for the protein injected 
or for one very closely allied to it. The specific ferment remains as a 
zymogen in the cells of the animal and in its blood and tissue fluids, 
and is activated when the same protein is again injected. 
All protein poisons contain a toxic and a non-toxic portion; the first 
is not specific, while the second is. The first injection of the protein 
produces no symptoms because there is no proteolytic ferment present. 
The second injection, if sufficient time has elapsed for the formation 
of the ferment, causes symptoms because, the specific ferment having 
been produced, the protein when reinjected is split up into its poisonous 
and non-poisonous portions, and the former causes symptoms of in- 
toxication or anaphylaxis. Those changes occur not only in the plasma, 
but also in the cells, and the symptoms depend on whether the second 
injection was made locally or into the general blood current. The 
second injection causes no symptoms unless, as in all other immunity 
reactions time enough is allowed to elapse for the cells to assimilate 
the proteins and for specific ferments to be produced. 
Gay and Southard applied the term anaphylactin to this ferment. 
Friedberger calls the poisonous part of the split protein anaphylatoxin. EFFECT 
225 
Amount Required to Sensitize: Very small amounts such as 0.0001 c.c. 
of horse serum sensitizes an animal by whatever way inoculated. 
Incubation.-This varies in different animals. In man it is usually 
seven to twelve days. It gradually reaches its maximum and lasts for 
four months to a year or more. Where the reaction is present after 
much longer periods there has probably intervened a later sensitization, 
just as must have happened where an anaphylactic shock occurs in 
persons after a primary serum injection. 
Quantity of Foreign Protein Required to Produce Symptoms.-This is 
much larger than the amount which sensitizes. Much smaller amounts 
produce symptoms when given intravenously than when given sub- 
cutaneously. Thus, in a guinea-pig, 0.01 c.c. of serum intraperitoneally 
has as much effect as 0.5 c.c. subcutaneously. 
The following proteins and others produce the antiferments: 
1. Animal proteins in solution: Foreign serum, hemoglobin, milk 
albumin, egg albumen, etc. 
2. Cellular animal proteins: Hed-blood cells, leukocytes, cells of 
organs and tumors. 
3. Vegetable proteins: Extracts of bacteria, seeds, etc. 
4. Cellular vegetable proteins: Living or dead bacteria and yeasts. 
Specificity.-When an animal is sensitized with one protein it reacts 
most strongly to one of the same chemical structure, but also slightly 
to others nearly allied; thus a guinea-pig sensitized to egg albumen 
of the hen reacts slightly to egg albumen of the duck. This is similar 
to the group reaction to agglutinins, precipitins, etc. 
Antianaphylaxis.-According to Besredka and Steinhardt animals 
recovering from a second injection of serum are immune for a variable 
time. 
The effect of protein sensitization may be manifest in many ways 
Thus von Pirquet and Schick in 1905 described the symptoms which 
frequently follow in man after an injection of horse serum. These 
symptoms follow almost as frequently primary as secondary injections, 
but the time of their appearance is late with the primary injections and 
early with the secondary ones. The probable explanation is that after 
the injection of the first serum the cells gradually assimilate it and in 
a few days develop the specific proteolytic ferment. This acts upon 
the portion of the serum not yet assimilated and splitting it into the 
poisonous and non-poisonous portions, the toxic symptoms of fever, 
rash, etc., develop. The development of the symptoms in infectious 
diseases is attributed by Vaughan and others to this same fact of 
protein cleavage, as in the case of serum injections. 
Danger.-About 1 in 20,000 primary injections of diphtheria anti- 
toxic sera causes the immediate appearance of anaphylactic shock, in 
which symptoms of respiratory embarrassment and convulsions develop. 
About 1 in 50,000 of these cases ends fatally. Friedberger and Ilartock 
have shown that with the occurrence of anaphylactic symptoms there 
is a diminution of complement. 
The best studied instance of anaphylaxis is that produced in the 
15 226 
PRINCIPLES OF MICROBIOLOGY 
guniea-pig by the injection of a foreign proteid, such as horse serum, 
egg white, milk, etc. For example, if a guinea-pig is injected with 
a small quantity (about 0.01 c.c.) of horse serum, and, after an 
interval of ten to twelve days, is again injected with horse serum, but 
with a comparatively large amount (3-5 c.c. subcutaneously; 0.25 c.c 
intravenously), it will probably die in a short time. In a very sen- 
sitive animal symptoms appear within ten minutes and death occurs 
within an hour. The chief symptoms are respiratory failure, clonic 
spasms, and paralysis. If a smaller dose of serum be given to the 
sensitized animal it may show only slight symptoms and recover with 
the production of immunity. This phenomenon was noticed simul- 
taneously in several laboratories but was first definitely described by 
Theobald Smith, and is frequently called the Smith phenomenon. 
Tuberculin and mallein reactions are well-known instances of ana- 
phylactic manifestations (see special subjects). 
Doerr, of Vienna,1 closes a most excellent review of recent work on 
anaphylaxis or protein sensitization with the following paragraph: 
"While it must be admitted that the action of those infective bac- 
teria, which are not known to produce specific toxins, remains with- 
out explanation, and while the theories which have been developed by 
Von Pirquet, Friedberger, Vaughan, Schittenhelm, Weicherdt, and 
others have opened up a new way to the understanding of incubation, 
fever, and crises, still it must be borne in mind that the premises of these 
theories do not possess the force of chemical facts. It has not been 
positively shown that the symptoms of anaphylaxis are due to the 
parenteral cleavage of proteins, that the true anaphylactic poison is 
identical with that produced in vitro, and that both come from the 
antigen. Even if we agree with Dold, Sachs, and Ritz, that so far as 
the role of anaphylaxis in the infectious diseases is concerned, it is 
irrelevant from what matrix and by what processes the hypothetical 
anaphylactic poison is produced, even then all the difficulties are not 
removed. Numerous infecting agents are not anaphylactogens; they 
do not differ in their effects upon sensitized and non-sensitized animals; 
and even when there are differences, they are slight compared with 
those seen when the protein antigens derived from the higher plants 
are employed. The relatively simple structure of the bacterial proteins 
is the cause of this. Therefore, it is questionable whether one has the 
right to explain the phenomena of the infectious diseases with serum 
sensitization as a starting point. Moreover, the infections are not so 
monomorphic as some suppose from a superficial consideration." 
Serum Sickness.-Besides these rare accidents there are the disa- 
greeable after-effects which we group under the name serum sickness. 
Under this name we now include the various clinical manifestations 
following the injection of horse serum into man. The principal 
symptoms of this disease are a period of incubation varying from 
eight to thirteen days, fever, skin eruptions, swelling of the lymph 
1 Handbuch d. path. Mikroorganismen, Zweite Auflage. Kolle and Wassermann. SERUM SICKNESS 
227 
glands, leukemia, joint symptoms, edema, and albuminuria. The 
term "serum sickness" was first used by von Pirquet and Schick,1 
from whose excellent monograph the following data are chiefly taken. 
In 1874 Dallera reported that urticarial eruptions may follow the 
transfusion of blood. In the year 1894 the use of diphtheria antitoxin 
introduced the widespread practice of injecting horse serum. In the 
same year several cases were reported in which these injections were 
followed by various skin manifestations, mostly of an urticarial char- 
acter. Following these came a great mass of evidence which made it 
clear that following the injection of antidiphtheric serum these sequelae 
were usually comparatively harmless. 
Due to Serum as Such.-Heubner in 1894 and von Bokay some- 
what later expressed the opinion that these manifestations were due 
to other properties than the antitoxin in the serum, and this has 
proved to be the case. It has also been shown that the skin eruptions 
and other symptoms follow in a considerable degree according to the 
amount of serum injected, and this has led to attempts to eliminate 
the non-antitoxic portion of the serum as much as possible.2 The 
serum reaction has been studied by many investigators, but is not yet 
fully understood. 
Von Pirquet and Schick believe that the phenomena classed under 
serum sickness should be classed with those of anaphylaxis. 
It is probable that man cannot be sensitized in the same way as the 
guinea-pig, the most susceptible of the laboratory animals. Children 
have, in numerous instances, been injected with antidiphtheric horse 
serum at short and long intervals without, so far as we are aware, 
causing severe symptoms. Certain serums-for example, the anti- 
tubercle serum of Maragliano-are habitually used by giving injections 
at intervals of days or weeks. The rare fatal cases so far reported 
have all followed primary injections. 
While it may be true that the sensitizing of guinea-pigs by a previous 
injection of serum is analogous to the condition present in man which 
gives rise to the sudden symptoms following an injection of antitoxic 
serum, there is, in our experience, no reason to avoid a second im- 
munizing injection of serum when it is really indicated. A subcu- 
taneous injection in man comparable to tKe amount required to produce 
sickness in a guinea-pig would be over 200 c.c. We should hesitate, 
however, to give a large intravenous injection in a sensitized child. 
Banzhaf and Famuleuer have recently shown that chloral in large 
doses will prevent sickness in sensitized guinea-pigs. 
1 v. Pirquet and Schick, Die Serum Krankheit, Wien, 1905. 
2 See Banzhaf, The Preparation of Antitoxin, Johns Hopkins Hospital Bulletin, 1911, 
xxii, No. 241. PART II. 
PATHOGENIC MICROORGANISMS INDIVIDU- 
ALLY CONSIDERED. 
CHAPTER XV. 
THE PATHOGENIC MOLDS (HYPHOMYCETES, EUMY- 
CETES) AND YEASTS (BLASTOMYCETES). 
THE HYPHOMYCETES. 
The majority of the molds are not pathogenic for human beings, 
and interest us more as organisms which are apt to infect foodstuffs 
and media. Some are, however, true parasites, and produce a number 
of rather common diseases; for example, ringworm, favus, thrush, 
and pityriasis versicolor. Certain of the commoner molds (mucor, 
aspergillus) have also been reported from time to time as present in 
pathologic conditions in man as well as in the lower animals. Many 
varieties have been found in plant diseases, and others may be a source 
of danger to man indirectly. Indeed, when they form poisonous 
substances, as in the infection of grain by claviceps purpurea (ergot 
poisoning), they are distinctly dangerous. 
Paltauf reported the case of a man who died after enteritis with 
secondary peritonitis. The autopsy showed multiple abscesses in 
brain and lungs, besides the lesions in the intestines and peritoneum, 
in all of which a species of mucor was' found. Two other cases of 
primary mucor infection in humans were reported by Furbringer. 
A number of species of mucor have been found in ear and eye infec- 
tions; for example, Mucor corymbifer (Fig. 89) has been found in 
ophthalmia. A number of varieties are pathogenic for lower animals. 
Aspergillus is found thus very frequently in lower animals, especially 
in birds, where a kind of pseudotuberculosis is often produced. 
Quite a number of similar cases have been reported in man, and it 
is supposed that the infection may be carried from birds to man. 
Aspergillus fumigatus (Fig. 90), is the most frequent variety found. 
Penicillium minimum (similar to glaucum, Fig. 88) has been found 
by Liebermann in inflammation of the ear. The more common patho- 
genic forms for man are those producing the various hair and skin 230 
PATHOGENIC MICROORGANISMS 
lesions mentioned above. These will now be described (see p. 27 for 
general-characteristics for molds; see also Plate I). 
Fig. 88 
Penicillium glaucum. Gelatin culture. Spread stained with gentian violet. 500 : 1 
(From Itzerott and Niemann.) 
Fig. 89 
Mucor corymbifer, Coh. Mycelium with underlying branched carriers, sporangia 
at o have burst, -y1. (After Lichtheim.) 
Trichophyton (Tinea, Ringworm Fungus).-Ringworm of the body 
or hairless parts of the skin, Tinea circinata, and ringworm of the hairy 
parts, Tinea tonsurans and Tinea barbae or Tinea sycosis, are due 
to fungoid organisms placed in the imperfecti group (p. 28). THE HYPHOMYCETES 
231 
Methods of Examination.-Living Specimens.-Place scales or hairs in hot 
liq. potassse for a few seconds. Examine under cover-glass. 
Permanent specimens.-Remove fat with chloroform, then place material in 
formic acid and heat until boil (2 to 3 minutes). Remove acid by washing 
in distilled water, stain with Loeffler's methylene blue. Wash, dehydrate in 
absolute alcohol, clear in xylol and mount in balsam. 
Sabouraud's media are best for growth of the organism. 
According to Sabouraud, whose conclusions are based on an exten- 
sive series of microscopic examinations of cases of tinea in man and 
animals, of cultivation in artificial media, and of inoculation on man 
and animals, there are two distinct types of the fungi causing ringworm 
in man-one with small spores (2/z to 3^) which are known as micro- 
spora, and one with large spores (7p to 8ju) which are called megalo- 
spora. They differ in their mode of growth on artificial media and in 
Fig. 90 
Aspergillus fumigatus. Gelatin culture. Spread stained with gen tian-violet. 500 : 1. 
(From Itzerott and Niemann.) 
their pathological effects on the human skin and its appendages. The 
small spored type is the common fungus of Tinea tonsurans of children, 
especially of those cases which are rebellious to treatment, and its 
special seat of growth is in the substance of the hair. T. megalosporon 
(Fig. 91) is essentially the fungus of ringworm of the beard and of 
the smooth part of the skin; the prognosis as regards treatment is good. 
One-third of the cases of T. tonsurans of children are due to megalospora. 
The spores of T. microsporon are contained in a mycelium; but this is 
not visible, the spores appearing irregularly piled up like zodglea masses; 
and, growing outside, they form a dense sheath around the hair. The 
spores of T. megalosporon are always contained in distinct mycelium 
filaments, which may either be resistant when the hair is broken up or 
fragile and easily breaking up into spores. The two types show distinct 
and contsant characters when grown in artificial cultures. The cultures 232 
PA THOGENIC MICROORGA NISMS 
of T. microsporon show a downy surface and white color; those of T. 
megalosporon a powdery surface, with arborescent peripheral rays, and 
often a yellowish color. Although the morphological appearances 
Fig. 91 
Hair riddled with ringworm fungus. Megalosporon variety. 
Fig. 92 
These two half-plates show three months' growth on peptone-maltose agar of two 
megalosporon varieties of the ringworm fungus. Natural size. THE HYPHOMYCETES 
233 
mode of growth, and clinical effects of each type of trichophyton show 
certain characters in general, yet there are certain constant minor 
differences which point to the fact that there are several different 
kinds of species of fungus included under each type. The species 
included under T. microsporon are few in number, and, with the excep- 
tion of one which causes the common contagious "herpes" of the horse, 
almost entirely human. The species of T. megalosporon are numerous 
and fall under several natural groups, the members of which resemble 
one another both from clinical and mycological aspects (Fig. 92). 
varieties of T. megalosporon. Cultures inoculated into guinea-pigs and 
other laboratory animals may produce infection. 
Achorion Schoenleinii (Favus).-Favus is due to a fungus dis- 
covered by Schoenlein in 1839, and called by Ilemak Achorion schoen- 
leinii. The disease is communicated by contagion, the fungus being 
often derived from animals, especially cats, mice, rabbits, and fowls; 
dogs also are subject to it. It grows much more slowly than the 
ringworm fungus, and is, therefore, not so easily transmitted. Want 
of cleanliness is a predisposing factor. The fungus seems to find a 
more favorable soil for its development on the skin of persons in weak 
health, especially from phthisis, than in others. 
Fig. 93 
A portion of a favus-infected hair; magnified. 
Pathologically, the disease represents the reaction of the tissues to 
the irritation caused by the growth of 'the fungus. The spores gen- 
erally find their way into the hair follicles, where they grow in and 
about the hair (Fig. 93). The favus fungus grows in the epider- 
mis, the density of the growth causing pressure on the parts below, 
thus crushing out the vitality of the hair and giving rise to atrophic 
scarring. The disease shows a marked preference for the scalp, but 
no part of the skin is exempt, and even the mucous membranes are 
liable to be attacked. Kaposi has reported a case in which a patient 
suffering from universal favus died, with symptoms of severe gastro- 
intestinal irritation, which was found after death to be due to the 
presence of the favus fungus in the stomach and intestines. On the 
scalp it first appears as a tiny sulphur-yellow disk or scutulum, depressed 
in the centre like a cup and pierced by a hair. This is the characteristic 234 
PATHOGENIC MICROORGANISMS 
lesion. The cup shape is attributed by Unna to the fact that growth 
is more vigorous at the sides than at the centre. 
The favus fungus is readily cultivated at the body temperature, 
and also at room temperature, in the ordinary culture media, as agar, 
blood serum, gelatin, bouillon, milk, infusion of malt, eggs, potato, etc. 
(Fig. 94). The growth develops slowly and shows a preference to 
growth beneath the surface of the medium-except on potato, upon 
which it develops on the surface in layers. 
The characteristic form of growth is that 
of moss-like projections from a central 
body. The color is at first grayish-white, 
then yellowish. As seen under the micro- 
scope, ray-like mycelium filaments are 
developed, which divide into branches. 
The ends are often swollen or club- 
shaped, and there are various enlarge- 
ments along the body of the filament. 
Several varieties have been discovered. 
Microsporon Furfur.-This organism, 
found in pityriasis versicolor, belongs to 
a group of fungi which, in contrast to 
the more parasitic fungi, favus and tri- 
chophyton, invades only the most super- 
ficial layers of the skin. It does not penetrate the deeper layers nor 
does it give rise to any considerable pathologic changes in the skin or 
hair. Although the vegetative elements of these fungi are much more 
numerous in the affected portions of the skin than is the case with the 
more parasitic species, they are not nearly as contagious as the latter. 
By preference the organism attacks the chest, abdomen, back, and 
axillae, less frequently neck and arms, while exceptionally it attacks 
also the face. The growth shows itself as scattered spots varying in 
color from that of cream-coffee to reddish-brown. The spots are 
readily scraped off and show fine lamellation or scaling. Occasionally 
the spots are confluent, and sometimes arranged in ring form like 
Herpes tonsurans. 
In spite of their slight contagiousness this is one of the most fre- 
quent dermatomycoses. Although it is distributed widely over the 
earth, it is more frequently observed in southern than in northern 
countries. 
Persons with a tender skin and a disposition to perspire freely are 
particularly affected by Pityriasis versicolor, and this is undoubtedly 
the only reason why the affection is so frequently observed in con- 
sumptives. Women are more frequently attacked than men, while 
children and old people are rarely affected. 
The source of infection is unknown, since the absence of contagion 
has frequently been demonstrated. It seems likely that the spores of 
this fungus are so widely distributed that susceptible individuals are 
easily infected. 
Fig. 94 
Five-month-old colony of favus 
on peptone-maltose agar; actual 
size. THE HYPHOMYCETES 
235 
The arrangement of the fungus in the scales of epidermis is char- 
acteristic. The short and thick-curved hyphae (7 m to 13m long and 
3m to 4m wide) surround large clumps of spores. The spores are 
coarse, doubly contorted (4m to 7m) or round. On staining with 
Ziehl's solution the spores are seen to contain deeply stained globules 
lying, in all probability, on the inner surface of the cell membrane. 
The rest of the protoplasm is but little stained, or not at all. One 
frequently finds that these globules have disintegrated into numerous 
fine granules. The globules are also found free; what their nature 
is does not appear; they are not found in cultures, the freshly de- 
veloped spores showing only a single globular mass of protoplasm 
possessing a fine blue lustre. 
Cultures are obtained with difficulty and the growths cannot be 
transferred for many culture generations. 
Fig. 95 
Inflammation of cornea by thrush, g, conidia; e, pus cells. (From Plaut in Kolle and 
Wassermann.) 
Thrush (Oispora Oidium) (Fig. 95).-Thrush, as is well known, occurs 
most frequently in the oral mucous membrane of infants during the 
early weeks of life. It is also found as a slight mycosis in the vagina, 
especially of pregnant women. In rare cases the disease attacks 
adults, and especially those whose system has been undermined by 
other diseases, such as diabetes, typhoid patients, etc. A few cases 
are recorded in the literature in which this fungus has given rise to 
constitutional disease. In these cases autopsy has shown abscesses 
in various parts of the body, such as in the lungs, spleen, kidney, and 
brain. 236 
PATHOGENIC MICROORGANISMS 
In the lesions of the disease as well as in cultures, this fungus appears 
both as a yeast and a mycelium. It thus seems to stand between the 
true molds and the yeasts. The yeast cells are oval in form, about 5g 
to 6g long and 4g wide, and can in no way be distinguished from other 
yeast cells either by their appearance or their method of propagation. 
The threads of the mycelium vary markedly in length and thickness, 
and show all intermediate forms between a typical and a budding 
mycelium. Cultures are easily obtained on artificial media, especially 
on those favorable for yeasts (p. 106). 
Thrush is not much influenced by acids or alkalies. On the other 
hand, it is very susceptible to the common disinfectants, especially 
salicylic acid, corrosive sublimate, phenol, etc. This fact is made use 
of in local treatment. 
Sporotricha.-Another of the molds classed with the Fungi imperfecti 
(family Mucedinese) has been found to produce disease in man and 
some of the lower animals. The first variety described was by Schenk 
in 1898. It was declared one of the sporotricha by F. Smith. In I960 
Hektoen and Perkins described minutely a culture isolated from 
another case and gave the organism the name Sporotrix (Sporotrichon) 
Schenki. 
Since the disease was made more thoroughly known by the studies 
of Beurmann and Gougerot and others, many cases have been reported 
in various parts of the world, and several new species of sporotricha 
have been described. Beurmann and Gougerot first described the 
species so far most frequently reported, that is, Sporotrichon beurmanni, 
which is so similar to Sporotrichon schenki that they are classed now 
by most observers as one species. So several of the other species 
described may prove on further study to be but one species. 
Earlier the disease was often mistaken for tuberculosis or syphilis 
because of the similarity of the lesions which in general are slowly 
growing granulomata ending in degeneration. When it was found 
that the disease responds readily to treatment with potassium iodide 
and other iodide combinations, but not to mercury salts, it became 
comparatively easy to corroborate the diagnosis. In 1912 Rudiger 
published a review of the cases reported to date. 
Morphology-In the pus from the lesions only oval, highly refractile 
conidia or spores (3g to 4g x 1.6g to 3g) are seen and these generally 
sparingly. They may lie within the tissue cells. They are Gram- 
positive. In cultures these spores grow out into a branching irregu- 
larly septate mycelium (the hyphae about 1g thick), and the new 
conidia are formed simply at the sides and ends of the hyphae without 
definite fruiting organs. (Plate I, Fig. 5). The conidia often occur in 
whorls. 
Cultivation.-They grow readily on ordinary media, but better when 
it is slightly acid. Sabouraud's medium (p. 106) is most favorable. 
They grow both at room and at incubator (36° C.) temperature. At 
20-25° C. growth is seen in about four days, and at 36° C. in about 
forty-eight hours. Minute, fluffy, snow-flake-like colonies appear which PATHOGENIC YEASTS 
237 
slowly become brown or brown-black, and, when in a mass, con- 
voluted. Gougerot found that by letting a drop of the pus flow 
over a glass slide and keeping in a moist atmosphere, the sprouting 
of the spores may be followed under the microscope in two or three 
days. Agglutination and complement fixation have been studied by 
Widal and Abrami (1908). Their results, which are given in the next 
paragraph, await further corroboration. 
Agglutination.-Homogeneous emulsions of the spores are made from 
cultures on Sabouraud's medium six to twelve weeks old. A large 
amount of the cultures is removed and rubbed until dry in a mortar. 
To this, drop by drop, is added several cubic centimeters of normal salt 
solution, while continuing to rub. The suspension is filtered through 
moistened filter paper. The filtrate contains only free spores which in 
positive cases agglutinate in from fifteen to sixty minutes in dilutions 
of 1 to 150 to 1 to 1800, generally 1 to 300 to 1 to 400. Agglutination 
with the sera of other mold and yeast infections may occur to less extent. 
Complement-fixation is said to be not specific for the different mold and 
yeast infections. 
Pathogenesis.-Cultures or pus injected subcutaneously or intra- 
peritoneally into lower animals such as mice, rats, and dogs produce 
granulomatous lesions similar to those found in man. 
In man the lesions vary from superficial, nonulcerative gummae to 
deep viseral abscesses. Sometimes the diagnosis is difficult to determine. 
According to Gougerot the following points help in differentiation: 
1. Many lesions with general good health. 
2. Lesions begin as indolent swellings which gradually become large 
gummae, on which one or several small ulcers with violet edges may 
appear. These ulcers discharge a thin, yellowish, shiny pus, and the 
centre gradually becomes cicatrized with a persistance of the abscess 
under the skin. Generally there is no adenitis. 
3. Microscopic and cultural examinations reveal the organisms. 
Large quantities of the pus should be inoculated into each culture 
tube which should not be closed with rubber cap. The dry method 
should also be tried. Direct smears are not satisfactory. 
4. Serum diagnosis may be tried. 
5. Animal inoculations give positive results. 
6. Treatment with potassium iodide. This produces a cure unless 
given very late in the disease or unless the mucous membranes are 
extensively affected. Some cases need treatment for two or three 
months before improvment is seen. Occasionally death occurs. 
The pathogenic blastomycetes (see p. 29 for general characteristics 
of yeasts; see also Plate I, Figs. 6 and 7), may be briefly summarized 
as follows: 
Saccharomyces busse is the name given to a yeast isolated in 1894 
by O. Busse from the tibia of a thirty-one-year-old woman who died 
PATHOGENIC YEASTS. 238 
PATHOGENIC MICROORGANISMS 
thirteen months after the first symptoms appeared. The autopsy showed 
numerous broken-down nodules on several of the bones, and in the lungs, 
spleen, and kidneys. The yeast was cultivated from all these foci. 
Saccharomyces tumefaciens was isolated in 1895 by Curtis. The patient 
was a young man showing multiple tumors on the lips and neck having 
the gross appearance of softened myxosarcomata. 
This yeast is pathogenic for rats, mice, and dogs, only slightly so for 
rabbits, and not at all for guinea-pigs. 
Various similar cases have since been described, a number of them 
becoming generalized, and ending fatally. In generalized blastomy- 
cosis the lung seems frequently to be the seat of primary infection. 
The cases described first by Rixford and Gilchrist as coccidiosis 
due to " Coccidioides imitis"-thought to be a protozoon-may be 
classed here, since Ophuls, Oliver, and others have shown that the 
"coccidium" form hyphae and elliptical forms on culture. Cultures 
from fresh tissue do not grow readily, but after they are once started 
they grow with ease on the usual laboratory media. The growth is 
more mold-like than yeast-like, except on potato, when budding yeast- 
like cells are produced. Dogs, rabbits, and guinea-pigs are susceptible 
to the fungus and show lesions similar to those in human beings. 
Fig. 96 
Section of lung. X 150; blastomycetes in large syncytial cell masses. (From Fontaine, 
Hasse, and Mitchell.) 
A typical case of systemic blastomycosis, reported by Fontaine, 
Hasse, and Mitchell is accompanied by very good illustrations of 
tissue sections. Fig. 96 shows the characteristic microscopic appear- 
ance of the lung lesion. PATHOGENIC YEASTS 
239 
Several cases of ophthalmia have been ascribed as due to yeasts, and 
even "trachoma" has been said to be caused by this organism. 
Buschke isolated a yeast from a cervical discharge in which no 
gonococci were present. The yeast was pathogenic for guinea-pigs. 
In 1895 Dr. G. Tokishige reported an epidemic quite common 
among horses in Japan, known as "Japanese worm," "benign worm," 
or "pseudoworm," which is caused by one of the saccharomyces. 
This disease begins in the skin in the form of hard, painless nodules 
from the size of a pea to that of a walnut. These break down and give 
rise to gradually extending ulcers. Pure cultures of the saccharo- 
myces are pathogenic only for horses, not for rabbits, guinea-pigs, or 
hogs. In the districts where the disease prevails among horses it is 
also frequently seen in cattle. / 
Shortly after Tokishige's publication a similar disease occurring 
in horses in Italy and southern France was identified as being caused 
by saccharomyces. Cultures of this yeast, however, differ somewhat 
from that obtained in Japan, so that Busse is inclined to regard the two 
as two different species of blastomycetes. 
Kartulis, in Alexandria, described about a hundred cases of a skin 
affection occurring in the gluteal regions of men and characterized by 
an elongated finger-like swelling, which breaks and emits a purulent 
discharge, forming an unhealed sinus. In the discharge and surrounding 
tissues are numerous blastomycetes which Kartulis after cultivation 
and study considered a variety of the ordinary fermenting yeast (Saccha- 
romyces cerevisise hansen). The cases were cured by excising the 
growth. 
Fig. 97 
Blastomycosis in infant aged 8 months, showing lesion on left cheek. (Kessler.) 
Kessler reported a skin lesion in an infant (Fig. 97) probably due 
to a similar blastomycete, since the lesions healed after treatment with 
potassium iodide. The description of the yeast isolated is too incomplete 
to identify it. 240 
PATHOGENIC MICROORGANISMS 
Some years ago the attempt was made to connect the develop- 
ment of cancerous growth with blastomycetes. This was due in a 
measure to a certain similarity between the yeasts and the cell in- 
clusions or so-called "parasites" of cancer, and further, to the fact 
that when yeasts are injected into the animal body tumor-like nod- 
ules are often developed at the site of inoculation and in the internal 
organs. But these nodules are not tumors in the pathological sense 
of the term, but merely masses of blastomycetes mixed to a very 
variable degree with inflammatory tissue proliferations. 
Yeasts are Gram-positive, or Gram-amphophile. They stain readily 
with other stains in the young state. Older forms stain very irregularly. 
They are cultivated-sometimes with difficulty directly from the tissues 
-on ordinary media, but best on media made slightly acid (p. 106). 
Some pathogenic varieties grow best at blood-heat. Most varieties grow 
easily at room temperature. 
The important part played by yeasts in certain industries is treated 
in Part III. 
Busse. In Kolle u. Wassermann's Handbuch d. path. Mikroorg., 1913, Jena. Ind. Ed. 
De Beurmann et Gougerot. Traite des Sporotrichoses, 1912, Paris. 
Fontaine, Hasse and Mitchell. The Arch, of Int. Med., 1909, iv, 101. 
Hektoen and Perkins. Jour. Exp. Med., 1900, v, 77. 
Kessler. Jour. Amer. Med. Assoc., 1907, xlix, 550. 
Klocker. Trans, by Allan and Millar, 1903, New York and London. 
Ricketts. Jour. Med. Res., 1901, vi, 377. 
Ruediger. Jour. Inf. Dis., 1912, xi, 193. 
Schenk. Johns Hopkins Hospital Bulletin, 1898, p. 286. 
References. CHAPTER XVI. 
THE PYOGENIC COCCI. 
THE STAPHYLOCOCCI (MICROCOCCI). 
Staphylococci were first obtained from pus by Pasteur in 1880. 
In 1881 Ogston showed that they frequently occurred in abscesses, 
and in 1884 Rosenbach fully demonstrated their etiological impor- 
tance in circumscribed abscesses, osteomyelitis, etc. Of the staphy- 
lococci those producing yellow and white pigments are by far the most 
important since they are the pathogenic varieties. 
The Staphylococcus (Pyogenes) Aureus.-The Staphylococcus au- 
reus is one of the commonest pathogenic bacteria, being usually present 
in the skin and mucous membranes, and is the organism most fre- 
quently concerned in the production of acute, circumscribed, sup- 
purative inflammations. 
Morphology.-Small, spherical cells, having a diameter of 0.7/z 
to 0.9/z, occurring solitary, in pairs as diplococci, in short rows of 
three or four elements, or in groups of four, but most commonly in 
irregular masses, simulating clusters of grapes; hence the name 
staphylococcus. (See Fig. 98.) 
Staining.-It stains quickly in aqueous solutions of the basic ani- 
line colors and with many other dyes. When previously stained 
with aniline gentian violet it is not decolorized by Gram's method. 
When slightly stained each sphere frequently is seen to be already 
dividing into two semispherical bodies. 
Biology.-The Staphylococcus pyogenes aureus is an aerobic, fac- 
ultative anaerobic micrococcus, growing at a temperature from 8° to 
43° C., but best at 25° to 35° C. The staphylococci grow readily 
on all the common laboratory media, su^h as milk, bouillon, nutrient 
gelatin, or agar. A slightly alkaline reaction to litmus is best for 
the growth of the staphylococci, but they also grow in slightly acid 
media. 
Cultivation. - Growth in Nutrient Bouillon.-The growth of the 
staphylococcus is rapid, reaching about 50,000,000 per c.c. at the end 
of twenty-four hours at 30° C. The bouillon is cloudy and frequently 
has a thin pellicle. Later a slimy sediment forms. The odor is dis- 
agreeable. In peptone-water, growth occurs with indol production. 
Growth on Gelatin.-Grown on gelatin plates it develops, at room 
temperature, within forty-eight hours, punctiform colonies, which when 
examined under a low-power lens, appear as circular disks of a pale 
brown color, somewhat darker in the centre, and surrounded by 
a smooth border. The colonies grow rapidly. The appearance of the 
1G 242 
PA THOGENIC MICROORGANISMS 
growth is most characteristic. Immediately surrounding the colo- 
nies, which are of a pale yellow color, there is a deepening of the sur- 
face of the gelatin, due to its liquefaction. By suitable light a num- 
ber of these shallow depressions with sharply defined outlines may be 
seen on the gelatin plate, having a diameter of from 5 to 10 mm., in 
the centres of which lie the yellow colonies. Later the liquefaction 
becomes general, the colonies running together. In stab cultures in 
gelatin a white confluent growth at first 
appears along the line of puncture, fol- 
lowed by liquefaction of the medium, 
which rapidly extends to the sides of 
the test-tube. At the end of two days 
the yellow pigmentation begins to form, 
and this increases in intensity for eight 
days. Finally, the gelatin is completely 
liquefied, and the staphylococci form a 
golden-yellow or orange-colored deposit 
at the bottom of the tube. Under 
unfavorable conditions the Staphylo- 
coccus aureus gradually loses its 
ability to make pigment and to 
liquefy gelatin. 
Growth on Agar.-In streak and stab cultures on agar a whitish 
growth is at first produced, and this at the end of a few days becomes 
a faint to a rich golden yellow on the surface. The yellow pigmenta- 
tion is produced only in the presence of oxygen; colonies formed at the 
bottom of a stab culture or under a layer of oil remain white. 
Milk.-Milk is coagulated at the end of from one to eight days. 
Potato.-The staphylococci grow readily on potato and produce 
abundant pigment. 
Growth on Leffler's Solidified Blood-serum.-Growth vigorous, with 
fairly good pigment production. Some varieties slowly liquefy the 
serum. 
Growth on Blood-agar.-If nutrient agar to which a little animal 
blood has been added is streaked with staphylococci there appears, at 
the end of twenty-four hours at 35° C., about the growth a clear zone, 
owing to the hemolytic effect of the staphylococcus products. 
Acids Produced.-In certain culture media there is, as a result of the 
growth of the Staphylococcus aureus, a production of acid in consider- 
able quantities, consisting chiefly of lactic, butyric, and valerianic 
acids. These acids have been supposed to play a part in the production 
of pus, in which, according to some observers, they are often present. 
Resistance.-The staphylococcus is distinguished from most other 
pon-spore-bearing pathogenic bacteria by its greater power of resistance 
to outside influences, desiccation, etc., as well as to chemical disin- 
fectants. Cultures of the Staphylococcus pyogenes in gelatin or agar 
retain their vitality for a year or more. Suspended in water its ther- 
mal death-point varies with different cultures and averages about 
Fig. 98 
Staphylococcus. X 1100 diameters. THE STAPHYLOCOCCI 
243 
two hours at 50° C., one-half hour at 60° C., ten minutes at 70° C., 
and five minutes at 80° C. Upon silk threads and in media rich 
in organic matter its resistance is greater, but subjected to 80° C. 
for thirty minutes or boiling for two minutes it is almost surely killed. 
Cold has but little effect. Thirty per cent, of the organisms remained 
alive after being subjected by us to freezing in liquid air for thirty 
minutes. These are average figures. Some cultures are more resistant 
than others. 
They are quite resistant to direct sunlight and drying. Dried 
pus contains living staphylococci for weeks and even months, and 
they can be found alive in the fine dust of the air in living rooms and 
operating rooms. 
To most disinfectants the staphylococci are rather resistant. The 
presence with staphylococci of organic substances, especially albumin, 
increases their resistance. In watery solution dissolved mercuric 
chloride, 1 to 1000, destroys the organisms in five to fifteen minutes, 
but when in pus not for several hours. Hydrogen peroxide in 1 per cent, 
solution kills in about one-half hour. 
Products of Growth.-Pigment Formation.-Pigment formation, 
already described, is considered within a limited species characteristic. 
Thus different strains of Staphylococcus aureus produce a pigment 
varying from a pale brown to a deep golden yellow. It usually becomes 
less intense upon prolonged cultivation. The pigment is classed as a 
lipochrome (Schneider). It is soluble in alcohol, chloroform and ether. 
In order to test the amount of color produced, Winslow and Rogers recommend 
the following method: A portion of the growth is removed on a loop needle and 
spread out on white drawing paper with a rough surface. After drying at room 
temperature the color is compared with a standard color chart. 
Toxic Substances.-Filtrates of cultures contain toxic substances. 
Injected into the peritoneal cavity they excite peritonitis. Under 
the skin they produce 'infiltration or abscess formation. In the blood 
they injure both the red and white corpuscles. 
Cultures of the staphylococcus, when sterilized by boiling and in- 
jected subcutaneously, produce marked positive chemotaxis and often 
local abscesses. Leber found also that 'sterilized cultures introduced 
into the anterior chamber of the rabbit's eye would bring about a 
fibropurulent inflammation, the cornea becoming infiltrated, and 
perforation alongside of the sclerotic ring finally taking place. This 
was followed by the formation of pus in the anterior chamber and 
recovery. These local changes follow the inoculation of small quan- 
tities only of the dead cultures; but when large amounts are injected 
into a vein or into the abdominal cavity, toxic effects are produced. 
The hemolytic effects of certain products of virulent staphylococci 
have recently been studied. In cultures they can be detected about 
the third or fourth day of incubation and reach their maximum on 
the ninth to fourteenth day. Virulent staphylococci are more apt to 
produce this substance than the non-virulent, but there is no definite rule. 244 
PATHOGENIC MICROORGANISMS 
The specific hemolysin, known as staphylolysin, is destroyed by heat- 
ing for twenty minutes at 56° C. An antibody for this is formed by inocu- 
lating animals with culture filtrates. A substance called leukocidin is 
produced which injures leukocytes. It also produces an antibody. 
The gelatin-liquifying enzyme-gelatinase-has already been mentioned. 
Pathogensis.-The pathogenic effect of the Staphylococcus {pyo- 
genes') aureus on test animals varies considerably, according to the 
mode of application and the virulence of the special culture employed. 
In man a simple rubbing of the surface of the unbroken skin with 
pus from an acute abscess is, as a rule, sufficient to produce a purulent 
inflammation, and the introduction of a few germs from a septic case 
into a wound may lead to a fatal pyemia. These conditions can only 
be reproduced in lower animals with difficulty, and by the inoculation of 
large quantities of the culture. Small subcutaneous injections, or the 
inoculation of open wounds in mice, guinea-pigs, and rabbits, are 
commonly without result; occasionally abscess formation may follow 
at the point of inoculation, which usually ends in recovery. The pus- 
producing property of the organism is exhibited in proportion to the 
virulence of the culture employed. Slightly virulent cultures, which 
constitute the majority of those obtained from pus taken from the 
human subject, when injected sucutaneously in large quantities 
(several c.c. of a fresh bouillon culture) into rabbits or guinea-pigs, give 
rise to local pathological lesions-acute abscesses. When virulent 
cultures are used-which are rarely obtainable-0.5 c.c. of a fresh 
bouillon culture is sufficient to produce similar results. The abscesses 
generally heal without treatment; sometimes the animals die from 
marasmus in consequence of the suppurative process. In intraperito- 
neal inoculations the degree of virulence of the culture employed is 
still more evident in the effects produced. The animals usually 
die in from two to nine days. The most characteristic pathological le- 
sions are found in the kidneys, which contain numerous small collec- 
tions of pus, and under the microscope present the appearances re- 
sulting from embolic nephritis. Punctiform, whitish-yellow masses of 
the size of a pea are found permeating the pyramids. Many of the 
capillaries and some of the smaller arteries of the cortex are plugged 
up with thrombi, consisting of micrococci. Metastatic abscesses 
may also be observed in the joints and muscles. The micrococci 
may be recovered in pure cultures from the blood and the various 
organs; but they are not numerous in the blood and are often difficult to 
demonstrate microscopically. Intravenous inoculations of animals are 
followed by similar pathological changes. Orth and Wyssokowitsch 
first pointed out that injection of staphylococci into the circulation of 
rabbits, whose cardiac valves have previously been injured, produced 
ulcerative endocarditis. Subsequently, Weichselbaum, Prudden, and 
Fraenkel and Sanger obtained confirmatory results, thus establishing 
the fact that when the valves are first injured, mechanically or chemic- 
ally, the injection into a vein of a pure culture of Staphylococcus aureus 
gives rise to a genuine ulcerative endocarditis. It has been further THE STAPHYLOCOCCI 
245 
shown by Ribbert that the same result may be obtained without 
previous injury to the valves by injecting into a vein the staphylococcus 
from a potato culture suspended in water. In his experiments not only 
the micrococci from the surface, but the sperficial layer of the potato 
were scraped off with a sterilized knife and mixed with distilled water 
and the successful result is ascribed to the fact that the little agglome- 
rations of micrococci and infected fragments of potato attach them- 
selves to the margins of the valves more readily than isolated cocci 
would do. Not infrequently, also, in intravenous inoculations of 
young animals there occurs a localization of the injected material in the 
marrow of the small bones. This may take place in full-grown animals 
when the bones have been injured or fractured. The experimental 
osteomyelitis thus produced has been demonstrated to be anatomically 
analogous to this disease in man. 
Occurrence in Man.-Practically all microorganisms have been 
shown by experiment to produce, under certain conditions, the for- 
mation of pus by their products when inoculated into the animal 
body; but, while this has been demonstrated, the researches of bac- 
teriologists show that only a few species are usually concerned in 
the production of acute abscesses in man. Of these the two most 
important, by reason of their frequent occurrence and pathogenic 
power, are Staphylococcus aureus and Streptococcus pyogenes. These 
two organisms, are often found in the same abscess; thus, Passet, in 
33 cases of acute abscess, found Staphylococcus aureus and albus 
associated in 11, albus alone in 4, albus and citreus in 2, Streptococcus 
pyogenes alone in 8, albus and Streptococcus in 1, and albus, citreus, and 
Streptococcus in 1. The staphylococcus is likely to enter as a mixed 
infection into most infections due to other bacteria, and is almost 
always met with in all inflammations of the skin and mucous mem- 
branes or in cavities connected with them. 
The staphylococcus {Staphylococcus aureus) has been demonstrated 
not only in furuncles and carbuncles, but also in various pustular 
affections of the skin and mucous membranes-impetigo, sycosis, 
purulent conjunctivitis and inflammation of the lacrimal sac; in acute 
abscesses formed in the lymphatic glands, ,the parotid gland, the tonsils, 
the mammae, etc.; in metastatic abscesses and purulent collections in 
the joints; in empyema, infectious osteomyelitis, ulcerative endocarditis, 
pyelonephritis, abscess of the liver, phlebitis, etc. It is one of the chief 
etiological factors in the production of pyemia in the various pathologi- 
cal forms of that condition of disease. It is remarkable how many 
staphylococci may be present in the blood without a fatal result, if the 
original source of infection is removed. We met with one case in which 
over 800 staphylococci were present in 1 c.c. of blood. A week later 
only 5 were found. The patient finally died from pneumonia. • 
Not all persons are equally susceptible to infection by the staphy- 
lococcus; those who are in a cachectic condition or suffering from 
constitutional diseases, like diabetes, are especially predisposed to 
infection. In healthy individuals certain parts of the body, as the 246 
PATHOGENIC MICROORGANISMS 
back of the neck and the buttocks, are more liable to be attacked than 
others, with the production of furuncles, carbuncles, etc. In persons in 
whom sores are readily caused, in consequence of disturbances of 
nutrition, as in exhausting diseases, the micrococci settle at the points 
of least resistance. Such conditions are present in the bones of debili- 
tated young children, in fractures, and in injuries in general. 
The pyogenic properties of the staphylococcus have been demon- 
strated upon man by numerous experiments. Garre inoculated a 
small wound at the edge of one of his finger nails with a minute quan- 
tity of a pure culture, and purulent inflammation, extending around 
the margin of the nail, resulted from the inoculation. Staphylococcus 
aureus was recovered in cultures from the pus thus formed. The same 
observer applied a considerable quantity of a pure culture obtained 
from this pus-third generation-to the unbroken skin of his forearm, 
rubbing it well into the skin. At the end of four days a large carbuncle, 
surrounded by isolated furuncles, developed at the point where the 
culture had been applied. This ran the usual course, and it was 
several weeks in healing. No less than seventeen scars remained to 
testify to the success of the experiment. 
Immunity.-Rabbits have been rendered immune by means of 
inoculations with either dead or living cultures. Unless the inoculations 
are carefully done the animals frequently succumb. The staphylococci 
injected into an immunized animal are more rapidly taken up by the 
leukocytes than when injected into an untreated animal. 
A serum having some protective power has also been elaborated. 
Hiss claims good results from the use of leukocyte extracts in animals 
infected with Staphylococcus aureus. 
Therapeutic Use of Vaccine.-The treatment of abscesses, boils, 
and other localized staphylococcus infections by injections of repeated 
doses of 100,000,000 to 2,000,000,000 staphylococci has given very suc- 
cessful results. Pyemias have also been treated, but with uncertain 
results. The serum has not been used with success. 
Staphylococcus (Pyogenes) Albus.-It is morphologically identi- 
cal with the Staphylococcus {pyogenes} aureus, and is probably the same 
organism which has lost the property of producing pigment. On the 
average it is somewhat less pathogenic and seldom produces pyemia 
or grave infections. The surface cultures upon nutrient agar and potato 
have a milk-white color. Its biological characters are not to be dis- 
tinguished from the Staphylococcus aureus. 
The majority of bacteriologists agree with Rosenbach, that the 
aureus is found at least twice as frequently in human pathological 
processes as the albus. 
Staphylococcus Epidermidis (Albus) (Welch).-Probably identical 
with*the Staphylococcus {pyogenes} albus. With reference to this micro- 
coccus, Welch says: "So far as our observations extend-and already 
they amount to a large number-this coccus may be regarded as 
nearly, if not quite, a constant inhabitant of the epidermis. It is now 
clear why I have proposed to call it the Staphylococcus epidermidis THE MICROCOCCUS TETRAGENUS 
247 
albus. It possesses such feeble pyogenic capacity, as is shown by its 
behavior in wounds as well as by experiments on rabbits, that the 
designation Staphylococcus pyogenes albus does not seem appropriate. 
Still, I am not inclined to insist too much upon this point, as very prob- 
ably this coccus-which has hitherto been unquestionably identified 
by others with the ordinary Staphylococcus pyogenes albus of Bosenbach 
-is an attenuated or modified form of the latter organism, although, 
as already mentioned, it presents some points of difference from the 
classical description of the white pyogenic coccus." 
According to Welch, this coccus differs from the Staphylococcus 
albus in the fact that it liquefies gelatin more slowly, does not so 
quickly cause coagulation in milk, and is far less virulent when in- 
jected into the circulation of rabbits. It has been shown by the ex- 
periments of Bossowski and of Welch that this microorganism is 
very frequently present in clean wounds, and that usually it does 
not materially interfere with the healing process, although some- 
times it appears to cause suppuration along the drainage-tube, and is 
the common cause of "stitch abscesses." 
Staphylococcus (Pyogenes) Citreus.-Isolated by Basset (1885) from 
the pus of acute abscesses, in which it is occasionally found in asso- 
ciation with other pyogenic cocci. It is distinguished from the other 
species only by the formation of a lemon-yellow pigment. 
Other Staphylococci.-Other varieties have been occasionally met 
with which differ in some respects from the typical varieties. This 
difference may be in the fact that they liquefy gelatin more slowly 
or not at all, or in pigment formation, or in agglutination, or in still 
other respects. None of these varieties are of great importance. 
The Micrococcus Tetragenus. - This 
organism was discovered by Gaffky (1881). 
It is not infrequently present in the saliva 
of healthy individuals and in the sputum 
of consumptive patients. In sputum it 
is sometimes an evidence of mouth con- 
tamination rather than lung infection. It 
has been observed repeatedly in the walls 
of cavities in pulmonary tuberculosis asso- 
ciated with other pathogenic bacteria, 
which, though playing no part in the 
etiology of the original disease, contribute, 
doubtless, to the progressive destruction 
of the lung. Its pyogenic character is 
shown by its occasional occurrence in 
the pus of acute abscesses. Its presence has also been noted in the 
pus of empyema following pneumonia. 
Morphology.-Micrococci having a diameter of about l^u, which divide 
in two planes, forming tetrads, and bound together by a transparent, 
gelatinous substance, enclosing the cell like a capsule. In cultures 
the cocci are seen in various stages of division as large, round, cells in 
Fig. 99 
Micrococcus tetragenus. 
X 1000 diameters. 248 
PATHOGENIC MICROORGANISMS 
pairs of oval elements, and in groups of three and four (Figs. 99 and 100). 
When the division is complete they remind one of sardine in appear- 
ance, except that they do not divide in three directions and are not 
built up like diminutive cotton bales (see also Plate II, Fig. 4, and 
Plate III, Fig. 11). 
Staining.-This micrococcus stains readily with the ordinary ani- 
line dyes; the transparent gelatinous envelope is only feebly stained. 
It is not decolorized by Gram's method. 
Biology.-The growth of this micrococcus is slow under all condi- 
tions. It grows both in the presence and absence of oxygen; it grows 
best from 35° to 38° C., but may be cultivated also at the ordinary 
room temperature-about 20° C. 
Growth on Gelatin.-On gelatin plates small, white colonies are 
developed in from twenty-four to forty-eight hours, which, when ex- 
amined under a low-power lens, are seen to be spherical or lemon- 
shaped, grayish-yellow disks, with a finely granular or mulberry- 
like surface, and a uniform but somewhat roughly dentated border. 
When the deep colonies push 
forward to the surface of the 
gelatin they form white, ele- 
vated, drop-like masses, hav- 
ing a diameter of 1 to 2 mm. 
In gelatin stick cultures the 
gelatin is not liquefied. 
Growth on Agar and Blood- 
serum.-The colonies appear 
as small transparent, round 
points, which have a grayish- 
yellow color and are slightly 
elevated above the surface of 
the medium. 
Pathogenesis.-Subcuta- 
neous injections of a culture 
of this micrococcus in minute 
quantity is usually fatal to 
white mice. The micrococci 
are found in comparatively 
small numbers in the blood 
of the vessels and heart, but are more numerous in the spleen, lungs, 
liver, and kidneys. Intraperitoneal injections given to guinea-pigs 
and mice are followed by purulent peritonitis, beautifully formed 
cocci in groups of four being obtained in immense numbers from the 
exudate. Rabbits and dogs are not affected by large doses of a 
culture subcutaneously or intravenously administered. 
In man it is generally non-pathogenic, except in the conditions 
already cited. It has been obtained by us as the only organism in a 
case of chronic conjunctivitis. 
The serum from immunized cases has not been used therapeutically 
in human infection. Vaccines may be employed as with staphylococci. 
Fig. 100 
Micrococcus tetragenus from peritoneal fluid. 
Stained with fuchsin. (Fraenk 1.) X 1000 
diameters. THE STREPTOCOCCI 
249 
THE STREPTOCOCCI. 
The streptococci in their relation to human infection outweigh 
in importance most other disease-producing organisms. Under this 
name must be included not only the streptococci which excite inflam- 
mation in man, but all non-motile, non-spore-bearing spherical bacteria 
which divide, as a rule, in one plane only and remain attached in longer 
or shorter chains. Owing to the variations in morphology, cultural 
characteristics and virulence of this group of bacteria it has so far been 
impossible to give absolutely satisfactory differentiation of varieties 
in the group. The relation between pathogenicity and other char- 
acteristics is not clear cut. The relation of the pneumococcus to the 
group has not been decided. The recent work of Rosenow on the 
transmutation of streptococci and pneumococci calls for conformation. 
Such a transformation has not been proved to occur in any other 
types as distinct as these have appeared to be. The first classification 
was based upon pathogenicity and morphology. The first pathogenic 
streptococci were discovered by Koch in stained sections of tissue 
attacked by septic processes, and by Ogston in the pus of acute abscesses 
(1882). Pure cultures were obtained by Fehleisen (1883) from a case 
of erysipelas. The cultural and pathological characters were studied 
by him and it was shown to be capable of producing erysipelas in man. 
Rosenbach (1884) and Krause and Passet (1885) isolated the strepto- 
coccus from the pus of acute abscesses and gave it the name of Strepto- 
coccus pyogenes. It was first thought that the streptococci of erysipelas, 
of acute abscesses, of septicemia, of puerperal fever, etc., belonged 
each to a different species, because they seemed to possess differences 
in their biological and pathological characteristics, according to the 
source from which they were obtained. But it is now thought that the 
slight differences between the majority of these streptococci are but 
acquired non-permanent variations of organisms derived from the same 
species. 
The first grouping of the pathogenic forms on biochemical char- 
acteristics was made by Schottmuller in 1903. His three types were: 
(1) Streptococcus pyogenes or erysipelatus which shows hemolysis on 
blood agar plates; (2) Streptococcus mitior or viridans, which produces 
a green halo about the colonies on blood agar plates, and (3) Strepto- 
coccus mucosus which produces a dark green zone. Park and Williams 
(1905) showed that Streptococcus mucosus should be placed with the 
pneumococci under the common name pneumococcus mucosus. Gordon 
(1903) and Andrews and Horder (1906) using nine test substances on 
over a thousand strains divided streptococci from various sources into 
nine groups. This work has been for the most part corroborated, but 
there has been much unfavorable criticism of such a classification based 
only on biochemical reactions. Serological reactions while encouraging, 
have not yet given more clear cut results. They have special importance 
as helping to indicate the proper serum to use in infections. Lyall 
recently has repeated the work with the carbohydrates and has tried 250 
PATHOGENIC MICROORGANISMS 
to show the relationship between the chractenstic carbohydrate reac- 
tion and the reactions on erythrocytes. The following table shows his 
grouping: 
Inulin 
Methemoglobin 
Indifferent 
Methemoglobin 
Bile soluble 
Salicin + 
Raffinose + 
Hemolysis 
Salicin + 
Raffinose + 
Salicin + 
Raffinose + or O 
Streptococcus 
pyogenes 
Streptococcus fecalis 
and 
other streptococci 
Pneumococcus 
and 
Pneumococcus mucosus. 
Salicin + 
Salicin O 
Streptococcus viridans. 
Lyall did not use dextrose, lactose, and saccharose in his differential 
tests, since practically all streptococci ferment these sugars. 
Streptococcus Pyogenes.-Under this heading are included all those 
streptococci that produce hemolyses and ferment dextrose, lactose, 
saccharose, and salicin. 
Fig. 101 
Fig. 102 
Streptococci in peritoneal fluid, partly 
enclosed in leukocytes. X 1000 dia- 
meters. 
Streptococcus growing in long chains 
in bouillon culture. X 1000 diameters. 
Morphology.-The cocci, when fully developed are spherical or 
oval. They have no flagella or spores. They vary from 0.4^ to l^u 
in diameter. They vary in dimensions in different cultures and 
even in different parts of a single colony. They multiply by binary 
division in one direction only, forming chains of eight, ten, twenty, 
and more elements, being, however, often associated distinctly in 
pairs. On solid media the cocci occur frequently as diplococci, but 
usually they grow in longer or shorter chains. Frequently certain 
cocci exceed their fellows greatly in size, especially in old cultures, 
when they may be considered the result of involution processes. 
Hueppe formerly called these arthrospores. Some varieties have THE STREPTOCOCCI 
251 
distinct capsules when growing in the blood and in blood-serum media 
(Hiss). 
Staining.-They stain readily by aniline colors and the pyogenic 
varieties positively by Gram's method. Some varieties, mostly 
saprophytic, growing in short chains are negative to Gram's stain. 
Biology.-Streptococci grow readily in various liquid and solid cul- 
ture media. The most favorable temperature for their development is 
from 30° to 37° C., but they multiply rather freely at ordinary room 
temperature-18° to 20° C. They are facultative anaerobes, growing 
both in the presence and absence of oxygen. 
Fig. 103 
Fig. 104 
Streptococci from solidified serum cul- 
ture appearing mostly in diplococci. 
X 1000 diameters. 
Streptococci in throat exudate 
smeared on cover-glass. X 1000 dia- 
meters. 
Cultivation.-Growth on Gelatin.-Tubes of gelatin which have been 
inoculated with streptococci by puncture with a platinum needle show 
on the surface no growth beyond the point of entrance. In the depth 
of the gelatin on the second or third day a distinct, tiny band appears, 
with granular edges or made up of granules. These granules may 
be very fine or fairly coarse. They are nearly translucent, with a 
whitish, yellowish, or brownish tinge. With characteristic cultures 
the gelatin is not liquefied. 
Growth on Agar.-On agar plates the colonies are visible after, 
twelve to thirty hours' growth at 37° C., and present a beautiful ap- 
pearance when magnified sufficiently to see the individual cocci in 
the chain. The colonies are small, not averaging over 0.5 mm. in 
diameter (pinhead). From different sources they vary in size, thick- 
ness, mottling, color, and in the appearance of their borders. The 
streptococcus growing in short chains in bouillon shows but little 
tendency to form true loops, but rather projecting rows at the edges 
of the colonies, while those growing in long chains show beautiful loops, 
which are characteristic of this organism. 
Growth on Bouillon.-Most streptococci grow well in slightly alka- 
line bouillon at 37° C., reaching their full development within thirty- 252 
PATHOGENIC MICROORGANISMS 
six to forty-eight hours. Those which grow in long chains usually 
give an abundant flocculent deposit and leave their liquid clear. The 
deposit may be in grains, in tiny flocculi, in larger flakes, or in tough, 
almost membranous masses, the differences depending on the strength 
of union between the pairs of cocci in the chains. Some of the strep- 
tococci growing in long chains, however, cause the broth to become 
cloudy. This cloudiness may be only temporary or it may be lasting. 
Those growing in short chains, as a rule, cloud the broth, this cloudiness 
remaining for days or weeks. A granular deposit appears at the 
bottom of the tube. An addition of 0.5 to 1 per cent, glucose aids the 
development of streptococci, but the acid produced tends later to hasten 
their death and make them lose virulence. A trace of calcium aids 
the growth. This is best added as a piece of marble, which has the 
additional advantage of neutralizing some of the acids produced. 
Growth in Ascitic or Serum Bouillon.-The development in this, 
which is the best medium for the growth of the streptococcus, is more 
abundant than in plain bouillon. The liquid is generally clouded, 
and a precipitate occurs after some days, the fluid gradually clearing. 
The addition of blood-serum frequently causes streptococci, growing 
in short chains in nutrient bouillon, to produce long chains. The 
reverse is also true, and in the blood all forms are usually found, 
some, at least, being diplococci or in short chains. 
Effect on Inulin.-This is not fermented by most varieties. 
Growth on Solidified Blood-serum.-This is also an excellent medium 
for the streptococcus. Tiny, grayish colonies appear twelve to eighteen 
hours after inoculation. 
Growth in Milk.-All streptococci grow well in milk. As a rule, 
when growth is luxuriant a marked production of lactic acid with 
coagulation of the casein occurs. 
Development of Hemolytic Substances.-Hemolysis is demonstrated 
within blood agar plates or in fluid containing blood in test-tubes. The 
plate method is as follows: If 1 c.c. of fresh or defibrinated blood is 
added to 6 c.c. of melted agar at 40° to 45° C., well shaken, inoculated 
with characteristic streptococci and poured in a Petri dish there will 
appear in twelve to twenty-four hours tiny colonies surrounded by 
clear zones of about | to | inch in diameter. 
The tube method is used for quantitative determination of hemolysis. 
The titration is made by adding decreasing amounts of a definite 
culture (eighteen-hour 2 per cent, peptone ascitic broth culture, accord- 
ing to Lyall), to a constant quantity of washed red-blood cells ( 1 c.c. 
of a 5 per cent, suspension of sheep's red cells, Lyall). The tubes are 
incubated in water bath at 37° C. for one hour and readings are then 
made. 
Pneumococci and many streptococci, which occur together with 
characteristic forms in the throat, lungs, and elsewhere, on the other 
hand produce only narrow zones of a green pigment. Anthony in our 
laboratory has found that from a streptococcus producing abundant 
hemolytic substances strains may be obtained, by selecting certain THE STREPTOCOCCI 
253 
colonies, which fail to make them. She has not been able to obtain 
from strains producing in first cultures the green pigment only any 
strains producing hemolytic substances. 
Duration of Life Outside of the Body.-This is not, as a rule, very 
great. When dried in blood or pus, however, they may live for several 
months at room temperature, and longer in an ice-chest, and in gelatin 
and agar cultures they live for from one week to three months. In 
order to keep streptococci alive and virulent, it is best to transplant 
them frequently and to keep them in serum or ascitic fluid bouillon 
in small sealed glass tubes in the ice-chest. 
Resistance to Heat and Chemicals.-The thermal death point of the 
streptococcus is between 52° and 54° C., the time of exposure being 
ten to twenty minutes. 
Mercuric chloride, 1 :5000; sulphate of copper, 1 : 200; trichloride 
of iodine, 1 :750; peroxide of hydrogen, 1 : 50; carbolic acid, 1 :100; 
cresol, 1 :250; lysol, 1 :300; creolin, 1 :130, all kill streptococci within 
a few minutes. 
Pathogenesis.-The majority of test animals are not very sus- 
ceptible to infection by' the streptococcus, and hence it is difficult to 
obtain any definite pathological alterations in their tissues through 
the inoculation into them of cultures of this organism by any of the 
methods ordinarily practised. White mice and rabbits, under simi- 
lar conditions, are the most susceptible, and these animals are, there- 
fore, usually employed for experimentation. Streptococci, how- 
ever, differ greatly in the effects which they produce in inoculated 
animals, according to their animal virulence, which is very different 
from human virulence. The most virulent when injected in the 
minutest quantity into the circulation or into the subcutaneous tissues 
of a mouse or rabbit, produce death by septicemia. Those of somewhat 
less virulence produce the same result when injected in considerable 
quantities. Those still less pathogenic produce septicemia, which is 
mild or severe, when injected into the circulation; but when injected 
subcutaneously, they produce abscess or erysipelas. The remaining 
streptococci, unless introduced in quantities of 20 c.c. or over, produce 
only a slight redness, or no reaction at all, when injected subcutaneously, 
and little or no effect when injected directly into the circulation. Many 
of the streptococci obtained from cases of cellulitis, abscess, empyema, 
and septicemia belong to this group. 
A number of varieties of streptococci have thus been discovered, 
differing in virulence and in their growth on artificial media; but all 
attempts to separate them into various classes, even with the use of 
specific serum, have largely failed, because the differences observed, 
though often marked, are not constant, many varieties having been 
found to lose their distinctive characteristics, and even to apparently 
change from one class to another. A further objection to any of the 
existing classifications of streptococci, which are based on the manner 
of growth on artificial culture media, is that it has been impossible 
to make any which would at the same time give even an approximate 254 
PATHOGENIC MICROORGANISMS 
idea of their virulence. Experiments have proved that streptococci 
originally virulent may become non-virulent after long cultivation on 
artificial media, and, again, that they may return to their original 
properties after being passed through the bodies of susceptible animals. 
The peculiar type of virulence which they may acquire tends to perpet- 
uate itself, at least for a considerable time. 
One important fact that experience teaches us is that those strepto- 
cocci which are the most dangerous are those which have come imme- 
diately from septic conditions, and the more virulent the case the 
more virulent the streptococci are apt to be for animals of the same 
species. There seems also to be a strong tendency for a streptococcus 
to produce the same inflammation, when inoculated, as the one from 
which it was obtained; for example, streptococci from erysipelas tend 
to produce erysipelas, from septicemia to produce septicemia, etc. 
Streptococci, however, obtained from different sources (abscesses, 
puerperal fever, erysipelas, etc.) are in many instances capable, under 
favorable conditions, of producing erysipelas when inoculated into the 
ear of a rabbit, as has been proved by experiment, provided they possess 
sufficient virulence. 
Occurrence in Man.-Streptococci have been found to be the primary 
cause of infection in the following diseases: Erysipelas, circumscribed 
and extensive acute abscesses, impetigo, cellulitis (circumscribed as well 
as diffused), sepsis, puerperal infection, acute peritonitis, angina, 
bronchopneumonia, periostitis, osteomyelitis, synovitis, otitis media, 
mastoiditis, enteritis, irregular cases of rheumatic fever, meningitis, 
pleurisy, empyema, and endocarditis. Associated with other bacteria 
in diseases of which they were the specific cause, they have also been 
found as the secondary infection in many diseases, such as in pulmonary 
tuberculosis, bronchopneumonia, septic diphtheria, and diphtheritic 
scarlatina. 
In cases of septic thrombus of the lateral sinus following mastoiditis 
there is almost certainly a streptococcus septicemia. Libman has shown 
that an examination of the blood may be useful in diagnosis. 
In diphtheritic false membranes this micrococcus is very commonly 
present, and is frequently the source of deeper infection, such as abscesses 
and septicemia; and in certain cases attended with a diphtheritic 
exudation, in which the Loffler bacillus has not been found by competent 
bacteriologists, it seems probable that the Streptococcus pyogenes, 
alone or with other pyogenic cocci, is responsible for the local inflamma- 
tion and its results. These forms of so-called diphtheria, as first pointed 
out by Prudden, are most commonly associated with scarlatina and 
measles, erysipelas, and phlegmonous inflammation, or occur in indi- 
viduals exposed to these or other infectious diseases. So uniformly 
are long-chained streptococci present in the pseudomembranes of patients 
sick with scarlet fever, that many investigators have suspected a special 
variety of them to be the cause of this disease. The same is true for 
smallpox. Many varieties are regularly found, however, in the throat 
secretion of healthy individuals (in 100 examinations by us we found THE STREPTOCOCCI 
255 
long-chained streptococci in 83, and probably could have found them 
in some of the others by longer search). Their abundance in scarlet 
fever and smallpox is most probably due to their increase in the injured 
mucous membrane and entrance into the circulation when the protective 
properties of the blood have been lowered. 
Septic Sore Throat--Streptococcic infection of the throat appears 
at times as a severe epidemic. Most of these epidemics have been 
traced to the milk supply. The streptococci may come either from a 
septic human throat or from the septic udder of a cow. The local 
infection is usually accompanied by severe general symptoms and by 
suppurative foci elsewhere. 
Occurrence in Animals.-Besides streptococci similar to those in 
man, animals are infected by strains that are negative to Gram and 
fluidify gelatin. Udder infections of the cow and glandular diseases 
of the horse are frequently due to these. The streptococcic inflamma- 
tions in animals are almost as frequent and serious as they are in man. 
Effect on Tumors.-Fehleisen inoculated cultures, obtained in the 
first instance from the skin of patients with erysipelas, into patients 
in the hospital suffering from inoperable malignant growths-lupus, 
carcinoma, and sarcoma-and he obtained positive results, a typical 
erysipelatous inflammation having developed around the point of 
inoculation after a period of incubation of from fifteen to sixty hours. 
This was attended with chilly sensations and an elevation of tempera- 
ture. Persons who had recently recovered from an attack of erysipelas 
frequently proved to be immune. These experiments were undertaken 
on the ground that malignant tumors had previously been found to 
improve or entirely disappear in persons who had recovered from 
accidental erysipelas. This fact was therapeutically applied to the 
treatment of malignant tumors. Then the mixed toxins of the strepto- 
coccus and B. prodigiosus were given, and it became apparent that 
the toxins of the latter organism were much the more important. 
Results from Injections in Tumors.-In some cases of sarcoma this 
method met with considerable success. Coley in this country makes 
the following statement: 
"The toxins injected subcutaneously into the tissues, either into 
the tumor substance or into parts remote from the tumor, exercise a 
distinctly inhibitory action upon the growth of nearly all varieties 
of sarcoma. This action is the least marked in melanotic sarcoma, 
and thus far no cases of this form of tumor have disappeared under 
the treatment. The influence of the toxins upon round-cell sarcoma 
is much more powerful than it is upon melanotic, although distinctly 
less than upon the spindle-celled variety. A number of cases of round- 
celled sarcoma in which the diagnosis was questioned disappeared, and 
the patients have remained well beyond three years. Nearly half of 
the cases treated show no appreciable decrease in size; the majority of the 
others which did show marked improvement at first, after decreasing 
in size for a few weeks, again began to increase and were no longer 
influenced by the treatment. 256 
PATHOGENIC MICROORGANISMS 
"I have now a number of cases of spindle-celled sarcoma which 
have remained well beyond three years; one case of mixed (round and 
spindle) celled, after remaining well three years, had a return in the 
abdomen, and died about eight months later. The result here certainly 
establishes the correctness of the early diagnosis. 
" It should be distinctly stated that all of the tumors under con- 
sideration were inoperable, as I have never advised treatment 
except in such cases." 
Some surgeons have not had nearly as favorable results as Coley. 
There seems to be no question about good results having been obtained 
in a small percentage of cases. The injections cause fever and loss 
of flesh and strength. 
Production of Toxic Substances.-There is no doubt that the strepto- 
coccus causes fever, general symptoms of intoxication, and death by 
means of toxic substances which it forms in its growth; but we know 
very little about these substances or how they are produced. The 
cell substance of streptococci possesses only slight toxicity. Ruediger1 
has shown that a specific streptolysin is formed which produces a true 
antibody. The poisons while partly extracellular are mostly contained 
in the cell substance. Heat destroys a portion of them. They appear 
to attack especially the red-blood cells, and this hemolytic action seems 
to be to some degree in proportion to the virulence of the organism. 
Susceptibility to Streptococcus Infection.-As with the ever-present 
staphylococci, whose virulence, as we have seen, is usually slight, 
the streptococci are more likely to invade the tissues, forming abscesses 
or erysipelatous and phlegmonous inflammation in man when the 
standard of health is reduced from any cause, and especially when 
by absorption or retention various toxic organic products are present 
in the body in excess. It is thus that the liability to these local infec- 
tions, as complications of operations or sequelae of various specific 
infectious diseases, in the victims of chronic alcoholism, and consti- 
tutional affections, etc., are to be explained. It seems established 
that the absorption of toxic products formed in the alimentary canal 
as a result of the ingestion of improper food, or in consequence of 
abnormal fermentative changes in the contents of the intestine, or 
from constipation predispose to infection. 
Immunity. - In none of the streptococcus inflammations do we 
notice much apparent tendency to the production of immunizing and 
curative substances in the blood by a single infection. 
Several general infections usually progress to a fatal termination 
after a few days, weeks, or months. It is true, however, that cases 
of erysipelas, cellulitis, and abscess, after periods varying from a few 
days to months, tend to recover, and to a certain extent, therefore, we 
may assume that protective agents have been produced. In these 
cases, however, we know from experience that faulty treatment, by 
lessening the local or general resistance, would, as a rule, cause the 
1 Jour. Amer. Med. Assoc., 1903, xli, 962. THE STREPTOCOCCI 
257 
subsiding infection again to progress perhaps even to a more serious 
extent than the original attack. Koch and Petruschky tried a most 
interesting experiment. They inoculated cutaneously a man suffering 
from a malignant tumor with a streptococcus obtained from erysipelas. 
He developed a moderately severe attack, which lasted about ten days. 
On its subsidence they reinoculated him; a new attack developed, 
which ran the same course and over the same area. This was repeated 
ten times with the same results. 
This experiment proved that in this case, at least, the immunizing 
substances produced by repeated attacks of erysipelas were insufficient 
to make the tissues and lymph sufficiently bactericidal to prevent 
infection. 
The severe forms of infection, such as septicemia following injuries, 
operations, and puerperal infections, show little tendency to be arrested 
after being well established. Having, then, in remembrance, the above 
facts, let us consider the results already obtained in the experimental 
immunization and treatment of animals and men suffering from or in 
danger of infection with streptococci. Knorr succeeded in producing 
a moderate immunity in rabbits against an intensely virulent strepto- 
coccus by injections of very slightly virulent cultures. Marmorek 
was the first to attempt the production of a curative serum on a large 
scale. 
Influence of Serum from Immunized Animals upon Streptococcus 
Infections in Other Animals.-In the table are given the results fol- 
lowing the injection of small amounts of a serum which represents 
in immunizing value what about' one-third of the horses are able to 
produce when given in gradually increasing doses the living, virulent 
streptococcus. In the following experiments the serum and culture 
were injected subcutaneously in rabbits at the same time, but in oppo- 
site side of the body: 
Showing Strength of Average Grade of Antistreptococcic Serum given 
by Selected Horses after Six Months of Injection of Suitable 
Amounts of Living Streptococci. 
Weight of 
rabbit. 
1. Inoculated together . . . 1430 
2. Inoculated together . . . 1350 
3. On opposite sides . . . 1770 
4. On opposite sides . . . 1630 
Controls: 
1. Rabbits iniected with culture 1750 
Amounts 
inoculated. 
0.25 c.c. 0.01 c.c. 
0.125 c.c. 0.01 c.c. 
0.1 c.c. 0.01 c.c. 
0.1 c.c. 0.01 c.c. 
0.001 c.c. 
Results. 
Lived 
Lived 
Lived 
Lived 
Died in 
Autopsy. 
Streptococc 
infection. 
Streptococcic 
infection. 
2. 
only. 
Rabbits injected with culture 
1870 
0.001 c.c. 
4 days 
Died in 
only. 
24 hrs. 
The above results have been repeatedly obtained, and are abso- 
lutely conclusive that the serum of properly selected animals, which 
have been repeatedly injected with living streptococci in suitable 
doses possesses bactericidal properties upon the same streptococcus 
when it comes in contact with it within the bodies of animals. 
17 258 
PATHOGENIC MICROORGANISMS 
Definite protection from the serum has been obtained by many 
reliable observers since Marmorek's first reports. 
Polyvalent Serum.-Results of investigators show that the majority 
but not all streptococci met with in cellulitis, erysipelas, and abscess 
will be influenced by the same serum. Those obtained from cases of 
pneumonia and endocarditis and other exceptional infections are apt 
to have individual characteristics. In order that the serum may have 
specific antibodies for the variety of streptococci causing each separate 
infection, each horse is now injected with a large number of different 
varieties of streptococci. This serum will not be quite as efficient as if 
made by the streptococcus infecting the treated case, but will be fairly 
efficient for all cases. 
Dosage.-It is found that the immune bodies to be effective must 
be in sufficient concentration. The intravenous injections are usually 
100 c.c. for an adult. 
Preparation of the Serum.-Antistreptococcus serum is obtained 
from the horse after treatment by repeated injections of living or 
dead streptococcus cultures derived from human sources. As a rule, 
a number of varieties are given at the same time so that the serum 
will be active against any variety causing the infection. If the serum 
is to be used in scarlet fever, the streptococci used should be from 
cases of scarlet fever. The procuring of a serum of the highest potency 
requires a considerable number of animals, for some produce with the 
same treatment a more protective serum than others. The serum must 
be sterile from streptococcus as well as from other contaminations. 
Stability of the Serum.-It is fairly stable but, after several months, 
the serum loses some of its protective value. It should be kept in a 
cool and dark place. 
Standardization of the Value of the Serum.-There is at present 
no satisfactory way. The value of the serum is sometimes measured 
by the amount required to protect against a multiple of a fatal dose 
of a very virulent streptococcus of the same type as the one used to 
inject the horses. The dose is usually a thousand times the average 
fatal amount of a very virulent streptococcus. 
Other methods of standarization, such as the estimation of the 
amount of opsonins or agglutinins present, are also used. 
Therapeutic Results.-To estimate the exact present and future 
value of antistreptococcus serum is a matter of the utmost difficulty. 
Many of the cases reported are of little or no help, because, no cultures 
having been made, we are in doubt as to the nature of the bacterial 
infection. 
In the cases of puerperal fever, erysipelas, and wound infection 
that we have seen, the apparent results under the treatment have not 
been uniform. We have frequently observed favorable results which 
appeared to be due to the serum when doses of 50 to 100 c.c. were given 
intravenously. 
In a number of cases of septicemia where for days chills had occurred 
daily they ceased absolutely or lessened under daily doses of 20 to THE STREPTOCOCCI 
259 
50 c.c. The temperature, though ceasing to rise to such heights, did 
not average more than one or two degrees lower than before the injec- 
tions. In some cases the serum treatment was kept up for four weeks. 
Some cases convalesced; others after a week or more grew worse and 
died. In some cases the temperature fell immediately upon giving 
the first injection of serum, and after subsequent injections remained 
normal, and the cases seemed greatly benefited. As a rule, in these 
cases no streptococci or any other organisms were obtained from the 
blood. In bronchopneumonia, laryngeal diphtheria, tonsillitis, smallpox, 
and phthisis, we have seen little effect. 
The results obtained here in New York by both physicians and sur- 
geons have not, on the whole, been very encouraging. 
In some of the cases where apparently favorable results were obtained 
other bacteria than streptococci were found to be the cause of the 
disease. We believe that the following conclusions will be found fairly 
accurate: 
The serum will in animals limit an infection already started if it 
has not progressed too far. The apparent therapeutic results in cases 
of human streptococcus infection are variable. In some cases the 
disease has undoubtedly advanced in spite of large injections, and here 
it has not seemed to have had any effect. In other cases good observers 
rightly or wrongly believe they have noticed great improvement from 
it. Except rashes, few have noticed deleterious results, although very 
large doses have been followed in several instances, for a short time, 
by albuminous urine. 
In suitable cases we are warranted, we believe, in trying it, but we 
should not expect very striking results. 
For our own satisfaction, and to increase our knowledge, we should 
always have satisfactory cultures made when possible, and the strep- 
tococci, if obtained, tested with the serum used in the treatment. In 
the cases where we want most to use the serum, such as puerperal fever, 
septicemia, ulcerative endocarditis, etc., we find that it is very difficult 
to make a bacteriological diagnosis from the symptoms, and in over 
one-half of the cases even the bacteriological examination carried out 
in the most thorough way will fail to detect the special variety of bacteria 
causing the infection. This is often a great hindrance to the proper 
use of curative antistreptococcic serum, for it, of course, has no specific 
effect upon the course of any infection except that due to the strepto- 
coccus and the full effect only on its own type. 
Care should be taken to get the most reliable serum; much on the 
market is worthless, and as it is weak, and the testing for strength is 
difficult or impossible, full doses (50 to 100 c.c.) of serum should be given 
if the case is at all serious, for the dose is limited only by the amount 
of horse serum which we feel it safe to give, not because we have given 
sufficient protective substance. Intravenous injections give better 
results than those given subcutaneously. Studdiford has obtained 
good results by adding to the intravenous injection the packing of the 
septic uterus with gauze impregnated with the serum. 260 
PATHOGENIC MICROORGANISMS 
Scarlet Fever.-In Vienna for some years the serum of horses treated 
at each injection with a number of strains of streptococci derived from 
scarlet fever cases has been used in this disease. The serum given in 
large doses of 100 to 200 c.c. has apparently given good results in about 
half of those treated. It is only used in severe cases. Moser has chiefly 
advocated its use. One of us had the opportunity to look over the 
histories of his cases. Although left in doubt as to its value, it appears 
to us as worth a trial. Our own results and Nicoll's, in cases occurring 
in the Health Department hospitals, have been rather favorable. 
Streptococcus Vaccine.-The use of streptococcus vaccines as a 
preventive of scarlet fever was originated by Gabritchewsky and has 
been practised on an extensive scale largely in Russia with apparently 
favorable results. Jesjakoff in a study of 15,000 vaccinated cases con- 
cludes that three inoculations extending over a period of two weeks 
protect against the disease. In this country and elsewhere small series 
of cases reported from time to time tend to confirm his observations 
and the continuance of the procedure with the hope that it may be of 
value in lessening the susceptibility to scarlet fever of persons constantly 
exposed to it. 
Complement Fixation.-The method and results of this test have been 
considered in Part I, p. 190. 
Bacteriological Diagnosis.-Streptococci, using the name in a broad 
sense, can often be demonstrated microscopically by simply making a 
smear preparation of the suspected material and staining with methy- 
lene-blue solution or diluted Ziehl's fluid. In order to demonstrate 
them microscopically in the tissues, the sections are best stained by 
Kiihne's methylene-blue method. In all cases, even when the micro- 
scopic examination fails, the cocci may be found by the use of culture 
media, such as broth or plated agar at 37° C. To obtain them from a 
case of erysipelas it is best to excise a small piece of skin from the margin 
of the erysipelatous area in which the cocci are most numerous; this is 
crushed up and part of it transferred to ascitic or serum bouillon, and 
part is streaked across freshly solidified agar in a Petri dish on which 
a drop of sterile rabbit's blood had previously been placed. Both are 
kept in the incubator at 37° C. 
In septicemia the culture method is always required to demonstrate 
the presence of streptococci, as the microscopic examination of specimens 
of blood is not sufficient. For this purpose from 10 to 15 c.c. of the blood 
should be drawn from the vein of the arm aseptically by means of a 
hypodermic needle, and to each of three tubes containing 10 c.c. of 
melted nutrient agar kept at about 43° C. 1 c.c. of blood is added. After 
thoroughly mixing the contents are poured into Petri dishes. The 
remainder is added to several flasks containing 100 c.c. of nutrient 
broth, in order to produce a development of the cocci, which are found 
in small numbers in the blood. Petruschky is of the opinion that the 
cocci can be best shown in blood by animal inoculation. Having with- 
drawn from the patient 10 c.c. of blood by means of a hypodermic 
syringe, under aseptic precautions, he injects a portion of this into the THE STREPTOCOCCI 
261 
abdominal cavity of a mouse, while the other portion is planted in 
bouillon. Mice thus inoculated die from septicemia when virulent 
streptococci are present in only very small numbers in the blood. If 
a successful inoculation takes place we can, through the absence or 
presence of the development of capsules, often differentiate between 
the pneumococcus and the streptococcus, which cultures may fail to 
do. The development of a wide, clear zone about the colonies (upon 
blood-agar), without a development of green pigment, indicates that 
the streptococci belong to the pyogenes type. The absence of a definite 
zone and the development of a green color indicates that they are pneu- 
mococci, or streptococci which in these two respects resemble pneu- 
mococci. The growth in the Hiss inulin serum medium will generally 
differentiate between the two, as the pneumococci usually coagulate 
the serum, while the great majority of streptococci do not. The mor- 
phological and cultural characteristics of the streptococcus give us, 
unfortunately, no absolute knowledge as to the influence which the 
protecting serum will have. The actual test is here our only method. 
The detection of the streptococcus in the blood is in itself an unfavorable 
prognostic sign. 
The blood-cultures in many cases of supposed septicemia give no 
results, for many of these cases develop their symptoms and even die 
from the absorption of toxins from the local infection, such as an 
amputation wound or an infected uterus or peritoneum, and the bac- 
teria never invade the blood. When we get negative results we are, 
as a rule, utterly unable to test, the case with curative serums with 
any accuracy, for the sepsis may be due to either the streptococcus, 
colon bacillus, staphylococcus, or a number of other pathogenic varieties 
of bacteria. CHAP.TER XVII . 
THE DIPLOCOCCUS OF PNEUMONIA (PNEUMOCOCCUS, 
STREPTOCOCCUS PNEUMONIAE, MICROCOCCUS 
LANCEOLATUS). 
THE DIPLOCOCCUS OF PNEUMONIA. 
The diplococcus of pneumonia was observed in 1880 almost simul- 
taneously by Sternberg and Pasteur in the blood of rabbits inoculated 
with human saliva. In the next few years Talamon, Friedlander, 
A. Frankel, Weichselbaum, and others subjected this microorganism 
to an extended series of investigations and proved it to be the chief 
etiological factor in the production of lobar or croupous pneumonia 
in man. The relationship of this organism to the streptococcus group 
is spoken of in the preceding chapter. 
The outcome of the various investigations proved that the acute 
lung inflammations, especially when not of the frank lobar pneumonia 
type, are not excited by a single variety of microorganism, and that 
the bacteria involved in the production of pneumonias are also met 
with in inflammations of other tissues. 
In any individual pneumonic inflammation it is also found that 
more than one variety of bacteria may be active, either from the start 
or as a later addition to the original primary infection. 
Among all the microorganisms active in exciting pneumonia, the 
diplococcus of pneumonia is by far the most common, being almost 
always present in primary lobar pneumonia and as frequently as any 
other germ in acute bronchopneumonia and metastatic forms. Besides 
the different varieties of pneumococci the following bacteria are capable 
of exciting pneumonia: Streptococcus pyogenes, Staphylococcus pyogenes, 
Bacillus pneumoniae, Bacillus influenzae, Bacillus pestis, Bacillus diph- 
therias, Bacillus typhi, Bacillus coli, and the Bacillus tuberculosis. Since 
the varieties of bacteria exciting acute pneumonia, with the exception 
of the pneumococcus, are met with more frequently in other inflamma- 
tions and have been described elsewhere, they will only be noticed in 
this chapter so far as their relation to pneumonia demand. 
Morphology.-Typically, the pneumococcus occurs as spherical or 
oval cocci, usually united in pairs, but sometimes in longer or shorter 
chains consisting of from three to six or more elements and resembling 
the streptococcus. The cells, as they commonly occur in pairs, are 
somewhat oval in shape, being usually pointed at one end-hence 
the name lanceolatus or lancet-shaped. When thus united the junc- 
tion, as a rule, is between the broad ends of the oval, with the pointed THE DIPLOCOCCUS OF PNEUMONIA 
263 
ends turned outward; but variation in form and arrangement of the 
cells is characteristic of this organism, there being great differences 
according to the source from which it is obtained. As observed in the 
sputum and blood it is usually in pairs of lancet-shaped elements, which 
are surrounded by a capsule. (See Fig. 105.) When grown in fluid 
culture media longer or shorter chains are frequently formed, which 
can scarcely be distinguished from chains of certain streptococci, 
except that, as a rule, the length of the chain is less and the pairs of 
diplococci are farther apart. In cultures the individual cells are almost 
spherical in shape, and except in certain varieties are rarely surrounded 
by a capsule. (See Fig. 106.) The pneumococcus is by some classed 
as a streptococcus. Rosenow claims that a typical pneumococcus may 
be easily changed into a typical streptococcus. (See preceding chapter.) 
Fig. 105 
Diplococcus of pneumonia from blood, with surrounding capsule stained by method of Hiss. 
The capsule is best seen in stained preparations from the blood and 
exudates of fibrinous pneumonia or from the blood of an inoculated 
animal, especially the mouse, in which it is commonly, though not 
always, present. It is seldom seen in preparations from cultures (except 
in pneumococcus mucosus) unless special media are employed. Flagella 
are not present. 
Staining.-It stains readily with ordinary aniline colors; it is not 
decolorized after staining by Gram's method. The capsule may be 
demonstrated in blood or sputum either by Gram's or Welch's (glacial 
acetic acid) method, or the copper sulphate method of Hiss. 
Biology.-It grows equally well with or without oxygen; its parastic 
nature is exhibited by the short range of temperature at which it usually 
grows-viz., from 25° to 42° C.-best at 37° C. In the cultivation of 
this organism neutral or slightly alkaline media should be employed. 264 
PATHOGENIC MICROORGANISMS 
The organism when freshly isolated grows feebly on the serum-free 
culture media ordinarily employed for the cultivation of bacteria-viz., 
on nutrient agar and gelatin, in bouillon. The best medium for its 
growth is a mixture of one-third human or animal blood serum or 
ascitic or pleuritic fluid and two-thirds bouillon, or nutrient agar 
streaked with human, horse, or rabbit blood. 
Growth on Agar.-Cultivated on plain nutrient agar, after twenty- 
four to forty-eight hours at 37° C., the deep colonies are hardly visible 
to the eye. Under the microscope they appear light yellow or brown 
in color and finely granular. The surface colonies are larger, equalling 
in size those of streptococci, but are usually more transparent. If 
blood serum or ascitic fluid be added to the agar the individual colonies 
are larger and closer together, and the growth is more distinct in con- 
sequence and of a grayish color. The surface colonies are almost circu- 
lar in shape under a magnification of 60 diameters, finely granular in 
structure, and may have a somewhat darker, more compact centre, 
surrounded by a paler marginal zone. With high magnification cocci 
in twos and short rows often distinctly separated are seen at the edges. 
Fig. 106 
Fig. 107 
Pneumococcus from bouillon culture, resem- 
bling streptococcus. 
Pneumococci in peritoneal pus. Stained 
with fuchsin. X 1000 diameters. Clear 
spaces indicate capsules. 
Growth on Blood Agar.-The colonies on blood agar are greenish and 
no hemolysis is present. This is a useful characteristic in isolating 
pneumococci from sputum, although many streptococci give similar 
colonies. 
Growth on Blood Serum.-The growth on Loffler's blood-serum 
mixture is very similar to that on agar, but somewhat more vigorous 
and characteristic, appearing on the surface as a delicate layer of dew- 
like drops. 
Growth in Bouillon.-In bouillon, at the end of twelve to twenty-four hours 
in the incubator, a slight cloudiness of the liquid will be found to have been THE DIPLOCOCCUS OF PNEUMONIA 
265 
produced. On microscopic examination cocci can be seen to be arranged ill 
pairs or longer or shorter chains. After one or two transplantations the pneu- 
mococci frequently fail to grow. 
Growth in Milk.-It grows readily in milk, causing coagulation with the 
production of acid, though coagulation is not constant with some forms inter- 
mediate between the streptococcus and pneumococcus. 
Growth on Gelatin.-The growth on gelatin is slow, if there is any develop- 
ment at all, owing to the low temperature-viz., 24° to 27° C.-above which 
even the most heat-resistant gelatin will melt. The gelatin is not liquefied. 
Special Media.-When cultures are grown on serum-free media the vitality 
of some cultures may indeed be indefinitely prolonged; but after transplanta- 
tion through several generations it is found that the cultures begin to lose in 
virulence, and that they finally become non-virulent. In order to restore this 
virulence, or to keep it from becoming attenuated, it is necessary to interrupt 
the transplantation and pass the organism through the bodies of susceptible 
animals. 
The vitality is prolonged and the virulence less rapidly lost if serum ascitic 
fluid or blood is present in the medium. Serum or ascitic broth, serum semi- 
solid (stab cultures) or blood-streaked agar are the most satisfactory media for 
the preservation of cultures. 
Action of Bile and Bile Salts.-If 0.1 c.c. of rabbits' bile be added to 1 or 2 c.c. 
of a broth culture of pneumococci the culture becomes clear and the cocci 
become dissolved. A 10 per cent, solution of sodium taurocholate has the same 
action. Streptococci are not dissolved by bile. Serum and glucose interfere 
with the reaction. 
Hiss Serum Water with and without Inulin.-These are very useful. 
The inulin is fermented by typical pneumococci with coagulation of 
the serum, while most streptococci fail to ferment the inulin. This 
medium is, therefore, of considerable diagnostic value. 
Calcium Broth with or without Dextrose.-This medium has proven 
of great value for the propagation of cultures where agglutination 
tests are to be carried out. The addition of a small piece of marble 
to each tube of broth is the most satisfactory way of preparing it. 
Marble broth for this purpose was suggested independently by Bolduan 
and Hiss. 
Resistance to Light, Drying, and Germicidal Agents.-On artificial 
culture media the pneumococci tend to die rapidly. This is partially 
due to the acid produced by their growth. In sputum they live much 
longer. 
Pneumonic sputum attached in masses to clothes, when dried in the 
air and exposed to diffuse daylight, retains its virulence, as shown by 
injection in rabbits, for a period of nineteen to fifty-five days. Exposed 
to direct sunlight the same material retains its virulence after but a 
few hours' exposure. This retention of virulence for so long a time 
under these circumstances is accounted for by the protective influence 
afforded by the dried mucoid material in which the micrococci were 
embedded. Guarnieri observed that the blood of inoculated animals, 
when rapidly dried in a desiccator, retained its virulence for months; 
and Foa found that fresh rabbit blood, after inoculation and cultivation 
in the incubator for twenty-four hours, when removed at once to a 
cool, dark place, retained its virulence for sixty days. There are many 
conditions, therefore, in which the virulence of the micrococcus is 266 
PATHOGENIC MICROORGANISMS 
retained for a considerable length of time. To germicidal agents pneu- 
mococci are very sensitive. The fine spray expelled in coughing and 
loud speaking that remains suspended in the air soon dries so com- 
pletely that probably no pneumococci survive after two hours. 
Attenuation of Virulence.-This may be produced in various ways. 
The loss of virulence which occurs when the micrococcus is trans- 
planted through several generations in culture fluid containing no 
blood has already been referred to. An attenuation of virulence, it has 
been claimed, takes place also spontaneously in the course of pneumonia. 
This attenuation is probably only apparent. If a little sputum be taken 
at different periods in the disease and planted in ascitic bouillon the 
resultant cultures do not vary greatly in virulence. The average 
virulence for rabbits of cultures made from pneumonic sputum is greater 
than in those from normal sputum. 
Restoration and Increase of Virulence.-The simplest and perhaps 
the most reliable method of restoring lost virulence for any susceptible 
animal is by passage through the bodies of highly susceptible animals 
of the same species. Growth in fresh blood also increases it for the 
homologous animal. 
Maintenance of Virulence -This is best done by drying the spleen 
of an infected animal in a dessicator. The spleen should be removed 
just before or immediately after death. The virulence is preserved in 
this way for a month or longer. 
Toxin Production.-We have little exact knowledge upon the nature 
of the substances produced by or through the growth of the pneumo- 
cocci in animal tissues or artificial media. Rosenow showed that 
the autolysis of virulent pneumococci in NaCl solution brings into the 
solution a group of substances which inhibits the action of the pneumo- 
cocco-opsonin. Such extracts are also toxic for animals. 
Occurrence in Man during Health.-It is probable that in crowded 
communities the pneumococcus is present on the mucous membranes 
of most persons. We have found it generally present not only in the 
throats of persons living in New York City, but also in those of persons 
living on farms and in the Adirondack Mountains. It is commonly 
present only on the mucous membranes of the bronchi, trachea, pharynx, 
and nostrils. The healthy lung seems to be generally free from it. 
Judging from animal tests it is very possible that the virulence for man 
of the organisms present in health is much less than the virulence of 
those in a pneumonic lung. 
Pathogenicity in Man.-Characteristic or atypical pneumococci are 
present in fully 95 per cent, of characteristic cases of lobar pneumonia. 
Usually no other bacteria are obtained from the lungs. Atypical cases 
usually show the same conditions, but they may be due to strepto- 
cocci; influenza bacilli, etc. The more recent the infection the greater 
is the number of bacteria found in the diseased lung area. As the dis- 
ease progresses these decrease in number until finally at the crisis they 
disappear from the tissues, though at this time and long after convales- 
cence they may be present in the sputum. In atypical forms of pneu- THE DIPLOCOCCUS OF PNEUMONIA 
267 
monia they may remain longer in the tissues, and in walking pneumonia 
they may be absent in the original centres of infection or present only 
as attenuated varieties, while the surrounding, newly formed foci may 
contain fully virulent cocci. It has been shown by Nctter that more 
than one-half of the cases of bronchopneumonia, whether primary or 
secondary to some other disease, as measles and diphtheria, both in 
children and adults, are due to the diplococcus of pneumonia. Others, 
such as Pearce, have found other microorganisms, expecially the strep- 
tococci, in the majority of cases. These findings will be considered at 
the end of the chapter. 
The pneumococci are found partly in the alveoli and bronchioles 
of the inflamed lung and partly in the lymph channels and blood-capil- 
laries. Most of the organisms are found free, but a few are found in 
the leukocytes. Through the lymph channels they find their way to 
the pleura and to adjacent lymph glands. From the capillaries they 
find their way to the general blood-current, and thus to distant parts 
of the body. In about 20 per cent, of cases the pneumococci are so 
abundant that they can be found in cultures made from 5 to 10 c.c. of 
blood. In a number of instances the fetus has been found infected. 
The pneumococci are also responsible for: 
Inflammations Complicating Pneumonia.-In every case of lobar 
pneumonia and in most cases of bronchopneumonia pleurisy is developed, 
which is excited by the same microorganism that was predominant 
in the pneumonia. With pneumococci the exudate is usually moderate 
and of a fibrinous character, but may be more abundant and of a sero- 
fibrinous or purulent character. When the pleurisy is marked it is 
more apt to continue after the cessation of the pneumonia. Pleurisy 
due to pneumococci is more apt to go on to spontaneous recovery than 
that due to streptococci or staphylococci. 
The most frequent pneumococcic infections next to pleurisy, fol- 
lowing a pneumonia, are those of the middle ear, pericardium, endo- 
cardium, and meninges, and these not infrequently arise together. 
Pneumococcic inflammations of the heart valves are apt to be fol- 
lowed by extensive necrosis and growth of vegetations. In these cases 
pneumococci can sometimes be found in the blood for many weeks. 
Pericarditis due to pneumococci is a frequent complication, but is 
usually very slightly developed. Meningitis due to pneumococci may 
be either fibrinous or purulent or both and is apt to be secondary to 
otitis, mastoiditis, or pneumonia. Arthritis, periarthritis, and osteo- 
myelitis are rarer complications of a pneumococcic pneumonia. Besides 
moderate parenchymatous inflammation of the kidney, which occurs 
in most cases of pneumonia, well-marked inflammation may occur in 
which pneumococci exist in the kidney tissues in large numbers. 
How is the pneumococcus conveyed from its original seat in the 
lungs to distant internal organs? Chiefly by means of the blood- 
vessels and lymphatics, in both of which it has been found in great 
numbers. Their presence in the blood after death has been amply 
proved by numerous investigations. In many instances, they have 268 
PATHOGENIC MICROORGANISMS 
been recovered from the blood during life. Lambert, as a rule, 
found them in all fatal cases twenty-four to forty-eight hours 
before death. The conveyance of the infective agent by means 
of the blood and the lymph to all parts of the body, explains the 
multiplicity of the affections complicating a pneumonia, which are 
caused by this micrococcus; and not only the secondary, but also 
the primary diseases, as of the brain and meninges, may be explained 
in the same way. Knowing that the saliva and nasal secretions under 
normal conditions so frequently afford a resting place for the micro- 
cocci, we have only to assume the production of a suitable culture 
medium for these parasites in the body, brought about by an abnormal 
condition of the mucous membranes from exposure to cold, or a reduc- 
tion of the vital resisting power of the tissue cells in any of the internal 
organs, caused by disease, traumatism, excess of various kinds, etc., 
to comprehend readily how an individual may become infected with 
pneumococci, either primarily affecting the lungs and secondarily other 
organs in the body, or primarily attacking the middle ear, the peri- 
cardial sac, the pleura, the serous cavities of the brain, etc. 
Presence in Inflammatory Process Not Secondary to Pneumonia. 
-It is now known that the pneumococcus may infect and excite dis- 
eases in many tissues of the body independent of any preliminary 
localization in the lung. As a rule, these processes are acute and usually 
run a shorter and more favorable course than similar inflammations 
due to the streptococci. 
The most frequent primary lesions excited by the pneumococcus 
after lobar pneumonia, bronchopneumonia, and bronchitis are prob- 
ably meningitis, otitis media with its complicating mastoiditis, endo- 
carditis, pericarditis, rhinitis, tonsillitis, conjunctivitis, and keratitis; 
septicemia, arthritis, and osteomyelitis; inflammations of the epi- 
didymis, testicles, and Fallopian tubes; peritonitis, etc. 
Pneumococcic peritonitis and appendicitis are not so very frequent. 
The exudate is usually seropurulent. 
Conjunctivitis due to pneumococci frequently occurs in epidemic 
form and is frequently associated with rhinitis. 
From statistics collected by Netter the following percentages of 
diseases were caused by the pneumococcus: 
Pneumonia 65.9 per cent, in adults. 
Bronchopneumonia 15.8 per cent, in adults. 
Meningitis 13.0 per cent, in adults. 
Empyema 8.5 per cent, in adults. 
Otitis media 2.4 per cent, in adults. 
Endocarditis 1.2 per cent, in adults. 
In 46 consecutive pneumococcus infections in children there were: 
Otitis media 29 cases. 
Bronchopneumonia • . . 12 cases. 
Meningitis 2 cases. 
Pneumonia 1 case. 
Pleurisy 1 case. 
Pericarditis 1 case. THE DIPLOCOCCUS OF PNEUMONIA 
269 
The pneumococcus and streptococcus are the two most frequent 
organisms found in otitis media. The cases due to the pneumococcus 
are apt to run the shorter course, but have a tendency to spread to 
the meninges and cause a meningitis. The pneumococci may also 
find their way into the blood-current. This usually follows after sinus 
thrombosis. 
In bronchitis the pneumococcus is frequently met with alone or in 
combination with the streptococcus, the influenza bacillus, or other 
bacteria. 
In certain epidemics pneumococcic bronchitis and pneumonia simu- 
late influenza very closely and cannot be differentiated except by bac- 
teriological examinations. 
Primary pneumococcic pleurisy is frequent in children: it is very 
often purulent, but may be serous or serofibrinous. Its prognosis 
is better than that in cases due to other organisms. Frequently we 
have streptococci and staphylococci associated with the pneumo- 
cocci. 
Pathogenesis in Lower Animals.- Most strains of the Micrococcus 
lanceolatus are moderately pathogenic for numerous animals; mice and 
rabbits are the most susceptible, indeed some strains are intensely 
virulent for these animals, while guinea-pigs and rats are much less 
susceptible. Pigeons and chickens are refractory. In mice and rabbits 
the subcutaneous injection of small or moderate quantities of pneu- 
monic sputum in the early stages of the disease, or of a twenty-four- 
hour ascitic broth culture from such sputum, or of a pure, virulent 
ascitic broth culture of the micrococcus, usually results in the death 
of these animals in from twenty-four to forty-eight hours. The course 
of the disease produced and the postmortem appearances indicate that 
it is a form of septicemia-what is known as sputum septicemia. After 
injection there is loss of appetite and great debility, and the animal 
usually dies some time during the second day after inoculation. The 
postmortem examination shows a local reaction, which may be of a 
serous, fibrinous, hemorrhagic, necrotic, or purulent character; or there 
may be combinations of all of these conditions. The blood of inoculated 
animals immediately after death often contains the micrococci in very 
large numbers. For microscopic examination they may be obtained 
from the blood, and usually from pleural and peritoneal exudates when 
these are present. 
True localized pneumonia does not usually result from subcutaneous 
injections into susceptible animals, but injections made through the 
thoracic walls into the substance of the lung may induce a typical 
fibrinous pneumonia. This was first demonstrated by Talamon, who 
injected the fibrinous exudate of croupous pneumonia, obtained after 
death or drawn during life from the hepatized portions of the lung, 
into the lungs of rabbits. Wadsworth showed that by injecting virulent 
pneumococci into the lungs of rabbits which had been immunized, a 
typical lobar pneumonia was excited, the bactericidal property of the 
blood being sufficient to prevent the general invasion of the bacteria. 270 
PATHOGENIC MICROORGANISMS 
Pneumonia may be produced in dogs, and in rabbits less easily, by 
intratracheal injections. 
Varieties of the Pneumococcus.-As among all other microorgan- 
isms minutely studied, different strains of pneumococci show quite 
a wide range of variation in morphology and virulence. Some of the 
variations are so marked and so constant that they make it necessary 
to recognize several distinct varieties of the pneumococcus, and to 
class as pneumococci certain varieties which have before this been 
classed as streptococci-e. g., the so-called Streptococcus mucosus 
capsulatus {Streptococcus mucosus Schottmuller), when first isolated 
from pneumonic exudate or elsewhere, and planted on artificial cul- 
ture media containing serum, grows as a rounded coccus with a small 
dense distinct capsule, principally in short or medium chains; it pro- 
duces a large amount of mucus-like zobglia, forming very large spread- 
ing colonies; it promptly coagulates fluid serum media containing 
inulin. It is also very virulent for mice, but only moderately virulent 
for rabbits. After a number of culture generations on ordinary nutrient 
agar it apparently loses most of these characteristics. It then grows 
in small colonies principally as naked diplococci which may be elon- 
gated and pointed, produces no zobglia, and loses most of its virulence 
for mice and rabbits. It still coagulates inulin serum media, and 
when transferred to serum media regains its former morphological 
characteristics. For these reasons we consider this organism a distinct 
variety of the pneumococcus. This variety of pneumococcus has been 
isolated by us from the lungs after death following lobar pneumonia, 
out of twenty consecutive autopsies, as the only organism present twice, 
and with another variety of pneumococcus once. Together with 
other varieties it was isolated from four out of twenty specimens of 
pneumonic sputum, and from sixty specimens of normal throat secre- 
tion five times. In 1905 Park and Williams showed that this variety 
should be placed with the pneumococci under the common name 
Pneumococcus mucosus. 
Agglutination Reactions.-That agglutinins are produced in animals 
by the injection of pneumococci was shown by Neufeld, Clairmont, and 
others. It was first thought that this test might be used as a means 
of diagnosis. We found (Collins), however, that when a large number 
of strains are tested, the variability of the reaction is so great as to 
render it impractical as a means of diagnosis. In the case of the pneu- 
mococcus mucosus Collins found greater uniformity of reaction, all 
strains tested reacting alike and the agglutinins of one member of the 
group were absorbed by the other members. Recently Hanes has 
corroborated this work. Neufeld, Cole and Dochez have shown that 
certain pneumococci may be grouped according to serum reactions. 
According to Cole and Dochez, the groups based upon their agglutina- 
tion reactions are as follows: Groups 1 and 2, typical pneumococci; 
Group 3, pneumococcus mucosus; Group 4, heterogeneous strains. 
The members of the first three groups agree with those in the group in 
their specific serum reactions. Each strain in the last group seems THE DIPLOCOCCUS OF PNEUMONIA 
271 
independent as far as serum reactions are concerned; it is really not 
a group in the sense of the first three, but rather an assembling together 
of isolated strains. 
The following table by Cole gives the types he found in pneumonia 
in a. series of cases occurring in New York City, and the relative mor- 
tality of the cases according to the infecting type: 
Infecting 
Strain 
Mortality 
type. 
Cases. 
per cent. 
Deaths. 
per cent. 
1 
. . 34 
47 
8 
24 
2 
. . 13 
18 
8 
61 
3 
. . 10 
14 
6 
60 
4 
. . 15 
21 
1 
7 
- 
- 
- 
- 
Total . 
. . 72 
100 
23 
32 
In different localities and at different times in the same locality, the 
type percentage will probably vary greatly. 
Pneumococci taken from normal throats or from slight inflammation 
fall usually into Group 4 among the heterogenous strains. The strains 
belonging to Group 1 are widely scattered, being found not only through- 
out the United States but also in Europe. 
Immunity.-Following an attack of pheumonia some immunity is 
established, but this lasts but a short time. Early in the history of 
this organism experiments were begun for the production of immunity 
in animals by means of preventive inoculations. Later it was found 
that after successive injections of gradually increasing doses of virulent 
pneumococci into certain animals (horse, sheep, goat, rabbit), a serum 
of some protective and curative power in experimental animals was 
obtained. The mode of action of this serum is still the subject of study. 
According to Wright, Neufeld, and others, its activity is due to the 
presence of certain substances called opsonins (Wright), or bacterio- 
tropic substances (Neufeld), which act on the bacteria in such a way 
as to prepare them for ingestion by the phagocytes. The extent to 
which phagocytosis brings about the crisis and healing in pneumonia 
is not known. 
Therapeutic Experiments.1-The number of cases reported in 
which the blood-serum of animals artificially immunized against pneu- 
monic infection has been used for the treatment of the disease in human 
beings, although numerous, has not led to the formation of a definite 
opinion as to the final value of this as a therapeutic agent. In the cases 
we have observed there has been, in some a slight immediate lowering 
of the temperature, in others no apparent change. As a rule, the cases 
did rather better than was expected, but certainly no striking curative 
effects were apparent. The apparent good results in some and the 
absence of these in others are probably explained by the facts brought 
out by Cole and Dochez that certain strains can be influenced and 
others cannot. It seems probable that the serum may be able to prevent 
1 See Harvey Lecture. Cole, Arch. Int. Med., 1914, xiv, 56. 272 
PATHOGENIC MICROORGANISMS 
a general infection from taking place from the diseased lung, even 
though it may fail to influence the local process. It has also been 
shown that these injections of antipneumococcic serum are practically 
harmless. 
In the case of infections with the pneumococcus mucosus, serum 
therapy seems hopeless, as passive immunity is not produced in animals. 
In the case of Group 4, heterogeneity of the group makes it unlikely 
that a serum would be of value. Infections by Groups 1 and 2 are 
the most hopeful types for serum therapy using either univalent or 
bivalent sera. 
As is shown, the preceding tables Group 2 causes a more serious 
disease and likewise serum treatment seems less beneficial than in the 
milder infections due to Type 1. 
To apply a univalent serum the type of infecting organism must be 
known or the proper serum cannot be selected. 
The following method is employed at the Rockefeller Institute 
Hospital: The sputum coughed up from the lung is injected intra- 
peritoneally into a mouse. After four to five hours the mouse is killed 
and the peritoneal cavity washed out with salt solution. The washings 
are then centrifuged slowly to throw down the fibrin and cells and the 
supernatant fluid is then mixed in equal parts with known Types 1 
and 2 sera, and the presence or absence of agglutination determined 
after incubation for one to two hours. Type 3 can usually be diagnosed 
by the mucoid character of the peritoneal exudate. 
In pneumococcus septicemia no marked results have been seen. 
Large injections of 100 to 150 c.c. of a polyvalent serum are given 
intravenously. 
Vaccines.-The use of injections of dead pneumococci in pneu- 
monia and other acute pneumococcic inflammations has not been 
followed by appreciable beneficial results in those cases which have 
come under our observation. In subacute cases the results appear 
to be more favorable. With better understanding of the varied infecting 
types better results may become possible. CHAPTER XVIII. 
MENINGOCOCCUS OR MICROCOCCUS (INTRACELLULARIS) 
MENINGITIDIS, AND THE RELATION OF IT AND 
OF OTHER BACTERIA TO MENINGITIS. 
While, as has already been stated, the pneumococcus is a cause of 
isolated cases of meningitis the meningococcus is the most frequent 
cause of purulent meningitis either sporadic or endemic. In 1887 
Weichselbaum1 discovered a micrococcus in the exudate of cerebro- 
spinal meningitis in six cases, two of which were not complicated by 
pneumonia. He obtained it in pure cultures, studied its characteristics, 
and showed that this organism was clearly distinguishable from the 
pneumococcus and especially by its usual presence in the interior of 
pus cells, on which account he called it Diplococcns intracellularis 
meningitidis. In 1895 Jaeger and Scheurer drew especial attention to 
the etiological relationship of the organism to the epidemic form of 
cerebrospinal meningitis. They also believed it to be very probable 
that in most cases of primary meningitis it is from the mucous mem- 
brane of the nasal cavities and the sinuses opening out from them that 
both the diplococcus of pneumonia- and the micrococcus intracellularis 
find their way through the blood or perhaps directly through the lymph 
channels to the meninges. The former we know to be almost constantly 
present in the nasal cavities, and the latter we have reason to believe 
is not infrequently there. The nasal secretion of carriers is more dan- 
gerous to the community than that of infected persons because carriers 
are not recognized as such usually and mingle freely with the general 
public. The prevalence of epidemics in winter and spring, a time favor- 
able to influenza and pneumonia, also suggests the respiratory tract 
as the place of the infection and where an increase in virulence takes 
place. We do not as yet know why meningitis follows in some persons 
and not in others after infection of the mucous membranes. 
Morphology.--This organism occurs as biscuit-shaped micrococci, 
usually united in pairs, but also in groups of four and in small masses; 
sometimes solitary and small degenerated forms are found. It has no 
well-defined capsule. Cultures resemble strongly those of gonococci. 
In the sediment from spinal fluids (see Fig. 108) meningococci are 
both intra and extracellular. When they are intracellular the prognosis 
is better and frequently in cases where they are extracellular at first 
they later become intracellular. 
Staining.-They stain with all the ordinary aniline dyes and are readily 
decolorized by Gram's solution. A smear from a culture shows charac- 
1 Weichselbaum, Fortschr, d. Med., 1887, p. 573, 
18 274 
PATHOGENIC MICROORGANISMS 
teristic irregularity in staining, some of the cocei taking the counter 
stain poorly and some staining deeply. The positive cocci described 
by Jaeger and others were prob- 
ably contaminating organisms. 
The cells have no definite capsule. 
Cultivation.-They grow be- 
tween 25° and 38° C., best at 
about 35° C. They are most 
easily isolated and kept on 2 per 
cent, glucose ascitic agar neutral 
to phenolphthalein. The reaction 
of the media is very important. 
A culture can rarely be isolated 
on plain nutrient agar. After 
several generations on glucose 
ascitic agar they will grow quite 
luxuriantly on neutral salt-free veal 
agar for a few generations but tend 
to die out. 
Different strains of meningococci 
vary in the ease with which they 
may be cultivated, their ability to ferment carbohydrates, virulence for 
animals, agglutinability, degree of digestibility in leukocytes, and power 
of resistance to immune serum. 
Fig. 108 
Diplococcus intracellularis meningitidis in 
pus cells. X 1100 diameters. 
When grown for some time, a tolerably good growth develops at the end of 
forty-eight hours in the incubator on nutrient agar or glucose agar. This 
appears as a flat layer of colonies, about one-eighth of an inch in diameter, 
grayish-white in color, finely granular, rather viscid, and non-confluent unless 
very close together. On Lbffler's blood serum the growth forms round, whitish, 
shining, viscid-looking colonies, with smooth and sharply-defined outlines; 
these may attain diameters of one-eighth to one-sixteenth of an inch in twenty 
four hours. The colonies tend to become confluent and do not liquefy the 
serum. From the spinal fluid in acute cases, where the organisms are apt to 
be more abundant, a great many minute colonies may develop instead of a few 
larger ones. On agar plates the deep-lying colonies are almost invisible to the 
naked eye; somewhat magnified they appear finely granular, with a dentated 
border. On the surface they are larger, appearing as pale disks, almost trans- 
parent at the edges, but more compact toward the centres, which are yellowish- 
gray in color. On blood-agar or serum agar the growth is much more luxuriant 
than on plain agar and larger than the gonococcus. Cultivated in artificial 
media, while it often lives for weeks, it may die within four days, and requires, 
therefore, to be transplanted to fresh material at short intervals. 
Resistance.-It is readily killed by heat, disinfectants, sunlight, 
and drying. A few cocci may remain alive for one to three days in the 
dried state. To maintain cultures it is necessary to make transfers 
every other day. 
Pathogenesis.-This organism has a low-grade and very variable 
pathogenicity for laboratory animals. We used over one hundred 
guinea-pigs one winter in trying to establish a fatal dose of live culture AGGLUTINATION CHARACTERISTICS 
275 
and as many another winter trying to find the fatal dose of meningo- 
coccic extract and were never able to arrive at any definite conclusion. 
There seemed to be no rhyme or reason to the results. Following the 
intraperitoneal injection the temperature of the pig falls, the hair 
stands out, the abdomen becomes distended and the muscles rigid. 
The animal hunches up in a corner and seems very sick. Not infre- 
quently there is prolapse of the rectum. If the dose is fatal death 
usually occurs in from ten to forty-eight hours. At autopsy there is 
fluid exudate in the abdomen and sometimes in the pleural cavity, 
congestion or hemorrhage of the adrenals, hemorrhages into the mesen- 
tery, central tendon of the diaphragm and into the whole peri- 
toneum. Frequently it is possible to recover the meningococcus from 
the heart's blood when live culture has been used. The organisms do 
not have to multiply to produce death and the autolysate or killed 
culture is just as. fatal as live culture. Death is probably due to a bac- 
terial poison freed by the disintegration of the meningococci. Rabbits 
injected either subcutaneously or intravenously lose weight rapidly. 
Presence in the Nasal Cavity of the Sick and Those in Contact with 
Them.-In 1 of his 6 cases Weichselbaum succeeded in obtaining dip- 
lococci from the nasal secretion. In 1901 Albrecht and Ghon demon- 
strated them in healthy individuals. Scheurer, in his 18 cases, found 
the diplococci in the nasal secretions of all of them during life. In 50 
healthy individuals examined they were found in the nasal secretions 
of only two, one being a man suffering at the time from a severe cold. 
This man, it is interesting to note, had been employed in a room 
which had just previously been occupied by a patient with cerebrospinal 
meningitis. Lately, there has been a tendency to throw doubt on these 
findings, but from our experience in the 1906 epidemic in New York, 
one can state that the meningococci are usually present in great numbers 
in the nose and nasopharynx in most cases of meningitis during the first 
twelve days of illness. After the fourteenth day they cannot usually 
be found. In one case Goodwin of our laboratory obtained them on the 
sixty-seventh day. She also found them in five persons out of sixty 
tested who had been in close contact with the sick, and in two of fifty 
medical students. 
Pathogenicity for Man.-The most marked lesions occur at the base 
of the brain. The cord is always affected. This is not true to the same 
extent in other bacterial infections. In some epidemics the course 
of the disease is very rapid. The mortality varies from between 50 and 
80 per cent. 
Complicating Infections.-Occasionally we find secondary to the cere- 
brospinal meningitis, and due to the micrococcus, pneumonia, cystitis, 
conjunctivitis, inflammation of the middle ear, arthritis, endocarditis, 
etc. The most frequent and serious complication of meningococcic 
meningitis is pneumonia probably due in many cases to the pneumo- 
coccus. 
Agglutination Characteristics.-A considerable percentage of cultures 
of meningococci are relatively inagglutinable. Strains that are agglu- 276 
PATHOGENIC MICROORGANISMS 
tinable respond to the agglutinins developed in an animal immunized 
with a true strain. Careful absorption tests are capable in almost, if 
not all, instances of separating true meningococci from other Gram- 
negative organisms. This serum reaction is practically never used for 
diagnosis because it is so variable and unreliable. 
Parameningococcus.-Doptor (1909) obtained from nasal secretions 
several strains of cocci which, though similar to other meningococci 
in morphology and cultures, were different from them in some serologic 
properties. These cocci he called parameningococci, and he considered 
that in some cases they might invade the system and cause meningitis. 
His work has been corroborated by other French observers, and, just 
now, by Wollstein in this country. Wollstein, testing many strains 
from different parts of the world, for agglutination, opsonization, 
complement fixation and serum protection, comes to the conclusion 
that while certain strains exhibit differences enough to warrant the 
employment of them in the production of a polyvalent serum, they show 
too many variations in serum reactions to be separated into a distinct 
class. They should rather be considered special strains among menin- 
gococci. This conclusion is in general accord with those obtained 
through the extensive researches of Elser and Huntoon (1909). 
Localization of Meningococci.-In addition to the situation already 
noted the meningococcus is frequently found in the blood in the early 
days of the disease. Elser found it in blood cultures in 10 out of 40 
cases. The meningococci have also been found in the herpes and in the 
urine, a fact to be considered in ordering the hygiene of the sick room. 
Serum Treatment.-It is difficult to apportion the credit for the pro- 
duction of the first protective serum. Bonhoff and Lepriere produced 
in animals a serum which showed definite protection. The world-wide 
epidemic beginning in 1904 stimulated a number of laboratories to pro- 
duce sera in horses with the idea of treating human cases. 
Thus Kolle and Wassermann, Jochmann, Flexner, and ourselves 
immunized horses. The usual method was to begin with cultures 
recently obtained from human cases and grow them on ascitic agar 
or plain nutrient agar in tubes. The growth was scraped off, added 
to physiological salt solution, and heated to 55° to 60° for one hour. 
Living cultures were often substituted later. 
The original injections were quite small, being only one or two mod- 
erate-sized platinum loopfuls. Each succeeding injection was doubled 
in size each time, until the maximum dose of the growth on two Petri 
dishes was given, when the size of the injection was not changed. The 
injections were given about every eight days. Horses give the best 
serum after eight months to one year. Kolle and Wassermann injected 
one horse with the watery extract of recent cultures. They also used 
both the intravenous and subcutaneous methods. 
The Therapeutic Use of Serum.-In 1905 there was inaugurated 
in Hartford, the use of subcutaneous injections of diphtheria anti- 
toxic serum in meningitis. This influenced us to prepare and try the 
subcutaneous injection of an antimeningococcus serum. The results SERUM TREATMENT 
277 
reported by the physicians in some 20 cases did not seem to establish 
that any beneficial effects were obtained, so no further serum was issued. 
Later Kolle and Wassermann reported somewhat favorable results 
in a number of cases from the subcutaneous injection of a serum pre- 
pared by them. Meanwhile a serum prepared by Jochmann was 
employed by the intraspinal method in a series of cases. This method 
soon supplanted the subcutaneous injections. 
The first successful use of an immune serum in cases of human 
cerebrospinal meningitis, by the intraspinal method, should, therefore, 
so far as we know, be credited to Jochmann and the physicians who 
used his serum in the winter of 1905 and 1906. He reported a series 
of cases treated by the intraspinal method before the Congress for 
Internal Medicine held in Munich in April, 1906, and published his 
paper on May 17, 1906. 
The serum was prepared by injecting horses with increasing doses 
of meningococcus killed at about 58° C. The doses were given every 
eight days, beginning with a loopful and increasing until the growth on 
the surface of ascitic agar covering two Petri dishes was used. After 
this dose was reached living cultures were given. 
Characteristics of Serum.-The serum was shown to possess 
both bactericidal and opsonic power. He reported forty cases of cerebro- 
spinal meningitis which had been treated, but gave details concerning 
17 patients which all occurred in one hospital and were treated by 
Kromer. Five of these patients died and twelve recovered, a mortality 
of 29 per cent. He directed that after lumbar puncture, 20 to 50 c.c. 
of fluid should be removed and then 20 c.c. of immune serum injected. 
These injections should be repeated once or twice if the fever did not 
abate or returned. He noticed in general a bettering of the headache, 
stiffness of neck, and mental condition. Jochmann showed that in 
animals colored fluids injected into the spinal canal in the lumbar 
region passed the full length of the canal. 
Although the serum prepared in different laboratories in Europe 
was regularly used after Jochmann's report, it did not receive much 
attention in this country until Flexner, through his important experi- 
ments on infected monkeys, which demonstrated the value of the intra- 
spinal injections of the serum, aroused medical interest and paved the 
way for him to try out the serum on a large scale. All cases treated 
by him were subjected to most careful bacterial tests and clinical 
observation. Eighteen months later, Flexner and Jobling published 
their report which fully corroborated the earlier results of Jochmann. 
The serum prepared at the Rockefeller Institute for Medical Research 
has been sent to many places, both in this country and in Europe. 
The results obtained have been of the utmost value in arriving at the 
value of the intraspinal treatment. The following is abstracted from 
their latest report. 
There were 1294 cases of the disease in which the bacteriologic diag- 
nosis was made and the serum treatment used. In the first table the 
cases are subdivided according to certain age periods, and in the second 278 
PATHOGENIC MICROORGANISMS 
the total cases of each age period are further subdivided according 
as the serum was injected in the three arbitrarily chosen periods of 
duration of the disease. 
Table I.-Cases of Epidemic Cerebrospinal Meningitis Treated with the Antimeningitis 
Serum. 
Cases Analyzed According to Age Groups. 
Age. 
No. of cases. 
Recovered. 
Died. 
Per cent, 
recovered. 
Per cent, 
died. 
Under 1 year 
129 
65 
64 
50.4 
49.6 
1 to 2 years 
87 
60 
27 
69.0 
31.0 
2 to 5 years 
194 
139 
55 
71.6 
28.4 
5 to 10 years 
218 
185 
33 
84.9 
15.1 
10 to 20 years 
360 
254 
106 
70.6 
29.4 
Over 20 years 
288 
180 
108 
62.5 
37.5 
Age not given 
18 
11 
7 
61.1 
38.9 
Totals .... 
1294 
894 
400 
69.1 
30.9 
The highest mortality 49.6 per cent, is shown to have occurred in the 
first year of life. Even so it is greatly reduced, for under the older 
forms of treatment it was between 90 and 100 per cent. The average 
mortality in all the age periods was 30.9 per cent. 
Table II.-Cases of Epidemic Cerebrospinal Meningitis Treated with the Antimeningitis 
Serum. 
Age. 
Injected 1st to 3rd days. 
Injected 4th to 7th day. 
Injected later than 7th day. 
Rec. 
Died. 
% 
Rec. 
Died. 
% 
Ree. 
Died. 
% 
Under 2 years 
12 
1 
7.7 
28 
9 
24.3 
81 
78 
49.1 
2 to 5 years 
24 
6 
20.0 
49 
17 
25.8 
63 
30 
37.3 
5 to 10 years 
49 
6 
10.9 
61 
8 
11.6 
61 
16 
20.8 
10 to 20 years 
58 
9 
13.4 
73 
73 
31.1 
115 
56 
32.7 
Over 20 years 
20 
14 
41.2 
39 
16 
40.0 
101 
63 
38.4 
Totals 
163 
36 
18.1 
250 
123 
27.1 
421 
243 
86.6 
Cases Analyzed According to Day of Injection. 
Table II is instructive in bringing out the importance of early 
injections of the serum. The results in the first two years of life are 
especially noteworthy. The extraordinary figures given under the first 
period of injection, namely, in the first three days of the disease, can 
hardly be maintained. But the influence of period of injection is shown 
by the rapid rise in mortality in the subsequent two periods. The 
rule of the effects of early injection is preserved in the age periods up 
to the period of twenty years, when it disappears. The discrepancy 
occurring in the highest age period cannot be wholly accounted for at 
present. The explanations which suggest themselves are that among 
older individuals there tends to be a large number of very severe, 
rapidly fatal or fulminating cases of the disease, or that older persons 
are less subject to the beneficial action of the serum. As regards the 
actual proposition, it may be stated that adults not infrequently respond SERUM TREATMENT 
279 
promptly to the serum injections by abrupt termination of the disease 
or ameliorated symptoms and pathologic conditions. 
The total figures do not, however, fail to indicate that the early 
injections are more effective than the later ones, as is shown by the 
percentage mortality in the first-to-third-day period of 18.1, in the 
fourth-to-seventh-day period of 27.1, and the period later than the 
seventh day of 36.6. 
Directions for Use of Serum.-The following directions are issued 
by the Research Laboratory of the New York City Board of Health. 
The antimeningitis serum is a specific immune serum of therapeutic 
value only in meningococcic meningitis. It is administered subdurally, 
the subcutaneous route being practically worthless. 
Perform a lumbar puncture under aseptic precautions in the third 
or fourth lumbar space. A general anesthetic should never be used. 
In hypersensitive patients a local anesthetic may be advisable. Have 
the patient lying on the side with the back arched so that their will be 
the greatest possible distance between the spines of the vertebra*. 
Find the notch nearest a line connecting the crests of the ilia. Intro- 
duce the needle, preferably a Quncke needle, in the midline and push 
forward and a little upward. The distance the needle goes in depends 
upon the age of the patient and the muscular development. It varies 
from | to 3 inches. Allow the cerebrospinal fluid to flow out until 
the pressure is so reduced that only 3 or 4 drops come per minute. If 
the fluid is cloudy, inject the serum immediately. 
Where possible, further injections are made only after bacteriological 
examination has determined the cause to be the meningococcus. The 
antimeningitis serum does no harm in meningitis due to other organisms. 
The serum is warmed to body temperature and injected very slowly 
under the least possible pressure. A funnel and the tube arrangement 
allowing it to run in by gravity should be used. The barrel of an 
ordinary syringe may be used as a funnel. The rubber tubing should 
be | to | of an inch in diameter and long enough so that the funnel 
can be raised 12 to 15 inches. In general, the average dose for an adult 
is 20 to 40 c.c. and for infants and children 2 to 20 c.c. The amount 
depends as much upon the quantity of cerebrospinal fluid withdrawn 
as upon the age. An infant will frequently stand 10 to 15 c.c. without 
difficulty. The dose should usually be at least 5 to 10 c.c. less than the 
amount of cerebrospinal fluid withdrawn. Where serum apparently 
runs in freely after a dry tap, it is advisable to proceed very slowly 
and to watch the patient carefully for the slightest change in pulse and 
respiration. In cases with very thick exudate which will not flow 
through the needle, gentle suction with a syringe may be tried. If 
that fails, a little serum injected will sometimes start the flow. In very 
severe cases it is best to inject the serum every twelve hours until 
there is improvement. In moderate and mild cases, it should be repeated 
each day for the first four days. Further administration depends upon 
the patient's general condition and the bacteriological examination of 280 
PATHOGENIC MICROORGANISMS 
the fluid. Usually four to six injections are necessary, but as many as 
fifteen or more may have to be employed. 
During or immediately after the injection of serum the respiration 
may entirely cease or the pulse may become very rapid and thready. 
Such an occurrence, while alarming, is not necessarily serious and is 
best treated by immediate withdrawal of some of the serum if the needle 
is still in place. If the needle has been withdrawn, or, if after some of 
the serum is removed the symptoms do not ameliorate, artificial respira- 
tion should be resorted to for the respiratory condition and adrenalin 
or other stimulants hypodermically for the heart. 
Hospitals and those having available blood-pressure apparatus will 
find it of service in administering the serum. 
The successful treatment of cases of meningitis must always depend 
upon experience and judgment. It cannot be reduced to a rule of thumb. 
In all cases with meningeal symptoms a lumbar puncture should 
be done. No ill effects follow, on the contrary the relief of the pressure 
frequently produces beneficial results. 
Bacteriological Diagnosis.-The fluid should be collected in a sterile 
container. It may be clear, cloudy, or bloody. If it is clear it 
may be a normal fluid from a case of poliomyelitis or tuberculous 
meningitis. 
If it is cloudy it may be due to the meningococcus; streptococcus 
pneumococcus, pneumococcus mucosus, influenza bacilli, or other 
rarer organisms. The blood in a bloody fluid may be due to a previous 
hemorrhage or to the accidental piercing of a vein. The two conditions 
may be differentiated by centrifuging the fluid. If the supernatant 
fluid is yellow or reddish the hemorrhage is old. Clear fluid indicates 
a recent hemorrhage. It is unfortunate to have much blood in the fluid 
for it obscures the microscopic picture and then unless the culture is 
positive it is difficult to make even a tentative diagnosis. Clear fluids 
should always be centrifuged preferably for one hour at high speed. 
Cloudy fluids showing no organisms should also be centrifuged. The 
sediment should be used to make cultures and smears. The smears 
are examined: (1) for pus cells and (2) for tubercle bacilli if the fluid 
is clear or for other organisms if it is cloudy. The sediment from clear 
fluids should always be stained with the tubercle stain and that from 
cloudy fluids with Gram's stain. Once in a long while a cloudy fluid 
will be found to be tuberculous or a clear fluid to be due to some pyogenic 
organism but these occurrences are so rare as to be practically negligible. 
Gram's stain differentiates influenza bacilli, and meningococcus from 
the streptococcus, pneumococcus, and pneumococcus mucosus. The 
finding of Gram-negative cocci either intra or extracellular is presump- 
tive evidence of meningogoccic meningitis, but it is always well to 
follow it up with a culture. In order that cultures may grow fluids 
should be examined at the earliest moment possible, for meningococci 
in fluids over twelve hours old are frequently autolysed so that they 
will not grow. OTHER ORGANISMS EXCITING MENINGITIS 
281 
The following table gives the main differential points in making 
diagnosis from spinal fluids: 
Pressure. Amount, 
o.c. 
Appear- 
ance. 
Cytology. 
Bact. 
Albumin. 
Globulin. 
Animal 
Inoc. 
Normal 
Normal. 5-10 
Clear. 
Very few cells. 
Sterile. 
- 
Negative. 
Meningismus 
Increased. 10-100 
Clear. 
Very few cells. 
Sterile. 
- 
Negative. 
Infantile 
Increased. 20-100 
Clear; 
Early polynu- 
Sterile. 
+ -++ 
4-1-4- 
Negative 
paralysis 
some- 
cleosis; later 
or pneu- 
times 
lymphocytosis 
monia. 
slight 
up to 95 per 
fibrin 
cent.; endo- 
1 
web. 
thelial cells 
Tuberculous 
Increased. | 30-120 
Clear; 
Lymphocytosis 
Tubercle 
4-1-1-1-F 
4-4-4-4-4- 
Tubercu- 
meningitis. 
fibrin 
up to 95 per 
bacilli. 
losis in 4 
web. 
cent. 
weeks. 
Epidemic cer- 
Increased. 5-120 
Cloudy. 
Polynucleosis 
Meningo- 
4-4-1-1-1-F 
4-4-4-4-4-4- 
ebrospinal 
up to 98 per 
COCCUS. 
meningitis. 
cent. 
Meningitis 
Increased. 20-100 
Cloudy. 
Polynucleosis 
Infecting 
4-4-1-1-1-F 
4-F-F4-4-F 
due to other 
up to 98 per 
organisms. 
organisms. 
cent. 
Differential Diagnosis from Gonococci.-As a rule, the portion of 
the body from which the organisms are obtained reveals their source. 
When this is insufficient careful cultural and serological tests are 
required. McNeil has obtained a specific complement-fixation 
reaction. 
Other Gram-negative Cocci Resembling Meningococci.- Micro- 
coccus pharyngis (siccus) (von Lingelsheim), Diplococcus mucosus, Chromo- 
genic Gram-negative cocci, Micrococcus catarrhalis.-These may all be 
differentiated by cultural characteristics. 
Other Organisms Exciting Meningitis.-1. The pneumococcus. This 
diplococcus is one of the most frequent exciters of meningitis, both as 
a primary and a secondary infection. 
2. The Streptococcus pyogenes, Pneumococcus and Staphylococcus. 
Meningitis due to these organisms is almost always secondary to some 
other infection, such as otitis, tonsillitis, erysipelas, endocarditis, 
suppurating wound of scalp and skull, etc. 
3. The Bacillus influenzae. Numerous reports have been published 
of the presence of influenza bacilli in the meningeal exudate. Those 
that are reliable state in almost every instance that the meningitis 
is secondary to infection of the lungs, bronchi, the nasal cavities of 
their accessory sinuses. 
4. The colon bacillus, the typhoid bacillus, that of bubonic plague and 
of glanders, all may cause a complicating purulent meningitis. 
5. In isolated cases of meningitis complicating otitis media and 
other infections, other bacteria, such as the Micrococcus tetragenus, the 
Bacillus pyocyaneus, the gonococcus, etc., may be found. 
6. The tubercle bacillus. This is a very frequent cause. It is after 
the meningococcus the most frequent cause. 
Meningitis due to other organisms than the meningococcus is almost 
invariably fatal. 282 
PATHOGENIC MICROORGANISMS 
MICROCOCCUS CATARRHALIS (R. PFEIFFER). 
Micrococci somewhat resembling meningococci are found in the mucous 
membranes of the respiratory tract. At times they excite catarrhal inflamma- 
tion of the mucous membranes and pneumonia. These are at present included 
under the designation of Micrococcus catarrhalis. 
Microscopic Appearance.-They usually occur in pairs, sometimes in fours; 
never in chains. The cocci are coffee-bean in shape and slightly larger than the 
gonococcus, and are negative to Gram's stain. 
The micrococci are not motile and produce no spores. 
Cultivation.-They grow between 20° and 40° C., best at 37° C. and less 
rapidly at somewhat lower temperatures, developing on ordinary nutrient 
agar as grayish-white or yellowish-white, circular colonies of the size of men- 
ingococci. The borders of the colonies are irregular and abrupt as though 
gouged out. They have a mortar-like consistency. On serum-agar media the 
growth is more luxuriant. Gelatin is not liquefied. Bouillon is clouded, often 
with the development of a pellicle. Milk is not coagulated, but dextrose serum 
media may be. Gas is not produced. 
Location of Organisms.-In the secretion of normal mucous membranes 
they are occasionally present. In certain diseased conditions of the mucous 
membranes they may be abundant. 
Pathogenic Effects in Animals.-For white mice, guinea-pigs, and rabbits, 
some cultures are as pathogenic as meningococci, while others are less so. 
Differential Points Separating them from the Meningococci.-These organisms 
have undoubtedly been at times confused. Some assert that the meningococci 
grow only above 25° C. Many cord cultures of meningococci grow below this 
point. Some assert that the meningococci will not grow on 5 per cent, glycerin 
agar. Many undoubted cultures do. Careful agglutinin absorption tests are 
of great differential value, but can only be carried out safely by one accustomed 
to them. The meningococci tested by us have removed all the agglutinins acting 
upon meningococci from a specific meningococcus serum while the allied 
organisms have removed only about 60 per cent, of them. The probability is 
that the organisms described by different writers as Micrococcus catarrhalis 
were not all the same variety, and some of them were meningococci. 
Vaccine Therapy.-Good reports have been made of the results of injecting 
the dead organisms in a number of infections due to this micrococcus. 
References. 
Elser and Huntoon. Jour. Med. Res., 1909, xx, 377. 
Flexner and Jobling. Jour. Exp. Med., May 1, 1913, xvii, No. 5, 260. 
A. Sophian, in the Journal of the American Medical Association for March 23, 1912. 
Wollstein. Jour. Exp. Med., 1914, xx, 201. CHAPTER XIX. 
THE GONOCOCCUS OR MICROCOCCUS GONORRHOEA 
-THE DUCREY BACILLUS OF SOFT CHANCRE- 
MICROCOCCUS MELITENSIS. 
The period at which gonorrhea began to inflict man is unknown. 
The earliest records make mention of it. Wherever civilized man 
has penetrated gonorrhea is prevalent among the people. Except for 
a period after the fifteenth century it was generally recognized as a 
communicable disease and laws were made to control its spread. The 
differentiation between the lighter forms of gonorrhea and some other 
inflammations of the mucous membranes was, however, almost impos- 
sible until the discovery of the specific microorganism by Neisser, 
in 1879. 
The organism was first observed in gonorrheal discharges, and, 
described by him under the name of "gonococcus;" but though several 
attempted to discover a medium upon which it might be cultivated, 
it was reserved for Bumm, in 1885, to obtain it in pure culture upon 
coagulated human-blood serum, and then after cultivating it for many 
generations to prove its infective virulence by inoculation into man. 
The researches of Neisser and Bumm established beyond doubt that 
this organism is the specific cause of gonorrhea in man. Gonorrhea 
is in almost all cases among adults transmitted through sexual inter- 
course. Gonorrheal ophthalmia is a frequent accidental infection at 
birth and vaginitis in the young child is frequently produced by the 
carelessness of the nurse or mother carrying infection. 
Microscopic Appearance.-Micrococci, occurring mostly in the 
form of diplococci. The bodies of the diplococci are elongated, and, 
as shown in stained preparations, have an unstained division or inter- 
space between two flattened surfaces facing one another, which give 
them their characteristic, "coffee-bean" or "kidney" shape. The 
older cocci lengthen, then become constricted in their middle portion, 
and finally divide, making new pairs (Fig. 109). The diameter of an 
associated pair of cells varies according to their stage of development 
from 0.8^ to 1.6m in the long diameter-average about 1.25m-by 
0.6m to 0.8m in the cross diameter. 
Extracellular and Intracellular Position of Gonococci.-In gonor- 
rhea, during the earliest stages before the discharge becomes purulent, 
the gonococci are found mostly free in the serum or plastered upon the 
epithelium cells, but later almost entirely in small, irregular groups 
in or upon the pus cells and epithelial cells, and always extranuclear. 
With the disappearance of the pus formation more free gonococci 
appear. Discharge expressed from the urethra usually contains more 284 
PATHOGENIC MICROORGANISMS 
free organisms than the natural flow. Gonococci are sometimes irregular 
in shape or granular in appearance, involution forms, found particularly 
in older cultures and in chronic urethritis of long standing. Single pus 
cells sometimes contain as many as one hundred gonococci ami seem to 
be almost bursting and yet show but slight signs of injury. These diplo- 
cocci are also found in or upon desquamated epithelial cells. There 
is still discussion as to whether the gonococci actively invade the pus 
cells or only are taken up by them. There is no evidence that the 
gonococci are destroyed by the pus cells (Fig. 110). In gonorrhea of the 
conjunctiva they are contained in the epithelial cells, sometimes in 
large numbers. They form dense groups which change as they do in 
older cultures, producing metachromate granules in round, swollen, 
pale blue bodies. These masses finally present an appearance somewhat 
like the cell inclusions found in "trachoma" (p. 439). 
Fig. 109 
Smear from pure culture of gonococcus on 
agar. X 1100 diameters. ' (Heiman.) 
Gonococcus in pus cells. X 100 diame- 
ters. 
Staining.-The gonococcus stains readily with the basic aniline 
colors. Loffler's solution of methylene blue is one of the best stain- 
ing agents for demonstrating its presence in pus, for, while staining 
the gonococci deeply, it leaves the cell protoplasm but faintly stained. 
Fuchsin is apt to overstain the cell substance. Beautiful double- 
stained preparations may be made from gonorrheal pus by treating 
cover-glass smears with methylene blue and eosin. Numerous methods 
for double staining have been employed, with the object of making 
a few gonococci more conspicuous. None of them have any specific 
characteristics such as the Gram stain. It is now established that 
gonococci from fresh cultures and from recent gonorrheal infections 
are, when properly treated by Gram's method, quickly and surely 
robbed of their color and take on the contrast stains. The removal 
of the stain from gonococci in old flakes and threads from chronic 
cases is not so certain. This difference is mostly due to the fact that 
equally uniform specimens cannot be prepared. The decolorized APPEARANCE OF COLONIES 
285 
gonococci are stained by dipping the films for a few seconds into a 1: 10 
dilution of carbol-fuchsin or a solution of bismarck brown. This stain- 
ing should be for as short a time as suffices to stain the decolorized 
organisms. This method of staining cannot be depended upon alone 
absolutely to distinguish the gonococcus from all other diplococci 
found in the urethra and vulvovaginal tract, for, especially in the female, 
other diplococci are occasionally found which are also not stained by 
Gram's method. It serves, however, to distinguish this micrococcus 
from the common pyogenic cocci, which retain their color when treated 
in the same way, and in the male urethra it is practically certain, as 
few organisms have been found in that location which in morphology and 
staining are identical with the gonococcus. It is certainly the most 
distinctive characteristic of the staining properties of the gonococcus, 
and it is a test that should never be neglected in differentiating this 
organism from others which are morphologically similar. 
Biology.- Grows best at blood-temperature; the limits being 
roughly 25° and 40° C. It is a facultative anaerobe. It is not motile 
and produces no spores. 
Culture Media.-The gonococcus requires for its best growth the 
addition to nutrient agar of a small precentage of blood-serum or some 
equivalent. The following media have proven of value: 
1. Human blood from the sterilized finger streaked on common 
nutrient agar. 
2. Human ascitic, pleuritic, or cystic fluid, 1 part added to and mixed 
with 2 parts melted 5 per cent, glycerin nutrient, 1.5 per cent, agar 
having a temperature of 55° to 60° C. The whole after mixing being 
poured into a Petri dish or cooled slanted in a tube. The same propor- 
tions of nutrient broth and ascitic fluid make a suitable fluid medium. 
One per cent, glucose may be added. 
3. Swine serum nutrose media. Wassermann strongly recommends 
this mixture. In our hands it has given good results. 
4. Nutrient or 5 per cent, glycerin agar. When considerable pus 
is streaked on simple agar media a good growth of gonococci is usually 
obtained. After continued cultivation gonococci cultures frequently 
grow on media containing no serum. Some strains grow on ordinary 
glycerin or glucose nutrient agar and even on plain nutrient agar from 
the start. Alkaline veal agar gives good growths of started cultures. 
Viability.-Cultures frequently die in forty-eight to seventy-two 
hours when kept at room temperature. In the ice-box they may live 
for several weeks. They frequently live for one week in the thermostat 
at 36° C. on plain nutrient agar. 
Appearance of Colonies.-A delicate growth is characteristic. At 
the end of twenty-four hours there will have developed translucent, 
very finely granular colonies, with scalloped margin. The margin is 
sometimes scarcely to be differentiated from the culture medium. In 
color they are grayish white, with a tinge of yellow. The texture is 
finely granular at the periphery, presenting punctated spots of higher 
refraction in and around the centre of yellowish color (Fig. 111). 286 
PATHOGENIC MICROORGANISMS 
Surface Streak Culture.-Translucent grayish-white growth, with 
rather thick edges. 
Resistance.-The gonococcus has but little resistant power against 
outside influences. It is killed by weak disinfecting solutions and by 
desiccation in thin layers. In comparatively thick layers, however, 
as when gonorrheal pus is smeared on linen, it has lived for forty-nine 
days, and dried on glass for twenty-nine days (Heiman). It is killed 
at a temperature of 45° C. in six hours and of 60° in about thirty 
minutes. 
Occurrence of Gonococci.-Outside of the human body or material 
carried from it gonococci have not been found. 
Toxins.-In the gonococcus cells substances are present which are 
toxic after heating and contact with alcohol. Injected in considerable 
amounts into rabbits, they cause 
infiltration and often necrosis. Ap- 
plied to the urethral mucous men- 
brane there is produced an inflam- 
mation of short duration. In gon- 
orrhea the secretion is believed to 
be due to these intracellular toxins. 
Repeated injections give only slight 
immunity. The filtrate of recent 
gonococcus cultures contains little 
toxin. 
Pathogenesis. - Non-transmissi- 
ble to all animals. Both the living 
and dead gonococci contain toxic 
substances which cause death or 
injury when injected in large quan- 
tities. 
The etiological relation of the 
gonococcus to human gonorrhea has been demonstrated beyond ques- 
tion by the infection of a number of healthy men with the disease 
by the inoculation of pure cultures of the microorganism. 
Disease Conditions Excited by Gonococci.-Affections due to this 
organism are usually restricted to the mucous membranes of the urethra, 
prostate, neck of bladder, cervix uteri, vagina, and conjunctiva. The 
conjunctival, vaginal, and rectal mucous membranes are much more 
sensitive in early childhood than in later life. The usual course of the 
inflammation is as follows: The gonococci first increase upon the mucous 
membranes which show congestion, infiltration with serous exudate 
and accumulation of leukocytes. The cocci then penetrate the epithe- 
lial layer down to the submucous connective tissue. Recovery or a 
prolonged chronic inflammation may then persist. The original infec- 
tion of the urethra or vagina and cervix may remain localized or spread 
to adjacent parts or through blood and lymph be carried to all parts 
of the body. Gonococci thus cause many cases of endometritis, metritis, 
salpingitis, oophoritis, peritonitis, prostitis, cystitis, epididymitis, and 
Fig. Ill 
Colonies of gonococci on pleuritic fluid 
agar. (Heiman.) DURATION OF INFECTIONS AND OF CONTAGIOUS PERIOD 
287 
arthritis. Abscesses of considerable size, periostitis, and otitis are occa- 
sionally due to the gonococcus. 
Endocarditis and Septicemia.-Cases of gonococcus endocarditis 
and septicemia are not infrequent. Gonococcus septicemia may occur 
in connection with other localizations or alone. Nearly every year 
one or two of these cases are met with in every general hospital. In 
a considerable number of cases where gonococci are obtained from the 
blood the patients recover. The fever is sometimes typhoid-like in 
character. 
Complications.-General infections with gonococci are often fol- 
lowed or accompanied by neuralgic affections, muscle atrophies, and 
neuritis. Urticaria occasionally occurs. 
Immunity.-Immunity in man after recovery from infection 
seems to be only slight in amount and for a short period if present 
at all. It is known that the urethra in man or cervix uteri in woman 
may contain gonococci which lie dormant and may be innocuous in 
that person for years, but which may at any time excite an acute 
gonorrhea in another individual or, under stimulating conditions, in 
the one carrying the infections. Animals may, however, be immunized, 
and their blood is both bactericidal and slightly antitoxic. 
Therapeutic Use of Serum and Vaccine.-The use of sera in acute 
gonorrheal joint inflammation has given in a considerable percentage 
of cases good results and seems to be worth trying. It seems to be 
useless in acute gonorrhea of the mucous membranes. Vaccines (heated 
cultures) have also been used with apparently real benefit in joint 
inflammations and even in very localized chronic infections of the 
urethra, bladder, and elsewhere. They have also been used in acute 
vaginitis in young children. In our cases the symptoms abated sooner 
than we expected, but the gonococci persisted. The dose is from 
twenty to a thousand millions given every three to seven days. The 
benefit of serum and vaccine in septicemia is doubtful. 
Sensitized serum seem to do good in some cases. 
Complement Fixation.-By the method of McNeil (p. 189) very good 
results have been obtained, and the method is used regularly in the 
New York City Health Department. 
Agglutination.-Torrey has shown that gonococci resemble pneu- 
mococci in that there are a number of different strains which have 
different specific and but little common agglutinins. The agglutina- 
tion test is of no practical value in diagnosis. 
Duration of Infections and of Contagious Period.-There is no 
limit to the time during which a man or woman may remain infected 
with gonococci and infect others. We have had one case under obser- 
vation where twenty years had elapsed since exposure to infection, 
and yet the gonococci were still abundant. It is now well established 
that most of the inflammations' of the female genital tract are due 
to gonococci, and the majority of such infections are produced in 
innocent women by their husbands who are suffering from latent 
gonorrhea. 288 
PATHOGENIC MICROORGANISMS 
Bacteriological Diagnosis of Gonorrhea.-In view of the fact that 
several non-gonorrheal forms of urethritis occasionally exist, and also 
that micrococci morphologically similar to Neisser's diplococcus 
are at times found in the normal vulvovaginal tract of adults, it be- 
comes a matter of importance to be able to detect gonococci when 
present, and to differentiate these from the non-specific organisms. 
Besides this, the gonococci which occur in old cultures and in chronic 
urethritis of long standing sometimes take on a very diversified appear- 
ance. From a medicolegal and social stand-point, therefore, the differ- 
ential diagnosis of the gonococcus has in certain cases a very practical 
significance. 
There are three methods of differential diagnosis now available- 
the microscopic, the cultural, and complement fixation. The method 
employed in the latter test is given on p. 189. Animal inoculations 
are of no value, as animals are not susceptible, and, of course, human 
inoculations are generally impossible. In the microscopic diagnosis 
it should be borne in mind that after the acute serous stage has passed, 
the specific gonococci in carefully made preparations are always found 
largely within the pus cells. Diplococci morphologically similar to 
gonococci occurring in other portions of the field and outside of the pus 
cells should not be considered specific by the test only. It should also 
be remembered that the gonococci are decolorized by Gram's method, 
while other similar micrococci which occur in the urethra are, as a rule 
at least, not so decolorized. Organisms having these characteristics 
can for all practical purposes be considered as certainly gonococci if 
obtained from the urethra. From the vulvovaginal tract the certainty 
is not so great, since other diplococci are found in gonorrheal pus 
from this area more frequently than from the urethra which stain as 
gonococci; here cultures should also be made. 
In chronic urethritis Heiman allows the patient to void his urine 
either immediately into two sterilized centrifugal tubes or first into 
two sterile bottles. The first tube will contain threads of the anterior 
urethra; the second tube will be likely to contain secretion from the 
posterior urethra and from the prostate gland if, while urinating, the 
patient's prostate be pressed upon with the finger. Tubes containing 
such urine are placed in the centrifuge and whirled for three minutes 
at twelve hundred or more, revolutions per minute, the threads are 
thrown down. The centrifuged sediment will be found to contan. 
most of the bacteria present, epithelial cells, and, at times, spermatozoia 
When the examinations are negative and it is important to be certain, 
either massage or injections of a solution of silver nitrate may be 
employed. The latter by causing a temporary irritation with increase 
of secretion will almost surely cause a discharge of gonococci if any 
infection was present. 
In acute cases where the pus is abundant the specimen for examina- 
tion may be collected, when the patient is before one, by passing a 
sterilized platinum-wire loop as far up into the urethra as possible 
and withdrawing some of the secretion. BACTERIA RESEMBLING GONOCOCCI 
289 
In vulvovaginitis the procedure should be as follows: 
For obtaining the vaginal material the labia are held well apart by an attend- 
ant wearing sterile rubber gloves. Sterile slender cotton swab is used which 
passes easily into the vagina without touching any external part but entrance. 
(If pus from the cervix is desired a speculum should be used.) The swab is rubbed 
gently about vaginal wall, then withdrawn and rolled (not rubbed) quickly over 
a slide (slide sterilized and held face down if culture is to be made). In making 
this smear care is used not to pass the swab over the same surface twice. In this 
way a beautifully spread film is made. The swab is then returned to its holder 
(and covered and numbered if culture is to be made). The air-dried slide is 
rewrapped in its filter paper and numbered. 
The Technique for Making Culture is as Follows'. A small amount of rich, 
sterile ascitic fluid is added to the tubes containing the swab. (If patient is at 
a distance from laboratory the ascitic fluid is sent in a separate tube, and is 
inoculated from swab just after smear is made; then swab is withdrawn from 
ascitic fluid tube, placed in its own tube, and both tubes are sent immediately 
to the laboratory.) After being stirred up in this fluid the swab is withdrawn 
and smeared in strokes radiating from the centre over ascitic-agar (1-4) plates 
containing 2 per cent, glucose. Other plates of the same medium are stroked 
with platinum loopfuls of the ascitic fluid emulsion. From two to four plates 
are made and placed in thermostat at 36° C. After twenty-four hours the 
plates are examined and if gonococcus-like colonies are seen they are fished. 
Then a smear is made from the whole of one of the most characteristic streaks, 
stained by Gram's method and examined for gonococcus-like organisms. 
Grouping Cases.-From the microscopic examination of well-made and well- 
stained smears (stained by Gram's method), and, where necessary, from cultures 
and from clinical appearance as well, the cases are divided into four groups, 
as follows: 
1. Positive cases, i. e., those showing leukocytes filled with morphologically 
typical gonococci in smear or showing typical cultures, or showing both. 
2. Suspicious cases, i. e., those showing in smears any suspicious intracellular 
diplococci and 50 per cent, or more of polymorphonuclear leukocytes. 
3. Observation cases, i. e., those showing in smears 50 per cent, or over of 
polymorphonuclear leukocytes, but no suspicious intracellular diplococci; or 
those having the clinical symptoms of discharge and inflammation and showing 
less than 50 per cent, of polymorphonuclear leukocytes. 
4. Negative cases, i. e., those showing in smears less than 50 per cent, poly- 
morphonuclears, and no suspicious intracellular diplococci, and no clinical 
evidence of the disease. 
5. Isolation of Groups.-Each group is kept isolated. 
6. Later Smears.-From the first three groups smears are made once a week 
until a negative smear is obtained. From negative groups smears are made if 
any suspicious symptoms appear. , 
7. Negative Diagnosis from Later Smears.-Three successive well-made and 
well-stained negative smears from first three groups, at intervals of not longer 
than three days, means a negative diagnosis. 
8. Later Cultures.-From cases where morphologically typical gonococci 
persist in smear, cultures may be made and gonococcus-like organisms isolated 
and studied to find out if they are true gonococci. 
Bacteria Resembling Gonococci.-A few micrococci which resembled 
gonococci in form and staining have been described. These assume 
importance largely because they may be confused with the gonococcus. 
They occur occasionally on the conjunctival and vaginal mucous mem- 
branes. One of these microorganisms, the Micrococcus catarrhalis (see 
p. 282), has an importance of its own. Others are probably unimpor- 
19 290 
PATHOGENIC MICROORGANISMS 
taut. When absolute certainty is demanded cultural and serologic tests 
must be applied. Differential diagnosis from meningococci is given on 
p. 281. 
MALTA FEVER. 
The Micrococcus Melitensis.-This microorganism was first dis- 
covered in the spleen in a case of Malta fever by Bruce in Malta in 
1887. The disease is mostly confined to the shores of the Mediter- 
ranean, but cases of it have been observed in Porto Rico, China, Japan, 
the Philippines, South America, and South Africa. This disease does 
not seem to be directly transmitted from person to persons. 
Clinical Symptoms.-Prodromal symptoms follow an incubation 
period of five to fourteen days. Headache, sleeplessness, loss of appetite, 
or vomiting accompany a high fever. The fever lasts for weeks, with 
intermissions and remissions. The fever period of one to three weeks 
may occur from time to time during a period of many months. The 
spleen and liver are enlarged. Neuralgic pains are severe. The fatal 
cases appear similar to severe cases of typhoid fever. 
Autopsy.-The spleen is large and very soft. The liver is also large 
and congested. Both organs show parenchymatous degeneration. 
Distribution of Micrococci.-These are abundant in the blood and all 
organs from the second day to the end of the disease. 
Morphology and Biology.-Very small rounded or slightly oval 
organisms, about 0.30/z in their greatest diameter. It is usually single 
or in pairs. In old cultures involution almost bacillary forms occur. 
They are not motile. 
Staining.-They stain readily with aniline dyes and are negative 
to Gram. 
Cultivation.-At 37° C. they grow rather feebly on nutrient agar 
and in broth. The colonies are not usually visible until the third 
day. They appear as small round disks, slightly raised, with a yellowish 
tint in the centre. The broth is slightly clouded after four to six days. 
The culture remains alive for several weeks or months. In gelatin 
the growth is very slow. Gelatin is not liquefied. 
Pathogenesis in Animals.-Monkeys are susceptible. They pass 
through the disease much like man. They can be infected by sub- 
cutaneous or mucous inoculation. Guinea-pigs and rabbits may also 
be infected but the disease progresses slowly in them. In Malta it has 
been found that about half of the goats pass the organisms in feces and 
so contaminate their milk. This is believed to be a source of infection. 
By safeguarding the milk the disease has been largely eliminated. 
Horses and cows are also susceptible. 
Therapeutic Results.-Injections of heated cultures have been 
thought to give good results. 
Methods of Diagnosis.-The diagnosis of Malta fever can frequently 
only be made with the help of bacteriological examination. Malaria, 
typhoid fever, and sepsis are the three diseases most apt to be con- 
founded with it. MICROCOCCUS ZYMOGENS 
291 
Cultures are made by spreading over the surface of a number of 
agar plates freshly drawn blood. Frequently no organisms develop. 
The agglutination test is then required. Many bloods of persons 
suffering from other infections agglutinate the micrococcus of Malta 
fever in low dilutions so that 1: 500 or over is required for a positive 
diagnosis. 
Animals injected with the coccus produce a serum agglutinating in 
high dilutions. Under suitable precautions this can be used to identify 
suspected cultures. 
Laboratory Infection.-A number of workers have infected them- 
selves with more or less serious results. 
MICROCOCCUS ZYMOGENS. 
MacCallum and Hastings1 observed this micrococcus in a case of 
acute endocarditis. It has since been found in a few other pathologic 
processes. It occurs in pairs and short chains. It grows well on agar, 
ferments lactose and glucose, and slowly liquefies gelatin. 
1 Jour. Exp. Med., 1899, iv, 521. CHAPTER XX. 
THE BACILLUS AND THE BACTERIOLOGY OF DIPHTHERIA. 
The lesions of diphtheria are caused by toxemia. The concen- 
trated poison at the seat of the exudate causes intense local inflam- 
mation, while in the more severe cases the absorbed poison diffused 
throughout the body causes widespread cellular injury, giving rise to 
definite injury of the cells of the muscle, nerve, and other tissues. 
Historical Notes.-This specific contagious disease can be traced 
back under various names to almost the Homeric period of Grecian 
history. From time to time during the following centuries we hear of 
epidemics both in Italy and in other portions of the civilized world 
which indicate that the disease never absolutely ceased. 
In 1771 Bard, an American, advanced strong reasons for believing 
that membranous croup and pharyngeal diphtheria were different 
manifestations of the same disease process. 
In 1821 Bretonneau published his first essay on diphtheria in Paris 
and gave to the disease its present name. His observations were so 
extensive and so correct that little advance in knowledge took place 
until the causal relations of the diphtheria bacilli and their associated 
microorganisms to the disease began to be recognized. 
Evidence of Causal Relationship.-As early as 1840 observers began to 
notice microorganisms in the pseudomembranes. Gradually the observa- 
tions became more exact. The most importance was attributed to 
micrococci. In the year 1883, however, bacilli which were very peculiar 
and striking in appearance were shown by Klebs to be of constant 
occurrence in the pseudomembranes from the throats of those dying 
of true epidemic diphtheria. He described the peculiar staining of the 
organisms. One year later Loffler separated these bacilli from the 
other bacteria and grew them in pure culture. When he inoculated the 
bacilli upon the abraded mucous membrane of susceptible animals 
more or less characteristic pseudomembranes were produced, and fre- 
quently death or paralysis followed with characteristic lesions. These 
animal experiments have been fortified by a number of accidental 
human infections in laboratories with pure cultures of bacilli with sub- 
sequent development of diphtheria. 
The Diphtheria Bacillus.-This bacillus is one of the most inter- 
esting of bacteria. Grown in the animal body or in suitable culture 
fluid, it produces a powerful toxin. Its morphology and staining are 
peculiar. Outside of the body it grows best on serum media. 
Morphology.-When cover-glass preparations made from the exu- 
date or from the cultures grown on blood serum are examined, the STAINING 
293 
diphtheria bacilli are found to possess the following morphological 
characteristics: The diameter of the bacilli varies from 0.3m to 0.8m 
and the length from Im to 6m- They occur singly and in pairs (see Figs. 
77 to 84) and very infrequently in chains of three or four. The rods 
are straight or slightly curved, and usually are not uniformly cylindrical 
throughout their entire length, but are swollen at the end, or pointed 
at the ends and swollen in the middle portion. The average length 
of the bacilli in pure cultures from different sources frequently varies 
greatly, and even from the same culture individual bacilli differ much 
in their size and shape. This is especially true when the bacilli are 
grown in association with other bacteria. The two bacilli of a pair 
may lie with their long diameter in the same axis or at an obtuse or 
an acute angle. The bacilli possess no spores, but have in them highly 
refractive bodies, some of which are the starting point for new bacilli 
(see p. 37). There are no flagella. For mode of division, see p. 37. 
Fig. 112 
Fig. 113 
One of the very characteristic forms of 
diphtheria bacilli from blood-serum cultures, 
showing clubbed ends and irregular stain. 
X 1100 diameters. Stain, methylene blue. 
Extremely long form of diphtheria 
bacillus. This culture has grown on 
artificial media for twenty years and pro- 
duces great amounts of toxin. X 1100 
diameters. 
Staining.-The Klebs-Loffler bacilli stain readily with ordinary 
aniline dyes, and retain fairly well their color after staining by Gram's 
method. With Lbffler's alkaline solution of methylene blue, and to 
a less extent with Roux's and dilute Ziehl's solutions, the bacilli from 
blood-serum cultures especially, and from other media less constantly, 
stain in an irregular and extremely characteristic way. (See Fig. 112.) 
The bacilli do not stain uniformly. In many cultures round or oval 
bodies, situated at the ends or in the central portions, stain much more 
intensely than the rest of the bacillus, usually showing metachromatism 
(metachromatic granules. See p. 37 and Plate III). Sometimes these 
highly stained bodies are thicker than the rest of the bacillus; again, 
they are thinner and surrounded by a more slightly stained portion. 
Other bacilli have barred staining. The bacilli stain in this peculiar 
manner at a certain period of their growth, so that only a portion of 
the organisms taken from a culture at any one time will show the 294 
PATHOGENIC MICROORGANISMS 
Fig. 114 
Fig. 115 
Diphtheria bacilli characteristic in shapes, 
but showing even staining. X 1000 dia- 
meters. Stain, methylene blue. 
Non-virulent diphtheria bacilli, show- 
ing stain with Neisser's solutions. This 
appearance was formerly supposed to be 
characteristic of virulent bacilli. Bodies 
of bacilli in smear, yellowish-brown; 
points, dark blue. 
Fig. 116 
Fig. 117 
B. diphtheria agar culture. Bacilli 
small and uniform in shape. X 1000 
diameters. 
B. diphtheriae. Forty-eight hours' agar 
culture. Thick, Indian-clubbed rods and 
moderate number of segments. One year 
on artificial culture media. X 1410 
diameters. 
Fig. 118 
Fig. 119 
B. diphtheriae. Forty-eight hours' agar 
culture. Many segments; long, Indian- 
clubbed ends. One year on artificial 
media. X 1410 diameters. 
B. diphtheriae. Twenty-four hours' 
agar culture. Coccus forms. Segmented 
granular forms on Loffler's serum. Only 
variety found; in cases of diphtheria at 
Children's Home. X 1410 diameters. THE MORPHOLOGY OF THE DIPHTHERIA BACILLUS 
295 
characteristic staining. The young cultures have the most regular forms, 
an eighteen-hour growth showing more clubbed forms than at twelve 
hours. After twenty-four hours the bacilli do not stain quite as well. 
In still older cultures it is often difficult to stain the bacilli, and the 
staining, when it does occur, is frequently not at all characteristic. 
The same round or oval bodies which take the methylene blue more 
intensely than the remainder of the bacillus are brought out still more 
distinctly by the Neisser stain. 
The Neisser stain is carried out by placing the cover-slip smear of diphtheria 
or other bacilli in solution No. 1 for from two or three seconds, and then, after 
washing, in No. 2 for from three to five seconds. The bacilli will then appear 
either entirely brown or will show at one or both ends a dark blue, round body. 
With characteristic diphtheria bacilli, taken from a twelve to eighteen hours' 
growth on serum, nearly all will show the blue bodies (Fig. 115), while with 
pseudo type (Fig. 121), to be described hereafter, few will be seen. 
The solutions are as follows: 
No. 1. 
Alcohol (96 per cent.) 20 parts. 
Methylene blue (Griibler) 1 part. 
Distilled water 950 parts. 
Acetic acid (glacial) 50 parts. 
No. 2. 
Bismarck brown 1 part. 
Boiling distilled water 500 parts. 
The Neisser stain has been advocated in order to separate the viru- 
lent from the non-virulent bacilli, without the delay of inoculating 
animals; but in our hands, with a very large experience, neither the 
Neisser stain not other stains, such as the modifications of the Roux 
stain, have given much more information as to the virulence of the 
bacilli than the usual methylene-blue solution of Lbffler. A few 
strains of virulent bacilli fail to show a marked characteristic stain, 
and quite a few pseudodiphtheria bacilli show the dark bodies. There 
are also in many throats bacilli which seem to have all the staining 
and cultural characteristics of the virulent bacilli, and yet have no 
relation to the disease diphtheria. They are therefore non-virulent in 
the sense that they produce no diphtheria toxin. As will be stated 
more fully later, nothing but animal inoculations with the suspected 
bacilli together with control injections of diphtheria antitoxin will 
separate harmless bacilli from those capable of producing diphtheria. 
The Morphology of the Diphtheria Bacillus on Serum-free Media.-This 
varies considerably with different culture media employed. On glycerin agar 
or simple nutirent agar there are two distinct types. One grows as smaller and, 
as a rule, more regular forms than when grown on serum culture media (Fig. 
116). The other type shows many thick, Indian-clubbed forms with a moderate 
number of segments (Figs. 117-119). Short, spindle, lancet, or club-shaped 
forms, staining uniformly, are all observed. The bacilli which have developed 
in the pseudomembranes or exudate in cases of diphtheria resemble in shape 
young bacilli grown on blood-serum, but stain more evenly. 296 
PATHOGENIC MICROORGANISMS 
Biology.-The Klebs-Loffler bacillus is non-motile and non-liquefy- 
ing. It is aerobic and facultative anaerobic. It grows most readily in 
the presence of oxygen. It does not form spores. It begins to develop, 
but grows slowly at a temperature of 20° C., or even less. It attains 
its maximum development at 37° C. In old cultures in fluid media, 
Williams has observed fusion of one bacillus with another. The fused 
forms live the longest. 
Growth on Culture Media.-Blood-serum.-Blood-serum, especially 
coagulated in the form of Lbffler's mixture, is the most favorable 
medium for the growth of the diphtheria bacillus, and is used particu- 
larly for diagnostic purposes in examining cultures from the throats 
of persons suspected of having diphtheria. For its preparation, see 
p. 315. If we examine the growth of diphtheria bacillus in pure culture 
on blood serum we shall find at the end of from eight to twelve hours 
small colonies of bacilli, which appear as pearl-gray, whitish-gray or, 
more rarely, yellowish-gray, slightly raised points. The colonies 
when separated from each other may increase in forty-eight hours 
so that the diameter may be one-eighth of an inch. The colonies lying 
together become confluent and fuse into one mass when the serum is 
moist. The diphtheria colonies after a growth of twelve hours become 
larger than those of the streptococci but remain smaller than those of 
the staphylococci. 
Growth on Agar.-On 1 per cent, slightly alkaline, nutrient or glycerin- 
agar the growth of the diphtheria bacillus is less certain and luxuriant 
than upon blood serum; but the appearance of the colonies when 
examined under a low-power lens, though very variable, is often far 
more characteristic. (Fig. 120). For this reason nutrient agar in Petri 
dishes is used to obtain diphtheria bacilli in pure culture. The diph- 
theria bacillus obtained from cultures which have developed for some 
time on culture media grows well, or fairly well, on suitable nutrient 
agar, but when fresh from pseudomembranes one prevalent type of 
bacilli grows on these media with great difficulty, and the colonies 
develop so slowly as to be frequently covered up by the more luxuriant 
growth of other bacteria when present, or they may fail to develop at all. 
If the colonies develop deep in the substance of the agar they , are 
usually round or oval, and, as a rule, present no extensions; but if 
near the surface, commonly from one, but sometimes from both sides, 
they spread out an apron-like extension, which exceeds in surface area 
the rest of the colony. When the colonies develop entirely on the 
surface they are more or less coarsely granular, and usually have a 
dark centre and vary markedly in their thickness. The colonies from 
some are almost translucent; from others are thick and almost as lux- 
uriant as the staphylococcus. The edges are sometimes jagged, and 
frequently shade off into a delicate lace-like fringe; at other times the 
margins are more even and the colonies are nearly circular. 
Peculiarites in the growth of the diphtheria bacillus upon agar are of prac- 
tical importance. If a large number of the bacilli from a recent culture are 
implanted upon a properly prepared agar plate a certain and fairly vigorous GROWTH IN ASCITIC OR SERUM BOUILLON 
297 
growth will always take place. If, however, the agar isr inoculated with an 
exudate from the throat, which contains but a few bacilli, no growth whatever 
may occur, while the tubes of coagulated blood-serum inoculated with the same 
exudate contain the bacilli abundantly. Because of the uncertainty, therefore, 
of obtaining a growth by the inoculation of agar with bacilli unaccustomed to 
this medium, agar is not a reliable medium for use in primary cultures for diag- 
nostic purposes. A mixture composed of two parts of a 1.5 per cent, nutrient 
agar and one part of sterile ascitic fluid makes a medium upon which the 
bacillus grows much more luxuriantly, but not so characteristically. 
Isolation of the Diphtheria Bacillus from Plate Cultures.-Nutrient plain or 
glycerin-agar should be freshly melted and poured in the Petri dish for this pur- 
pose. After it has hardened, the medium in a number of plates is streaked 
across with bacteria from colonies on the serum culture, which appear in size 
and color like the diphtheria bacilli. 
Other plates are made from a general 
mixture of all bacteria, selected, as a 
rule, from the drier portion of the 
serum. Still others are- inoculated 
from the pellicles of ascitic broth 
cultures. The plates are left in the 
incubator for about sixteen hours at 
37° C. In the examination of the 
plates one should first seek for typical 
colonies, then, if these are not found, 
for any that look most nearly like the 
characteristic picture. Diphtheria col- 
onies are very apt to be found at the 
edges of the streaks of bacterial growth. 
The pickings from the colonies are 
inoculated upon Lbffler's blood-serum, 
or into ascitic bouillon. 
Growth in Bouillon.-The diphtheria 
bacilli from about one-half the cul- 
tures grow readily in broth slightly 
alkaline to litmus; the other cultures 
grow feebly. The characteristic growth in neutral bouillon is one showing fine 
grains. These deposit along the sides and bottom of the tube, leaving the broth 
nearly clear. A few cultures in neutral bouillon and many in alkaline bouillon 
produce for twenty-four or forty-eight hours a more or less diffuse cloudiness, 
and frequently a film forms over the surface of the broth. On shaking the 
tube this film breaks up and slowly sinks to the bottom. This film is apt to 
develop during the growth of cultures which have long been cultivated in bouil- 
lon, and, indeed, after a time the entire development may appear on the surface 
in the form of a friable pellicle. The diphtheria bacillus in its growth causes a 
fermentation of meat-sugars and glucose, and thus, if these are present, changes 
the reaction of the bouillon, rendering it distinctly less alkaline within forty- 
eight hours, and then, after a variable time, when all the fermentable sugars 
have been decomposed, more alkaline again through the progressing fermenta- 
tion of other substances. Among the products formed by its growth is the 
diphtheria toxin. 
Fig. 120 
Colonies of diphtheria bacilli. X 200 
diameters. 
Growth in Ascitic or Serum Bouillon.-All varieties of diphtheria 
bacilli grow well in this medium, even when first removed from the 
throat. They almost always form a slight pellicle at the end of twenty- 
four to forty-eight hours. This culture medium is, as pointed out 
by Williams, of the greatest value in attempts to get pure cultures 
of the diphtheria bacillus from solidified serum cultures containing 298 
PATHOGENIC MICROORGANISMS 
few bacilli among many other bacteria. Plate cultures are made from 
the pellicle. The fluid is prepared by adding to the nutrient bouillon 
25 per cent, ascitic fluid or blood-serum. 
Growth on Gelatin.-The growth on this medium is much slower, more 
scanty, and less characteristic than that on the other media mentioned. This 
largely is on account of the lower temperature at which it must be used. 
Growth in Milk.-The diphtheria bacillus grows readily in milk, 
beginning to develop at a comparatively low temperature (20° C.). 
The milk remains unchanged in appearance as lactose is not fermented 
by the diphtheria bacillus. 
Pathogenesis.-In Lower Animals.-The diphtheria bacillus through 
its toxins is, when injected into their bodies, pathogenic for guinea- 
pigs, rabbits, chickens, pigeons, small birds, and cats; also in a lesser 
degree for dogs, goats, cattle, and horses, but hardly at all for rats and 
mice. In spite of its pathogenic qualities for these animals true diph- 
theria occurs in them with extreme rarity. As a rule, supposed diph- 
theritic inflammations in them are due to other bacteria which cannot 
produce disease in man. The cat is the only animal that we have 
known to contract true diphtheria from contact with the disease. 
Cobbett reports a case in a colt. At the autopsy of animals dying from 
the poisons produced by the bacilli, the characteristic lesions described 
by Loftier are found. At the seat of inoculation there is a grayish 
focus surrounded by an area of congestion; the subcutaneous tissues for 
some distance around are edematous; the adjacent lymph nodes are 
swollen; and the serous cavities, especially the pleura and the peri- 
cardium, frequently contain an excess of fluid usually clear, but at times 
turbid; the lungs are generally congested, the suprarenals are markedly 
congested. In the organs are found numerous smaller and larger masses 
of necrotic cells, which are permeated by leukocytes. The heart and 
certain voluntary muscular fibers and nervous tissues usually show 
degenerative changes. Occasionally there is fatty degeneration of the 
liver and kidneys. The number of leukocytes in the blood is increased. 
From the area surrounding the point of inoculation virulent bacilli 
may be obtained, but in the internal organs they are only occasionally 
found, unless an enormous number of bacilli have been injected. Par- 
alysis, commencing usually in the posterior extremities and then grad- 
ually extending to the whole body and causing death by paralysis of 
the heart or respiration, is also produced in many cases in which the 
inoculated animals do not succumb to a too rapid intoxication. In a 
number of animals we have seen recovery take place three to six weeks 
after the onset of the paralysis. 
Tissue Changes in Natural (Human) Infection.-The characteristic 
lesions are a pseudomembranous inflammation on some of the mucous 
membranes or occasionally on the surface of wounds and the general 
hyperplasias and parenchymatous inflammations produced by the 
absorbed toxic substances. Pneumonia is apt to occur as a compli- 
cation of laryngeal diphtheria. The membrane may be simply a thin THE PRODUCTION OF TOXIN IN CULTURE MEDIA 
299 
pellicle, which is easily removed without causing bleeding or it may 
be thick and firmly attached and leaving when removed a ragged 
bleeding surface. The tissue beneath the pseudomembrane is always 
intensely injected and often hemorrhagic. The cells show marked 
degenerative changes. 
Causes of Death.-These are chiefly toxemia, laryngeal obstruction 
and bronchopneumonia. Septicemia due to other bacteria is frequently 
an additional factor. 
Diphtheria Toxin.-This poison was assumed by Loftier (1884) to 
be produced by the bacilli, but it was first partially isolated by Roux 
and Yersin, who obtained it by filtration through porous porcelain 
from cultures of the living bacilli. It has not yet been successfully 
analyzed, so that its chemical composition is unknown, but it has 
many of the properties of proteid substances, and can well be desig- 
nated by the term active proteid. It resembles in many ways the 
ferments. After injection into the body there is a latent period before 
its poisonous action appears. The poison produced is probably com- 
posed of a mixture of several nearly related toxins. Diphtheria toxin 
is totally destroyed by boiling for five minutes, and loses some 95 per 
cent, of its strength when exposed to 75° C. for the same time; 60° C. 
destroys very little. Lower temperatures only alter it very gradually. 
Kept cool and from light and air, it deteriorates very slowly. Desiccated 
at low temperatures and kept dry in a vacuum it keeps unaltered for 
long periods. 
The Production of Toxin in Culture Media.-The artificial production of toxin 
from cultures of the diphtheria bacillus has been found to depend upon definite 
conditions, which are of practical importance in obtaining toxin for the inocu- 
lation of horses, and also of theoretic interest in explaining why cases of appar- 
ently equal local severity have such different degrees of toxic absorption. The 
researches of Roux and Yersin laid the foundation of our knowledge. Their 
investigations have been continued by Theobald Smith, Spronck, ourselves, 
and others. After an extensive series of investigations we (Park and Williams) 
came to the following conclusions: Toxin is produced by fully virulent diph- 
theria bacilli at all times during their life when the conditions are favorable. 
Under less favorable conditions some bacilli are able to produce toxin while 
others are not. Diphtheria bacilli may find conditions suitable for luxuriant 
growth, but unsuitable for the production of toxin. The requisite conditions 
for good development of toxin, as judged by the behavior of a number of cul- 
tures, are a temperature from about 32° to 37° C., a suitable culture medium, 
such as a 2 per cent, peptone nutrient bouillon made from veal, of an alkalinity 
which should be about 9 c.c. of normal soda solution per liter above the neutral 
point to litmus, and prepared from a suitable peptone (Witte) and meat. The 
culture fluid should be in comparatively thin layers and in large-necked Erlen- 
meyer flasks, so as to allow of a free access of air. The greatest accumulation 
of toxin in bouillon is after a duration of growth of the culture of from five to 
ten days, according to the peculiarities of the culture employed. At a too 
early period toxin has not sufficiently accumulated; at a too late period it has 
begun to degenerate. In our experience the amount of muscle-sugar present 
in the meat makes no appreciable difference in the toxin produced when a vigor- 
ously growing bacillus is used, so long as the bouillon has been made sufficiently 
alkaline to prevent the acid produced by the fermentation of the sugar from 
producing in the bouillon an acidity sufficient to inhibit the growth of the bacilli. 300 
PATHOGENIC MICROORGANISMS 
With the meat as we obtain it in New York, we usually get better results with 
unfermented meat than with fermented. In Boston, with the same bacillus, 
Smith gets more toxin from the bouillon in which the sugar has been fermented 
by the colon bacillus. Instead of colon bacilli, yeast may be added to the 
soaking meat, which is allowed to stand at about 25° C. The preliminary fer- 
mentation of the meat sometimes produces poisonous substances which are 
deleterious to the horses. -We have obtained especially good results with veal 
broth made from calves two to four weeks old (bob veal). When strong toxin 
is desirable the muscle is separated from all fat, tendon and fibrous tissue before 
being chopped. 
Under the best conditions we can devise, toxin begins to be produced by 
bacilli from some cultures when freshly sown in bouillon some time during the 
first twenty-four hours; from other cultures, for reasons not well understood, 
not for from two to four days. In neutral bouillon the culture fluid frequently 
becomes slightly acid and toxin production may be delayed for from one to 
three weeks. The greatest accumulation of toxin is on the fourth day, on the 
average, after the rapid production of toxin has commenced. After that time 
the number of living bacilli rapidly diminishes in the culture, and the con- 
ditions for those remaining alive are not suitable for the rapid production of 
toxin. As the toxin is not stable at 35° C., the deterioration taking place in the 
toxin already produced is greater than the amount of new toxin still forming. 
Bacilli, when repeatedly transplanted from bouillon to bouillon, gradually 
come to grow on the surface only. This characteristic keeps the bacilli in 
contact with the oxygen and seems to aid in the development of toxin. 
Comparative Virulence as Estimated by Toxin Production of Dif- 
ferent Cultures.-The virulence of diphtheria bacilli from different 
sources, as measured by their toxin production, varies considerably. 
Thus, as an extreme instance, 0.002 c.c. of a forty-hour bouillon culture 
of our most virulent bacilli will kill a guinea-pig, which it would require 
0.1 c.c. of culture of our least virulent bacillus to kill. This difference 
frequently depends on the unequal growth of the bacilli, one culture 
having fifty times as many bacilli as the other. When the different 
strains are grown on ascitic broth, upon which their growth is usually 
good, the majority of cultures are nearly equal in virulence, but some 
still show marked differences. Moreover, the diphtheria bacilli differ 
in the tenacity with which they retain their virulence when grown 
outside the body. The bacillus that we have used to produce toxin in 
the laboratory of the Board of Health has retained its virulence unaltered 
for twenty years in bouillon cultures. Other bacilli have apparently 
lessened their capacity for toxin production after being kept six months. 
Brown and Smith1 report on a culture in which 5 c.c. was the minimum 
toxic dose of the filtrate. The passage of diphtheria bacilli through 
the bodies of susceptible animals does not increase their toxin production 
to any considerable extent. 
Comparative Virulence of Bacilli and Severity of Case.-From the 
severity of an isolated case the virulence of the bacilli cannot be deter- 
mined. The presence of slight amounts of antitoxin in the blood of 
the person attacked and the association of other bacteria are at least 
two of the disturbing factors. The most virulent bacillus wTe have ever 
found was obtained from a mild case of diphtheria simulating tonsillitis. 
1 Jour. Med. Research, vol. xxx, No. 3, p. 443. PERSISTENCE OF DIPHTHERIA BACILLI IN THE THROAT 
301 
Another case, however, infected by the bacillus proved to be very 
severe. In localized epidemics the average severity of the cases prob- 
ably indicates roughly the virulence of the bacillus causing the infection, 
as here the individual susceptibility of the different persons infected 
would, in all likelihood, when taken together, be similar to that of other 
groups; but even in this instance special conditions of climate, food, 
or race may influence certain localities. It must be remembered that 
bacilli of like toxic power may differ in their liability to infect the mucous 
membrane. Virulence has thus two distinct meanings when used in 
connection with diphtheria bacilli. 
Virulent Bacilli in Healthy Throats.-Fully virulent bacilli have 
frequently been found in healthy throats of persons who have been 
brought in direct contact with diphtheria patients or diphtheria carriers 
without contracting the disease. It is, therefore, apparent that infec- 
tion in diphtheria, as in other infectious diseases, requires not only 
the presence of virulent bacilli, but also a susceptibility to the disease, 
which may be local or general. We now know that 50 per cent, of 
all persons are protected from infection because of having antitoxin 
present in their blood. Among the predisposing influences which con- 
tribute to the production of diphtheritic infection may be mentioned 
the breathing of foul air and living in overcrowded and ill-ventilated 
rooms, impure food, certain diseases, more particularly catarrhal 
inflammations of the mucous membranes, and depressing conditions 
generally. Under these conditions an infected mucous membrane 
may become susceptible to disease. In connection with Beebe (1894) 
we made an examination of the throats of 330 healthy persons who had 
not come in contact, so far as known, with diphtheria, and we found 
virulent bacilli in 8, only 2 of whom later developed the disease. In 
24 of the 330 healthy throats non-virulent bacilli similar to the viru- 
lent diphtheria bacillus were found. Very similar observations have 
since been made in Boston and by others in many widely separated 
countries. In 1905 Von Sholly in our laboratory examined 1000 throats 
of those who had not knowingly been in contact with diphtheria and 
found virulent diphtheria bacilli in 0.5 per cent, of the cases. We have 
found virulent bacilli in about 5 per cent, of cases of scarlet fever. 
This indicates they are more prevalent in throats than a single culture 
from normal cases indicate. 
Persistence of Diphtheria Bacilli in the Throat.-The continued 
presence of virulent diphtheria bacilli in the throats of patients who 
have recovered from the disease has been demonstrated by all inves- 
tigators. In the investigations of 1894 we found that in 304 of 605 
consecutive cases the bacilli disappeared within three days after the 
disappearance of the pseudomembrane; in 176 cases they persisted for 
seven days, in 64 cases for twelve days, in 36 cases for fifteen days, in 
12 cases for three weeks, in 4 cases for four weeks, and in 2 cases for 
nine weeks. Since then we have met with a case in which they persisted 
with full virulence for eight months. It is safe to say that in over 10 
per cent, of the cases a few bacilli persist two weeks after the disappear- 302 
PATHOGENIC MICROORGANISMS 
ance of the exudate and in over 1 per cent, four weeks. It is extremely 
difficult to prove that a case is absolutely clear as the bacilli may remain 
hidden in the epithelial cells of some tonsillar crypt and not be detected 
by cultures. 
Diphtheria-like Bacilli Not Producing Diphtheria Toxin.-In the 
tests of the bacilli obtained from hundreds of cases of suspected diph- 
theria which have been carried out during the past twenty years in 
the laboratories of the Health Department of New York City, in over 
95 per cent, of cases the bacilli derived from exudates or pseudomem- 
branes and possessing the characteristics of the Loffler bacilli have 
been found to be virulent, that is, producers of diphtheria toxin. But 
there are, however, in inflamed throats as well as in healthy throats, 
either alone or associated with the virulent bacilli, occasionally bacilli 
which, though morphologically and in their behavior on culture media 
identical with the Klebs-Loffler bacillus, are yet producers, at least in 
artificial culture media and the usual test animals, of no diphtheria 
toxin. Between bacilli which produce a great deal of toxin and those 
which produce none we find few minor grades of virulence. We believe, 
therefore, in accordance with Roux and Yersin these non-virulent 
bacilli should be considered as possibly attenuated varieties of the 
diphtheria bacillus which have lost their power to produce diphtheria 
toxin. This supposition is, however, not proven and it may be that 
the ancestors of these bacilli were never toxin producers. These ob- 
servers, and others following them, have claimed that the virulent 
bacilli can be artificially attenuated; but the reverse has not been proven 
that bacilli which produce no specific toxin have later been found to 
develop it. In our experience some cultures hold their virulence even 
when grown at 41° C. for a number of months, while others become 
slightly attenuated rather quickly. We have never yet been able to 
change a virulent culture into an absolutely non-virulent one, and we 
have never accomplished the reversed process. We believe therefore that 
for practical purposes bacilli which produce no toxin in animals can be 
disregarded as a possible source of human diphtheria. It may be added 
here that no facts have come to light which indicate that bacilli which 
do not produce diphtheria toxin in animals ever produce it in man. 
It must also be borne in mind, however, that such proof is necessarily 
very difficult to obtain. 
Bacilli Resembling the Diphtheria Bacilli.-Diphtheria-like bacilli 
are also found which resemble diphtheria bacilli very closely except 
in toxin production, but differ in one or more particulars. Both these 
and the characteristic non-virulent bacilli are found occasionally upon 
all the mucous membranes both when inflamed and when apparently 
normal. 
Diphtheria-like Bacilli Pathogenic to Guinea-pigs Producing no 
Diphtheria Toxin.-These bacilli are obtained frequently from normal 
or slightly inflamed throats or from other mucous membranes, and may 
be slightly pathogenic in guinea-pigs, since they may kill, as we found, 
in doses of 2 to 5 c.c. of broth culture subcutaneously or intraperi- HOFMANN'S PSEUDODIPHTHERIA BACILLI 
303 
toneally injected. Animals are not protected by diphtheria antitoxin 
from the action of these bacilli showing that their poisonous action is 
not due to diphtheria toxin. At autopsy the bacilli are usually found 
more or less abundantly in the blood and internal organs. The fact 
that large injections of antitoxic serum hastens the death of guinea- 
pigs injected with these bacilli has given rise to the notion that injections 
of antitoxin might be dangerous in persons in whose throats these bacilli 
were present, either as saprophytes, ui possibly, as inciters of slight 
disease. It is not the antitoxin, but the serum, which in large doses 
injures the vitality of the guinea-pigs and so slightly hastens death. 
The amount of serum required to produce this effect is far in excess 
of that properly given in man. These bacilli were first described by 
Davis1 from our laboratory and later by Hamilton in 1904. The pos- 
sibility of their being present affords no reason to avoid giving antitoxin 
in suspected cases. When pathogenic in man they are usually only 
feebly so. 
Hofmann's Pseudodiphtheria Bacilli.-Besides the typical bacilli 
which produce no diphtheria toxin, but which, so far as we can deter- 
mine, are otherwise identical with the Loffler bacillus, there are other 
bacilli found in positions similar to those 
in which diphtheria bacilli abound, which, 
though resembling these organisms in many 
particulars, yet differ from them as a class 
in others equally important. The variety 
most prevalent is rather short, plump, 
and more uniform in size and shape than 
the true Loffler bacillus (Fig. 121). On 
blood-serum their colony growth is very 
similar to that of the diphtheria bacilli. 
The great majority of them in any young 
culture show no polar granules when stained 
by the Neisser method, and stain evenly 
throughout with the alkaline methylene 
blue solution. They do not produce acid 
by the fermentation of glucose, as do all known virulent and many 
non-virulent diphtheria bacilli; therefore, there is no increase in 
acidity in the bouillon in which they are growm during the first twenty- 
four hours from the fermentation of the meat-sugar regularly present. 
They are found in varying abundance in different localities in New York 
City, in about 1 per cent, of the normal throat and nasal secretions, 
and seem to have now at least no connection with diphtheria; whether 
they were originally derived from diphtheria bacillus is doubtful; they 
certainly seem to have no connection with it now. They have been 
called pseudodiphtheria bacilli, and more properly, B. hofmanni? In 
bouillon they grow, as a rule, less luxuriantly than the diphtheria bacilli. 
Some of the varieties of the pseudodiphtheria bacilli are as long as the 
Fig. 121 
Psuedodiphtheria bacilli. 
(B. hofmanni.) 
1 Medical News, April 29, 1899. 
2 Clark, Jour, of Inf. Dis., 1910, vii, 335. 304 
PATHOGENIC MICROORGANISMS 
shorter forms of the virulent bacilli. When these are found in cultures 
from cases of suspected diphtheria they may lead to an incorrect diag- 
nosis. These bacilli are found occasionally in all countries where search 
has been made for them. There are also some varieties which resemble 
the short pseudobacilli in form and staining, but which produce acid 
in glucose bouillon. 
Bacillus Xerosis.-Diphtheria-like bacilli were found by Kutschert 
and Neisser (1884) in the condition known as xerosis conjunctivae. 
Since then, they have been found frequently in normal conjunctivae. 
These bacilli went by the name of xerosis bacilli. Under this name, no 
doubt, different observers have placed several different varieties of 
bacilli morphologically somewhat similar to the diphtheria bacilli. 
Knapp first limited the name to bacilli that ferment saccharose and not 
dextrin, in contradistinction to diphtheria bacilli which ferment dextrin 
and not saccharose. The fermentation tests for three of the diphtheria 
group, according to Knapp, may be shown as follows: 
Species. 
Dextrose. 
Levulose. 
Galactose. 
Maltose. Saccharose. Dextrin. 
B. 
diphtheria . 
. . + 
+ 
+ 
+ - + 
B. 
xerosis 
. • + 
+ 
+ 
+ + - 
B. 
hofmanni 
- 
- 
- 
- - - 
Note.-One per cent, sugar in Hiss serum-water media. 
These reactions do not separate a large number of the non-virulent 
bacilli morphologically like the diphtheria bacilli, which are found in 
throats under varying conditions. 
Persistence of Varieties of the Bacillus Diphtheria and of Diph- 
theria-like Bacilli.-The fact that there are distinct differences between 
strains of bacilli producing diphtheria toxins which are as great as 
between these and some strains of bacilli producing no diphtheria 
toxins has, we think, been fully established. 
But that such varieties are true sub-species with constant charac- 
teristics, one variety not changing into another of the established forms, 
has not been accepted by all. On the contrary, the opinion is held 
by some investigators that all of the various forms of diphtheria-like 
bacilli are the result of more or less transitory variations of the same 
species, and hence that the virulent forms are the result of a rapid 
adaptation to environment and consequent pathogenesis of the non- 
virulent forms, both typical and atypical. This question is of great 
practical importance in methods of handling the persons harboring these 
bacilli. 
Wesbrook, Wilson, and McDaniel,1 make a provisional classification 
based upon the morphology of the individual bacilli, into three groups, 
called granular, barred, and solid, two of the groups into seven types 
and the other into five, two of the types corresponding with those in 
the other groups not having been seen. They state that there is generally 
a sequence of types in the variations which appear throughout the 
course of the disease, the granular types, as a rule, predominating at 
1 Transactions of the Association of American Physicians, 1900. RESISTANCE TO HEAT, DRYING, AND CHEMICALS 
305 
the outset of the disease, and these giving place wholly or in part to 
the barred and solid types shortly before the disappearance of diphtheria- 
like organisms. 
The inference drawn from this work is that the diphtheria bacillus 
may be rather easily, especially in the throat, converted into non- 
granular, solidly staining forms of the "pseudodiphtheria" type, and 
that the converse may occur, and that therefore all diphtheria-like 
bacilli must be considered a possible source of danger. 
In studying the subject Williams1 (1902) came to the following con- 
clusions: Though some cultures change on some of the media, each 
changes in its own way, and each culture still has its distinct individ- 
uality. After many culture generations, especially when transplanted 
at short intervals, the different varieties of virulent diphtheria bacilli 
tend to run in lines parallel with a common norm, which seems to be a 
medium-sized, non-segmented bacillus, producing granules in early 
cultures on serum and growing well on all of the ordinary culture media. 
The non-virulent morphologically typical bacilli must be classed with 
the virulent varieties as one species, though there is little doubt that 
more minute study would show that the former constitute a distinct 
group. The atypical pseudo forms, however, which show no tendency 
to approach the norm of the typical forms, must be classed as distinct 
species. All of the pseudo and the non-virulent morphologically typical 
varieties when inoculated into the peritoneum of guinea-pigs in immense 
doses cause death. In studying successive direct smears from the 
throats of diphtheria patients no evidence of change from one type to 
the other was noted. Attempts have been made to give more virulence 
to some of these varieties by successive peritoneal inoculations, and by 
growing the organisms in symbiosis with several other organisms, but 
in no instance has any increase of virulence or decided change in mor- 
phological or cultural characteristics been noted. 
Since there are so many different forms or varieties of diphtheria- 
like bacilli, it is quite possible that some of them are derived from 
strains of the diphtheria bacillus and that under certain conditions 
they readily regain its characteristics. This seems to be the only, 
way to explain the apparent discrepancies in the results obtained by 
different observers. Such closely related varieties, however, do not 
appear to be common and we have up to the present been unable to 
obtain them. So we may safely say that in this region, at least, non- 
virulent diphtheria-like organisms retain their characteristics under 
various artificial and natural conditions, and that they may be regarded 
from a public health standpoint as harmless. 
Resistance to Heat, Drying, and Chemicals.-The thermal death point 
with ten minutes' exposure is about 60° C., with five minutes 70° C. 
Boiling kills almost instantly. It has about the average resistance of 
non-spore-bearing bacteria to disinfectants. In the dry state and 
exposed to diffuse light diphtheria bacilli usually die in from a few hours 
1 Journal of Med. Research, June, 1902. 306 
PA T HOG ENIC MICROORGANISMS 
to a few days, but they may live for months; when in the dark, or pro- 
tected by a film of mucus or albumin, they may live for even longer 
periods. Thus we found scrapings from a dry bit of membrane to con- 
tain vigorous and virulent living bacilli for a period of four months 
after removal from the throat, and if the membrane had not been at 
that time completely used, living bacilli could probably have been 
obtained for a much longer period. On slate- and lead-pencils, toys, 
tumblers, as well as on paper money, they .may live for several weeks, 
while on metal coins they die in twelve to thirty-six hours. In culture 
media, when kept at the blood-heat, they usually die after a few weeks; 
but under certain conditions, as when sealed in tubes and protected 
from heat and light, they retain their life and virulence for years. 
The bacillus is not sensitive to cold, for we found about 10 per cent, 
of the bacilli to retain their vitality and virulence after exposure for 
two hours to several hundred degrees below zero. At temperatures 
just below freezing they may remain alive for a number of weeks. 
Transmission of Diphtheria.-The possibility of the transmission 
of diphtheria from animals to man cannot be disputed; we have met 
with two instances where cats had malignant diphtheria, and many 
other animals can be infected, but there are no authentic cases of 
such transmission on record. So-called diphtheritic disease in animals 
and birds is usually, if not always, due to other microorganisms than 
the diphtheria bacilli. 
The virulent bacilli have been found on soiled bedding or clothing 
of a diphtheria patient, or drinking-cups, candy, shoes, hair, slate- 
pencils, etc. These sources of infection by which the disease may be 
indirectly transmitted are, however, the less important ones. The usual 
source of the bacilli are the discharges of diphtheria patients; the secre- 
tions of the nose and throat of convalescent cases of diphtheria in which 
the virulent bacilli persist from the healthy throats of individuals 
who acquired the bacilli from being in contact with others having 
virulent germs. When we consider the number of healthy carriers and 
that it is only the severe types of diphtheria that remain isolated during 
their actual illness, the wonder is not that so many, but that so few, 
persons contract the disease. 
Susceptibility to and Immunity against Diphtheria.-An individual 
susceptibility, both general and local, to diphtheria, as in all infec- 
tious diseases, is necessary to contract the disease. Age has long been 
recognized to be an important factor in diphtheria. Children within 
the first six months of life are but little susceptible, but exceptionally 
infants of a few weeks are attacked, the greatest degree of susceptibility 
being between the second and tenth year. After that age susceptibility 
decreases. Young animals born of mothers immune to diphtheria 
possess nearly the same degree of immunity as their mothers. They 
gradually lose this but retain traces up to six to twelve months. The 
human infant is now known to receive immunity from its mother. 
Diphtheria Antitoxin.-As the result of animal experiments, it is now 
known that an artificial immunity against diphtheria can be produced, 
by the action of diphtheria toxin on the cells causing the development USE OF ANTITOXIN IN TREATMENT AND IMMUNIZATION 
307 
of substances directly antidotal to the diphtheria toxin. The blood- 
serum of about 50 per cent, of persons who have recovered from 
diphtheria has been found to possess to some degree this protective 
property, which it acquires about a week after the beginning of the 
disease, and loses again before the end of a year. Those not possessing 
antitoxin probably have protection from bactericidal substances which 
have developed because of the infection. Moreover, the blood-serum 
of more than one-half of all individuals, usually adults, who have never, 
so far as known, had diphtheria, has a slight amount of antitoxin. 
Neutralizing Characteristics of Antitoxin.-Diphtheria antitoxin 
has the power of neutralizing diphtheria toxin, so that when a certain 
amount is injected into an animal before or together with the toxin 
it overcomes its poisonous action. There is a direct action of the anti- 
toxin upon its corresponding toxin. 
The various attempts to separate the toxin and antitoxin from 
neutral mixtures have been failures, and it is found that neutralization 
takes place according to the law of multiple proportions, i. e., to save 
an animal from 1000 fatal doses of diphtheria toxin requires little more 
than a hundred times as much antitoxin as is required for ten fatal 
doses, the resistance of the animal itself accounting for the differ- 
ence. The general characteristics of antitoxin are considered in Part I. 
Use of Antitoxin in Treatment and Immunization.-The antitoxin 
in the higher grades of globulin solution or serum is identical with that 
in the lower grades; there is simply more of it-in each drop. In treat- 
ment, however, for the same amount of antitoxin we have to inject 
less foreign proteids with the higher grades, and, therefore, have some- 
what less danger of rashes and other deleterious results. The amount 
of antitoxin required for immunization is 300 to 500 units for an infant, 
500 to 1000 for an adult, and proportionately for those between these 
extremes. The larger doses are advised when the danger of infection 
is very great. After the observation of the use of antitoxin in the 
immunization of several thousand cases, we have absolute belief in its 
power to prevent an outbreak of diphtheria when given in the amounts 
advised for at least two weeks, and also of its almost complete harmless- 
ness in the small doses required. When double the above quantities are 
given the immunity is prolonged on the average for abour one week. 
If it is desired to prolong the immunity the antitoxin injection is repeated 
every ten days. For treatment, mild cases should be given 2000 
units, moderate cases 2000 to 8000 units, and severe cases 10,000 to 
20,000 units. One injection suffices, if the proper amount is given. 
A glance at the charts will make this clear. The large amount of anti- 
toxin given in bad cases is for the purpose of quickly reaching the cells 
already attacked by the toxin and not because of the amount of toxin 
to be neutralized. Intravenous injections give most rapid effect, and 
should be used in all malignant cases. It takes forty-eight to seventy- 
two hours for the absorption into the blood of the greater part of the 
antitoxin from the subcutaneous tissues. This in bad cases may be 
a fatal delay. Antitoxin is only to a very slight extent absorbed when 
given by the mouth. 308 
PATHOGENIC MICROORGANISMS 
Results of the Antitoxin Treatment of Diphtheria.-The conclu- 
sions arrived at by Biggs and Guerard, after a review of a large amount 
Fig. 122 
Amount of antitoxin in 1 c.c. of serum from persons, at different intervals of time, 
after a single subcutaneous injection of 10,000 units. 
of the statistics and opinions published since the beginning of the anti- 
toxin treatment in 1892, were as follows: 
"It matters not from what point of view the subject is regarded, 
if the evidence now at hand is properly weighed, but one conclusion is 
Fig. 123 
Amount of antitoxin in 1 c.c. of serum from persons, at different intervals of 
time, after a single intravenous injection of 10 000 units. 
or can be reached-whether we consider the percentage of mortality 
from diphtheria and croup in cities as a whole, or in hospitals, or in 
private practice; or whether we take the absolute mortality for all the DETERMINATION OF THE PRESENCE OF ANTITOXIN 
309 
cities of Germany whose population is over 15,000, and all the cities 
of France whose population is over 20,000; or the absolute mortality 
for New York City, or for the great hospitals in France, Germany, 
and Austria; or whether we consider only the most fatal cases of diph- 
theria, the laryngeal and operative cases; or whether we study the 
question with relation to the day of the disease on which treatment 
is commenced, or the age of the patient treated; it matters not how 
the subject is regarded or how it is turned for the purpose of comparison 
with previous results, the conclusion reached is always the same, 
namely, there has been an average reduction of mortality from the 
use of antitoxin in the treatment of diphtheria of not less than 50 
Fig. 124 
Amount of antitoxin in 1 c.c. of serum from persons, at different intervals of time 
(days), after a single subcutaneous injection of 10,000 units. 
per cent., and under the most favorable conditions a reduction to one- 
quarter, or even less, of the previous death rate. This has occurred 
not in one city at one particular time, but in many cities, in differet 
countries, at different seasons of the year, and always in conjunction 
with the introduction of antitoxin serum and proportionate to the 
extent of its use." The combined statistics of deaths in 19 of the chief 
cities of the world show there were in the last four years not over 30 
per cent, as many deaths as in the four years preceding the introduction 
of antitoxin. Except where immunization has been practised on a 
large scale, no marked reduction in the number of cases of diphtheria 
has been evident. 
Determination of the Presence of Antitoxin in the Living Body.- 
The Schick Reaction.1-Recently Schick published a method by which 
1 Schick (B.) Die, Diphtheritoxin-Hautreaktion des Menschen als Vorprobe der Prophy- 
lactischen Diphtheric heilserum injection, Munch med. Woch., 1913, lx, 2608-2610. 
Park (W. H.), Zingher (A.), and Serota (H. M.), Archives of Pediatrics, 1914. 310 
PATHOGENIC MICROORGANISMS 
the presence of antitoxin in the blood and tissue can be determined very 
easily. A minute quantity of toxin is injected intracutaneously, and a 
local reaction follows if there is less than 3^ of a unit of antitoxin per 
c.c. of blood. This amount is considered sufficient to protect against 
diphtheria. The explanation of the test is that when no antitoxin is 
present the toxin acts on the cells of the skin; when antitoxin is present 
it neutralizes the toxin so that no poisoning occurs. A negative reaction 
therefore indicates the presence of antitoxin. 
A standard diphtheria toxin is diluted at first 1: 10 in 0.5 per cent, 
phenol; this dilution will keep in the ice-box with little deterioration 
for at least two weeks. For use further dilutions are made in normal 
saline, of such strength that 0.1 c.c. contains of the minimum lethal 
dose for the guinea-pig. This amount is injected intracutaneously on 
the flexor surface of the arm or forearm. If the injection has been made 
properly, a definite wheal appears which lasts for several minutes. 
A positive reaction appears in twenty-four to thirty-six hours, and is 
characterized by a circumscribed area of redness and slight infiltration 
which measures from 1 to 2 cm. in diameter. It persists for seven to 
ten days, and on fading shows superficial scaling and a persistant 
brownish pigmentation. The test represents a true irritant action of 
non-neutralized toxin. Pseudoreactions are seen occasionally in young 
adults, who may have a large amount of antitoxin. These are local 
sensitization phenomena of a general protein character, and can. be 
distinguished from the true reaction. They appear earlier, are more 
infiltrated, less sharply circumscribed and disappear in twenty-four 
to forty-eight hours. On fading they leave a faintly pigmented area 
which may show superficial scaling. They can also be obtained with 
dilutions of plain broth. 
By the use of this test a large number of individuals have been shown 
to be naturally immune. Negative reactions were obtained in 93 per 
cent, of the newborn, in 57 per cent, during the first year of life, in 37 
per cent, between two and five years, and 50 per cent, between five 
and fifteen years. In adults the negative reactions were as high as 
90 per cent. By applying the test, therefore, passive immunization 
with antitoxin of a large number of individuals can be omitted and the 
disagreeable symptoms of sensitization avoided. Of 400 cases of scarlet 
fever, showing a negative Schick reaction and who received no immunizing 
dose of anitoxin, not one developed clinical diphtheria. Of these cases 
25 per cent, were carriers of virulent diphtheria bacilli. 
Active Immunization with Toxin-antitoxin Mixtures.-Immunization 
of horses with mixtures of toxin and antitoxin has been used by us for 
years, but for human beings it was first recommended by Theobald 
Smith in 1907, and carried out by v. Behring in 1912. Mixtures of 
toxin and antitoxin which are neutral or slightly toxic to the guinea- 
pig are injected in small and repeated doses, subcutaneously or intra- 
cutaneously. Local signs, like redness, infiltration and tenderness 
and occasionally constitutional symptoms, such as fever, headache, 
and malaise, are noted. An increase of antitoxin is generally found RESULTS FROM USE OF ANTITOXIC GLOBULIN SOLUTION 
311 
in individuals who have natural immunity. This increase is noted 
often within six days after one injection of the vaccine. Non-immune 
patients, as determined by the Schick reaction, react less readily to 
active immunization. We found that only 22 per cent, of susceptible 
individuals will produce sufficient antitoxin to surely protect them 
against diphtheria. Another practical objection is that the immunity 
in this group is slowly produced so that during the first two weeks after 
the injection it is practically negligible. Usually this is the period during 
which we desire the greatest immunity, because it is then that the danger 
of infection is greatest in places like the measles and scarlet fever wards 
of a contagious disease hospital. Individuals who are definitely exposed 
to infection should therefore be passively immunized, even if the toxin- 
antitoxin mixture has been given. 
The mixtures of toxin and antitoxin used for active immunization 
represent from 50 to 90 per cent, of the L+ dose of toxin to each unit 
of antitoxin. Mixtures containing from 80 to 90 per cent, of the L+ 
dose of toxin will cause paralysis in guinea-pigs, but are still harmless 
to man. Within these limits they can be injected in doses of 0.25 to 
1 c.c. and repeated at the end of seven to ten days. The injections 
are given subcutaneously, or combined subcutaneously and intracu- 
taneously in the interscapular region. 
The production of sufficient antitoxin in susceptible cases after 
active immunization can be determined clinically by the Schick test. 
A negative reaction indicates that the person has become immune. 
According to our animal tests appreciable antitoxin will last from 
one to two years. In horses we found it disappeared in some within 
nine months, while in others it persisted for more than a year. The 
homologous antitoxin is produced in some cases in sufficient amount 
to be used in the passive immunization of individuals who are especially 
susceptible to anaphylactic symptoms like cases of asthma. 
Danger in Giving Subcutaneous Injections of Antitoxin.-About 1 
in 10,000 persons develop, within a few minutes after an injection of 
serum, alarming symptoms. About 1 in 50,000 of the injected die. 
About thirty deaths in all have been reported. In 140,000 persons 
injected by New York City Health Department Inspectors there have 
been two deaths due to serum. About the same proportion is reported 
from Boston. The persons suffering severe symptoms have usually 
been subject to asthma, while the fatal cases usually have the patho- 
logical changes known as status lymphaticus. A few of these rare cases 
die almost instantly. As a rule, when death occurs it takes place within 
a few minutes after the development of symptoms. Usually the respira- 
tory rather than the circulatory centre seems to be affected. Persons 
who have not reacted badly to a first injection do not need to fear a 
second. So far as known all fatal results have followed the first injection. 
Results from the Use of Antitoxic Globulin Solution.-The curative 
effect proved to be identical with that of the whole serum. Our tests 
showed clearly that not only the toxin, but also the poisons produced 
in the animal by injections with virulent bacilli are neutralized as com- 312 
PATHOGENIC MICROORGANISMS 
pletely by the globulin solution as by the antitoxic serum from which 
they are separated. The injections of the globulin solution were found 
to be followed by decidedly less severe rashes than the whole serum, and 
it was especially noted that there were very few who had any constitu- 
tional disturbances even when the development of the rashes did occur. 
The following comparative table gives a summary of the constitu- 
tional and local reactions obtained in the treatment of 50 cases of diph- 
theria in young children, with a lot of antitoxic serum received from 
three horses which was found to produce an excessive amount of dis- 
turbance, and of an equal number of similar cases treated with a solution 
of the antitoxic globulins derived from a portion of the same lot of serum 
is as follows: 
Children treated with 
the whole serum. 
Children 
treated 
antitoxic 
who were 
with the 
: gobulins. 
Marked constitutional symptoms accom- 
panied by a severe and persistent rash 
28 
per 
cent. 
0 per 
cent. 
Moderate constitutional symptoms accom- 
panied by a well-developed erythema or 
urticaria 
18 
per 
cent. 
4 per 
cent. 
Very slight constitutional disturbance 
accompanied by a more or less general 
urticaria or erythema 
20 
per 
cent. 
8 per 
cent. 
No appreciable constitutional disturbance 
but more or less general urticaria or 
erythema 
4 
per 
cent. 
34 per 
cent. 
No appreciable deleterious after-effects 
whatever 
30 
per 
cent. 
54 per 
cent. 
DURATION OF RASHES. 
Days. 
1 
2 
3 
4 
•5 
6 
7 
8 Totals 
Antitoxic globulin cases . 
. . . 5 
7 
5 
2 
3 
1 
-23 
Whole serum cases .... 
. . . 1 
4 
10 
1 
10 
3 
2 
5-36 
The concentration of antitoxin made possible by the elimination 
of the non-antitoxic substances is not only a convenience but is of a 
distinct importance, as it tends to encourage large doses. Some pro- 
ducers supply a product which is so rich in proteid as to be almost 
semisolid. This is not quite so well absorbed as the less concentrated 
product. The total solids in the globulin solution should not be more 
than twice those in the serum. 
The antitoxic globulin solution tends to become slightly cloudy 
when kept at moderate or high temperatures and substances such as 
solutions of carbolic acid and tricresol precipitate it. 
Development of Agglutins for Diphtheria Bacilli.-By the in- 
jections of the bodies of diphtheria bacilli into animals agglutinins 
have been developed in sufficient amount to act in 1: 5000 dilutions 
of the serum. The serum produced from diphtheria bacilli does not 
agglutinate pseudodiphtheria bacilli in high dilutions. The serum 
of patients convalescent from diphtheria has, as a rule, little agglu- 
tinating power. This test is not used in diagnosis. 
The Persistence in the Man's Blood of Injected Antitoxin Produced 
in the Horse.-Antitoxins and other antibodies produced in an animal PSEUDOMEMBRANOUS EXUDATIVE INFLAMMATIONS 
313 
disappear more rapidly when introduced into the blood of another 
species than into one of the same species. In man an alien serum must 
be used in all except exceptional cases. 
In our experiments in guinea-pigs we have found that the homol- 
ogous antitoxin was retained in appreciable amounts for at least six 
months, while the heterologous antibodies were noticeable to the same 
extent for only four weeks (see Human Chart, p. 309). There is a rather 
rapid loss of horse produced antitoxin during the first few days and then 
a slow loss becoming more and more gradual until final elimination at 
the end of ten days to three weeks. The larger the amount of anti- 
bodies injected the longer will be the time before the elimination of 
effective amount. 
Mixed Infection in Diphtheria.-Virulent diphtheria bacilli are 
not the only bacteria present in human diphtheria. Various cocci 
and bacilli more particularly streptococci, staphylococci, pneumococci, 
and influenza bacilli, are also found actively associated with Leffler's 
bacilli in diphtheria, playing an important part in the disease and lead- 
ing often to serious complications (sepsis and bronchopneumonia). 
Investigations indicate that when other pathogenic bacteria are asso- 
ciated with the diphtheria bacilli they mutually assist one another in 
their attacks upon the mucous membrane, the streptococcus being- 
parti cularly active in this respect, often opening the way for the invasion 
of the Loftier bacillus into the deeper tissues of supplying needed con- 
ditions for the development of its toxin. In most fatal cases of broncho- 
pneumonia following laryngeal diphtheria we find not only abundant 
pneumococci or streptococci in the inflamed lung areas, but also in 
the blood and tissues of the organs. As these septic infections due to 
the pyogenic cocci are in no way influenced by the diphtheria antitoxin, 
they frequently are the cause of the fatal termination. Other bacteria 
cause putrefactive changes in the exudate, producing alterations in 
color and offensive odors. 
Pseudomembranous Exudative Inflammations Due to Bacteria 
other than the Diphtheria Bacilli.-The diphtheria bacillus, though 
the most usual, is not the only microorganism that is capable of pro- 
ducing pseudomembranous inflammations. There are numerous 
bacteria present almost constantly in the throat secretions, which, under 
certain conditions, can cause local lesions very similar to those in the 
less-marked cases of true diphtheria. The streptococcus and pneu- 
mococcus are the two forms most frequently found in these cases, 
but there are also others, such as the Vincent's bacillus, which, under 
suitable conditions, excite this form of inflammation, but without 
serious constitutional symptoms. 
The pseudomembranous angina accompanying scarlet fever, and 
to a less extent other diseases, may not show the presence of diph- 
theria bacilli, but only the pyogenic cocci, especially streptococci, or, 
more rarely, some varieties of little-known bacilli. The deposit cover- 
ing the inflamed tissues in these non-specific cases is, it is true, usually 
but not always, rather an exudate than a true pseudomembrane. 314 
PATHOGENIC MICROORGANISMS 
Relation of Bacteriology to Diagnosis.-We believe that all expe- 
rienced clinicians will agree that, when left to judge solely by the 
appearance and symptoms of a case, there are certain mild exudative 
inflammations of the throat of similar appearance some of which trans- 
mit diphtheria while others do not. 
The doubtful cases that have the diphtheria bacilli in the exudate, 
are capable of giving true characteristic diphtheria to others, or later 
develop it characteristically themselves, while those in whose throats 
no diphtheria bacilli exist can under no condition give true charac- 
teristic diphtheria to others or develop it themselves. It is, indeed, 
true, as a rule, that cases presenting the appearance of ordinary folli- 
cular tonsillitis in adults are not due to the diphtheria bacillus. On the 
other hand, in small children mild diphtheria very frequently occurs 
with the semblance of rather severe ordinary follicular tonsillitis, due 
to the pyogenic cocci, and in large cities where diphtheria is prevalent 
all such cases must be watched as being more or less suspicious. 
Without doubt most observers agree with us in thinking that if in any 
case exposure to diphtheria is known to have occurred, even a slightly 
suspicious sore throat should be regarded as probably due to the diph- 
theria bacilli. If, on the other hand, no cases of diphtheria have been 
known to exist in the neighborhood, even cases of a more suspicious 
nature would probably not be regarded as diphtheria. Now that we 
know about 50 per cent, of children have a fairly constant supply of 
antitoxin in their blood, we have reason to believe that many of these 
doubtful cases are simply diphtheria carriers in which the bacilli are 
taking little or no part in making the lesions. Like any carriers they 
are dangerous to others. 
The presence of irregular-shaped patches of adherent grayish or 
yellowish-gray pseudomembrane on some other portions than the tonsils 
is, as a rule, an indication of the activity of the diphtheria bacilli. 
Restricted to the tonsils alone, their presence is less certain. 
Occasionally, in scarlatinal angina or in severe phlegmonous sore 
throats, patches of exudate may appear on the uvula or borders of the 
faucial pillars, and still the case may not be due to the diphtheria 
bacilli; these are, however, exceptional. Thick, grayish pseudomem- 
branes which cover large portions of the tonsils, soft palate, and nostrils 
are almost invariably the lesions produced by diphtheria bacilli. 
The very great majority of cases of pseudomembranes of exudative 
laryngitis, in the coast cities at least, whether an exudate is present 
in the pharynx or not, are due to the diphtheria bacilli. Nearly all 
membranous affections of the nose are true diphtheria. When the 
membrane is limited to the nose the symptoms are, as a rule, very slight; 
but when the nasopharynx is involved the symptoms are usually grave. 
Most cases of pseudomembranes and exudates, entirely confined 
to portions of the tonsils in adults, are not due to the diphtheria bacilli. 
Cases presenting the appearances found in scarlet fever, in which a 
thin, grayish membrane lines the borders of the uvula and faucial 
pillars, are rarely diphtheritic. As a rule, pseudomembranous inflam- TECHNIQUE OF THE BACTERIOLOGIC DIAGNOSIS 
315 
mations complicating scarlet fever, syphilis, and other infections dis- 
eases are due to the activity of the pathogenic cocci and other bacteria, 
induced by the inflamed conditions of the mucous membranes due to 
the scarlatinal or other poison. The possibility of these persons being 
carriers of diphtheria bacilli must always be kept in mind. 
Paralysis following a pseudomembranous inflammation is an almost 
positive indication that the case was one of diphtheria, although slight 
paralysis has followed in a very few cases in which careful cultures 
have revealed no diphtheria bacilli. These, if not true diphtheria, must 
be considered very exceptional cases. 
Bacteriologic Diagnosis.-From the above it is apparent that 
fully developed characteristic cases of diphtheria are readily diag- 
nosticated, but that many of the less marked, or at an early period 
undeveloped, cases are difficult to differentiate the one from the other. 
In these cases cultures are of the utmost value, since they enable us 
to isolate those in which diphtheria-like bacilli are found, and to give 
preventive injections of antitoxin to both the sick and those in contact 
with them, if this has not already been done. As a rule, cultures do 
not give us as much information as to the gravity of the case as the 
clinical appearances, for before the lapse of the twelve hours required 
for the laboratory report the extent of the disease is usually possible 
of determination. The reported absence of bacilli in a culture must be 
given weight in proportion to the skill with which the culture was made, 
the suitableness of the media, the location of the disease, and the 
knowledge and experience of the one who examined it. 
Diphtheria does not occur without the presence of the diphtheria 
bacilli; but there have been many cases of diphtheria in which, for 
one or another reason, no bacilli were found in the cultures by the 
examiner. In many of these cases later cultures revealed them. The 
reverse is also true, the presence of diphtheria bacilli in throats without 
clear clinical signs of diphtheria in no sense makes it a case of diphtheria. 
In a convalescent case the absence of bacilli in any one culture indicates 
that there are certainly not many bacilli left in the throat, but even 
repeated cultures cannot absolutely prove their total absence, for in 
some deep tonsillor crypt a few bacilli may remain in the epithelial cells. 
The physician must have intelligence to use advantageously laboratory 
findings. 
Technique of the Bacteriologic Diagnosis.-Collection of the Blood-serum and 
its Preparation for Use in Cultures.-A covered glass jar, which has been thor- 
oughly cleansed with hot water, is taken to the slaughter-house and filled with 
freshly shed blood from a calf or sheep. The blood is received directly in the 
jar as it spurts from the cut in the throat of the animal. After the edge of the 
jar has been wiped it is covered with the lid and set aside, where it may stand 
quietly until the blood has thoroughly clotted. The jar is then placed in an 
ice-chest. If the jar containing the blood is carried about before the latter has 
clotted, very imperfect separation of the serum will take place. The blood is 
allowed to remain twenty-four hours on the ice, and then the serum which sur- 
rounds the clot is siphoned off by a rubber tube and mixed with one-third its 
quantity of nutrient beef-broth, to which 1 per cent, glucose has been added. 316 
PATHOGENIC MICROORGANISMS 
This constitutes the Loffler blood-serum mixture. This is poured into sterilized 
tubes. Care should be taken in filling the tubes to avoid the formation of air 
bubbles, as they leave a permanently uneven surface when the serum has been 
coagulated by heat. About 3 c.c. are sufficient for each tube if the small size is 
employed; if not, 5 c.c. are required. The tubes, having been filled are placed 
slanted at the proper angle and then kept for two hours at a temperature just 
below 95° C. The degree of heat must be carefully watched, as otherwise the 
temperature may go too high, and if the serum is actually boiled the culture 
medium will be spoiled. After sterilization by this process the tubes contain- 
ing the sterile, solidified blood-serum can be placed in covered tin boxes, or 
stopped with sterile paraffined corks and kept for months. The serum thus 
prepared is quite opaque and firm. 
Swab for Inoculating Culture Tubes.-The swab we prefer to use to inoculate 
the serum is made as follows: A stiff, thin, iron rod, six inches in length, is 
roughened at one end by a few blows of a hammer, and about this end a little 
absorbent cotton is firmly wound. Each swab is then placed in a separate 
glass tube, and the mouths of the tubes are plugged with cotton. The tubes 
and rods are then sterilized by dry heat at about 150° C. for one hour, and stored 
for future use. These iron rods have proved more serviceable for making inocu- 
lations than platinum-wire needles or wooden sticks, especially in young chil- 
dren and in laryngeal cases. It is easier to use the cotton swab in such cases, 
and it gathers up so much more material for the inoculation that it has seemed 
more reliable. The wood unless very carefully selected is apt to break and is*too 
thick to use in the nose. 
For convenience and safety in transportation "culture outfits" have been 
devised, which consist usually of a small wooden box containing a tube of blood- 
serum, a tube holding a swab, and a record blank. These "culture outfits" may 
be carried or sent by messenger or express to any place desired. 
Directions for Inoculating Culture Tubes with the Exudate.-The 
patient is placed in a good light, and, if a child, properly held. The 
swab is removed from its tube, and, while the tongue is depressed with 
a spoon, is passed into the pharynx (if possible, without touching the 
tongue or other parts of the mouth), and is rubbed gently but firmly 
against any visible membrane on the tonsils or in the pharynx, and 
then, without being laid down, the swab is immediately inserted in 
the blood-serum tube, and the portion which has previously been in 
contact with the exudate is rubbed a number of times back and forth 
over the whole surface of the serum. This should be done thoroughly, 
but it is to be gently done, so as not to break the surface of the serum. 
The swab should then be placed in its tube, and both tubes, thin cotton 
plugs having been inserted, are reserved for examination or sent to 
the laboratory or collecting station (as in New York City). If sent 
to the health department laboratories for examination the blank forms 
of report which usually accompnay each "outfit" should be filled out 
and forwarded with the tubes. 
Where there is no visible membrane (it may be present in the nose 
or larynx) the swab should be thoroughly rubbed over the mucous 
membrane of the pharynx and tonsils, and in the nasal cavities, and a 
culture made from these. In very young children care should be taken 
not to use the swab when the throat contains food or vomited matter, 
as then the bacteriological examination is rendered more difficult. 
Under no conditions should any attempt be made to collect the material TECHNIQUE OF THE BACTERIOLOGIC DIAGNOSIS 
317 
shortly after the application of strong disinfectants (especially solutions 
of corrosive sublimate) to the throat. Cultures from the nostrils are 
often more successful if the nostrils are first cleansed with a spray of 
sterile normal salt solution. 
Examination of Cultures.--The culture tubes which have been 
inoculated, as described above, are kept in an incubator at 37° C. for 
twelve hours, and are then ready for examination. When great haste 
is required, even five hours will often suffice for a sufficient growth of 
bacteria for a skilled examiner to decide as to the presence or absence 
of the bacilli. On inspection it will be seen that the surface of the 
blood serum is dotted with numerous colonies, which are just visible. 
No diagnosis can be made from simple inspection; if, however, the 
serum is found to be liquefied or shows other evidences of contamina- 
tion the examination will probably be unsatisfactory. 
In order to make a microscopic preparation a clean platinum needle 
is inserted in the tube and quite a large number of colonies are swept 
with it from the surface of the culture medium, a part being selected 
where the most suitable colonies are found. A sufficient amount of 
the bacteria adherent to the needle is washed off in a tiny droplet of 
water previously placed on the cover-glass and smeared over its surface. 
The bacteria on the glass are then allowed to dry in the air. The 
cover-glass is then passed quickly through the flame of a Bunsen burner 
or alcohol lamp, three times in the usual way, covered with a few drops 
of Leffler's solution of alkaline methylene blue,- and left without heating 
for five or ten minutes. It is then rinsed off in clear water, dried, and 
mounted in balsam. When other methods of staining are desired they 
are carried out in the proper way (see Methods of Staining). 
In the great majority of cases one of two pictures will be seen with 
the oil-immersion lens-either an enormous number of character- 
istic Loffler bacilli, with a moderate number of cocci, or a pure cul- 
ture of cocci, mostly in pairs or short chains. (See Streptococcus.) 
In a few cases there will be an approximately even mixture of Loffler 
bacilli and cocci, and in others a great excess of cocci. Besides these, 
there will be occasionally met preparations in which, with the cocci, 
there are mingled bacilli more or less resembling the Loffler bacilli. 
These bacilli, which are usually of the pseudodiphtheria type of bacilli 
(see Fig. 121), are especially frequent in cultures from the nose. 
In not more than one case in twenty will there be any serious diffi- 
culty in making the diagnosis, if the serum in the tube was moist and 
had been properly inoculated. In such a case another culture must 
be made or the bacilli plated out and tested in pure culture. 
Direct Microscopic Examination of the Exudate.--An immediate 
diagnosis without the use of cultures is often possible from a micro- 
scopic examination of the exudate. This is made by smearing a slide 
or cover-glass with a little of the exudate from the swab, drying, heating, 
staining, and examining it microscopically. This examination, however, 
is much more difficult, and the results are more uncertain than when 
the covers are prepared from cultures. The bacilli from the membrane 318 
PATHOGENIC MICROORGANISMS 
are usually less typical in appearance than those found in cultures, and 
they are mixed with fibrin, pus, and epithelial cells. They may also 
be very few in number in the parts reached by the swab, or bacilli may 
be met with which closely resemble the Loftier bacilli in appearance, 
but which differ greatly in growth and in other characteristics, and have 
absolutely no connection with them. When in a smear containing 
mostly cocci a few of these doubtful bacilli are present, it is impossible 
either to exclude or to make the diagnosis of diphtheria with certainty. 
Although in some cases this immediate examination may be of the 
greatest value, it is not a method suitable for general use, and should 
always be controlled by cultures. 
When carried out in the best manner an experienced bacteriologist 
may obtain markedly accurate results. Higley in New York in a 
series of consecutive throat cases made the same diagnosis from the 
direct examination of smears as the Health Department laboratory 
made from the culture. To get the exudate he used a probe armed 
with a loop of heavy copper wire which has been so flattened as to 
act as a blunt curette. He makes thus thin smears from the exudate. 
After drying and fixing by heat the smears are stained for five seconds 
in a solution made by adding five drops of Kiihne's carbolic methylene 
blue to 7 c.c. of tap-water. After washing and drying stain for one 
minute in a solution of 10 drops of carbol-fuchsin in 7 c.c. of water. 
The dilute solution should be freshly prepared. The diphtheria bacilli 
will appear as dark red or violet rods, and their contour, mode of division, 
and arrangement are manifest. 
Animal Inoculation as a Test of Virulence.--If the determination 
of the virulence of the bacilli found is of importance, animal inocu- 
lations must be made. Experiments on animals form the only method 
of determining with certainty the virulence of the diphtheria bacillus. 
For this purpose, alkaline broth cultures of forty-eight hours' growth 
should be used for the subcutaneous inoculation of guinea-pigs. The 
amount injected should not be more than one-fifth per cent, of the 
body-weight of the animal inoculated, unless controls with antitoxin 
are made. In the large majority of cases, when the bacilli are virulent, 
this amount causes death within seventy-two hours. If a good growth 
is not obtained in nutrient bouillon, ascitic broth should be used. At 
the autopsy the characteristic lesions already described are found. 
Bacilli which in cultures and in animal experiments have shown them- 
selves to be characteristic may be regarded as true diphtheria bacilli, 
and as capable of producing diphtheria in man under favorable 
conditions. 
For an absolute test of specific virulence antitoxin must be used. 
A guinea-pig is injected with antitoxin, and then this and a control 
animal, with 2 c.c. of a broth culture of the bacilli to be tested; if the 
guinea-pig which received the antitoxin lives, while the control dies, 
it was surely a diphtheria bacillus which killed by means of diphtheria 
toxin-or, in other words, not simply a virulent bacillus, but a virulent 
diphtheria bacillus. When the bacilli to be tested grow poorly in a simple VINCENT'S ANGINA 
319 
nutrient bouillon they should be grown in bouillon to which one-third 
its quantity of ascitic fluid has been added. Quite a number of bacilli 
have been met with which killed 250 gram, guinea-pigs in doses of 2 to 
15 c.c., and yet were unaffected by antitoxin. These bacilli, though 
slightly virulent to guinea-pigs, produce no diphtheria toxin, and so 
cannot, to the best of our belief, produce diphtheria in man (see p. 302). 
Fig. 125 
Vincent's bacillus with accompanying spirochetes. 
Vincent's Angina.-The local symptoms are similar to a slight 
case of diphtheria. Exudate or pseudomembrane forms on the tonsils 
and tends to become necrotic leaving a superficial ulcer, which is slow 
in healing. The general disturbance, outside a little fever, is usually 
slight. The disease runs its course in from one to two weeks. It has 
been frequently noticed that the disease begins with an eruption of 
vesicles as in aphthous stomatitis. Paralysis never follows from this 
infection. The bacilli found by Vincent in the lesions are 6^4 to 12/4 
long by 0.6/x to O.8/4 broad. Their ends are tapering. They are fre- 
quently bent like the letter S and resemble spirillse. 
The bacilli stain with methyl blue irregularly so that light and dark 
bands alternate (see Fig. 125). 
Stained by the method of Romanowsky there appear sharply defined 
chromatin bodies in the blue stained protoplasm. 
The bacilli are not motile. 
These spindle-shaped bacilli have not been grown in pure culture, 
indeed there is doubt as to their nature. When direct smears are 
made from the exudate tiny spirochetes are usually found mixed with 
the bacilli. 
Certain necrotic conditions of the mucous membrane of the cheek 
and about the teeth are accompanied by microorganisms very similar 
to those described by Vincent. CHAPTER XXL 
THE BACILLUS AND THE BACTERIOLOGY OF TETANUS. 
Tetanus is a disease which is characterized by a gradual onset of 
general spasm of the voluntary muscles, commencing in both man and 
the horse most often in those of the jaw and neck, and extending in 
severe cases to the muscles of the body. The disease is usually associted 
with a wound received from four to fourteen days previously. It has 
long afflicted man. The writings of Hippocrates clearly describe the 
symptoms. 
In 1884 Nicolaier, under Fliigge's direction, produced tetanus in 
mice and rabbits by the subcutaneous inoculation of particles of gar- 
den earth. The Italians, Carle and Rattone, had just before demon- 
strated that the pus of an infected wound from a person attacked with 
tetanus could produce the same disease in rabbits. Finally, Kitasato, 
in 1889, obtained the bacillus of tetanus in pure culture and described 
his method of obtaining it and its biological characters. 
Occurrence in Soil, etc.-The tetanus bacillus occurs widely through- 
out the world as a common inhabitant of the soil, especially in places 
where manure has been thrown, being abundant in many localities, 
not only in the superficial layers, but also at the depth of several feet. 
It has been found in many different substances and places-in hay-dust, 
in horse and cow manure (its normal habitat is the intestine of the 
herbivora), in the mortar of old masonry, in the dust from horses' hair, 
and in the dust in rooms of houses, barracks, and hospitals. 
The tetanus bacilli are more numerous in certain localities than in 
others-for example, some parts of Long Island and New Jersey have 
become notorious for the number of cases of tetanus caused by small 
wounds-and they are fairly common in New York City. As a rule 
they are more abundant in regions where the temperature is high. 
In some islands and countries in the tropics cases of puerperal tetanus 
and tetanus in the newborn are very frequent. Tetanus bacilli are 
found in the intestines of about 15 per cent, of horses and calves living 
in the vicinity of New York City. They are also present to a somewhat 
less extent in the intestines of other animals and of man. 
Morphology.-From young gelatin cultures the bacilli appear as 
motile, slender rods, with rounded ends, 0.3m to 0.5m in diameter by 
2m to 4m in length, usually occurring singly, but, especially in old 
cultures, often growing in long threads. They form round or nearly 
round spores, thicker than the cell (from Im to 1.5m in diameter), occupy- 
ing one of its extremities and giving to the rods the appearance of small 
pins (Fig. 126). RESISTANCE OF SPORES TO DELETERIOUS INFLUENCES 
321 
Staining.-It is stained with the ordinary aniline dyes, and is not 
decolorized by Gram's method. The spores are readily stained and 
may be demonstrated by double-staining with Ziehl's method. The 
flagella are fairly easily stained on freshly developed bacilli taken from 
cultures which have been at short intervals several times transplanted. 
Biology.-An anaerobic, liquefying, moderately motile bacillus. It 
has abundant peritrichic flagella. Forms spores, and in the spore 
stage it is not motile. It grows slowly at temperatures from 20° to 
24° C., and best at 38° C., when, within twenty-four to thirty hours 
it forms spores. At temperatures of 20° to 24° C. spores form in from 
six to ten days. It will not in pure culture grow in the presence of 
oxygen, but grows well in an atmosphere of hydrogen gas. With certain 
other bacteria the tetanus bacillus grows luxuriantly in the presence 
of oxygen. 
Growth in Media.-The bacillus of tetanus grows in ordinary nutrient gela- 
tin and agar of a slightly alkaline reaction. The addition to the media of 1.5 
per cent, of glucose causes the development 
to be more rapid and abundant. It also 
grows abundantly in alkaline bouillon in an 
atmosphere of hydrogen. On gelatin plates 
the colonies develop slowly; they resemble 
somewhat the colonies of the Bacillus sub- 
tilis, and have a dense, opaque centre sur- 
rounded by fine, diverging rays. Lique- 
faction takes place more slowly, however, 
than with Bacillus subtilis, and the resem- 
blance to these colonies is soon lost. 
The colonies on agar are quite character- 
istic. To the naked eye they present the 
appearance of light, fleecy clouds; under 
the microscope, a tangle of fine threads. 
The stab cultures in gelatin exhibit the 
appearance of a cloudy, linear mass, with 
prolongations radiating into the gelatin from 
all sides (arborescent growth). Liquefaction 
takes place slowly, generally with the production of gas. In stab cultures in 
agar a growth occurs not unlike in structure that of a miniature pine tree. 
Alkaline bouillon is rendered somewhat turbid by the growth of the tetanus 
bacillus. In all cases a production of gas results, accompanied by a char- 
acteristic and very disagreeable odor. It develops in milk without causing 
coagulation. 
Fig. 126 
Tetanus bacilli with spores in dis- 
tended ends. X 1100 diameters. 
Resistance of Spores to Deleterious Influences.-The spores of the 
tetanus bacillus are very resistant to outside influences; in a desic- 
cated condition they may retain their vitality for several years, and 
are not destroyed in two and a half months when present in putrefy- 
ing material. They withstand an exposure of one hour to 80° C., but 
are usually killed by an exposure of ten minutes at 105° C. to live steam. 
They resist the action of 5 per cent, carbolic acid for ten hours. A 
5 per cent, solution of carbolic acid, however, to which 0.5 per cent, 
of hydrochloric acid has been added, destroys them in two hours. 
They are killed when acted upon for three hours by bichloride of 322 
PATHOGENIC MICROORGANISMS 
mercury (1 : 1000), and in thirty minutes when 0.5 per cent. HC1 is 
added to the solution. Silver nitrate solutions destroy the spores of 
average resistance in one minute in 1 per cent, solution and in about 
five minutes in 1 : 1000 solution. 
With regard to the persistence of tetanus spores upon objects where 
they have found a resting place, Henrijean reports that by means 
of a splinter of wood which had once caused tetanus he was able after 
eleven years again to cause the disease by inoculating an animal with 
the infective material. 
Isolation of Pure Cultures.-The growth of the tetanus bacillus in the ani- 
mal body is comparatively scanty, and is usually associated with that of other 
bacteria; hence, the organism is difficult to obtain in pure culture. The 
method of procedure which is most successful, consists in inoculating freshly 
boiled slightly alkaline nutrient agar or glucose bouillon with the tetanus-bear- 
ing material (pus or tissue from the inoculation wound), keeping the culture 
under anaerobic conditions for twenty-four to forty-eight hours at a temperature 
of 37° C., and, after the tetanus spores have formed, heating it for one-half an 
hour at 80° C., to destroy the associated bacteria. The spores of the tetanus 
bacillus are able to survive this exposure, so that when anaerobic cultures are 
then made in the usual way the tetanus colonies develop. When the tetanus 
bacilli are the only spore-bearing bacteria present, pure cultures are readily 
obtained; when other spore-bearing anaerobic bacteria are present, the isola- 
tion of a pure culture may be a matter of difficulty, but even then the pres- 
ence of tetanus toxin in the culture fluid will indicate the presence of tetanus 
bacilli. 
Pathogenesis.-In mice, guinea-pigs, rabbits, horses, cattle, goats and 
a number of other animals inoculations of pure cultures of the tetanus 
bacillus cause tetanus after an incubation of from one to four days. In 
the smaller animals tetanus usually develops first in the muscles nearest 
the point of inoculation. A mere trace of an old culture-only as much 
as remains clinging to a platinum needle-is often sufficient to kill 
very susceptible animals like mice and guinea-pigs. Other animals 
require a larger amount. Rats and birds are but little susceptible, and 
fowls scarcely at all. These never develop tetanus from natural infec- 
tion. It is a remarkable fact that an amount of toxin sufficient to kill 
a hen would suffice to kill 500 horses. It is estimated that if 1 gram 
of horse requires 1 part of toxin to kill, then 1 gram of guinea-pig requires 
6 parts, 1 of mouse 12, of goat 24, of dog 500, of rabbit 1500, of cat 
6000, of hen 360,000. Horses frequently develop tetanus after injuries 
or operations. Cultures from different cases vary in their toxicity. 
On the inoculation of less than a fatal dose in test animals a local tetanus 
may be produced, which lasts for days and weeks and then ends in 
recovery. On killing the animal there is found at autopsy, just at the 
point of inoculation, a hemorrhagic spot, and no changes other than 
these here or in the internal organs. A few tetanus bacilli may be 
detected locally with great difficulty, often none at all; possibly a few 
may be found in the region of the neighboring lymphatic glands and 
even in the blood. From this scanty occurrence of bacilli the conclusion 
has been reached that the bacilli of tetanus, when inoculated in pure 
culture, do not multiply to any great extent in the living body, but TOXINS OF THE TETANUS BACILLUS 
323 
only produce lesions through the absorption of the poison which they 
develop at the point of infection. It has been found that pure cultures 
of tetanus, after the germs have sporulated and the toxins been de- 
stroyed by heat, can be injected into animals without producing tetanus. 
But if pathogenic streptococci or staphylococci or even non-pathogenic 
organisms are injected simultaneously with the spores, or if there is an 
effusion of blood at the point of injection, or if there was a previous or 
later bruising of the tissues, the animals surely die of tetanus. 
Natural Infection.-Here the infection may be considered as prob- 
ably produced by the bacilli in their spore state, and the conditions 
favoring infection are almost always present. A wound of some kind 
has occurred, penetrating at least through the skin, though perhaps 
of a most trivial character, such as might be caused by a dirty splinter 
of wood, and the bacilli or their spores are thus introduced from the 
soil in which they are so widely distributed. If in any given case, 
the tissues being healthy, the ordinary saprophytic germs are killed 
by proper disinfection at once, a mixed infection does not take place, 
and tetanus will not develop. If, however, other bacteria, especially 
pathogenic or putrefactive types accompany the tetanus bacilli, or if 
the tissues infected be bruised or lacerated, the spores may develop and 
produce the disease. Gelatin and catgut are occasionally found to 
contain tetanus spores. 
Tetanus in Man.-Man and almost all domestic animals are sub- 
ject to tetanus. It is a comparatively rare disease in the United States, 
during times of peace, except after the Fourth of July celebration, 
when a considerable number of cases develop. Injuries causing crushing 
of tissues with accompanying contamination are the most likely to be 
followed by tetanus. Until immunization became the practice more 
than one hundred persons yearly developed tetanus after blank cart- 
ridge wounds. On examination of an infected individual very little 
local evidence of the disease can be discovered. Generally at the point 
of infection, if there is an external wound, some pus is to be seen, in 
which, along with numerous other bacteria, tetanus bacilli or their 
spores may be found. Although rather deep wounds are usually the 
seat of infection, at times such superficial wounds as an acne pustule 
or a vaccination may give the occasion for infection. In rare cases 
tetanus has developed from the infection of necrotic mucous membranes 
as in diphtheria. Not only undoubted traumatic tetanus, but also 
all the other forms of tetanus, are now conceded to be produced by the 
tetanus bacillus-puerperal tetanus, tetanus neonatorum, and idio- 
pathic tetanus. In tetanus neonatorum infection is introduced through 
the navel, in puerperal tetanus through the inner surface of the uterus. 
It should be borne in mind that when there is no external and visible 
wound there may be an internal one. The lesions in the nervous system 
are still obscure. Congestion, cellular exudate into the perivascular 
spaces, and chromatolysis of the ganglion cells are common. 
Toxins of the Tetanus Bacillus.-It is evident from the localization 
of the tetanus bacilli almost wholly at the point of inoculation and 324 
PATHOGENIC MICROORGANISMS 
their moderate multiplication at this point that they exert their action 
through the production of powerful toxins. These toxins are named, 
according to their action, the tetanospasmin and the tetanolysin. The 
first only is of importance. One ten-thousandth of a cubic centimeter 
of the filtrate of an eight-day bouillon culture of a fully virulent bacillus 
is sufficient to kill a mouse. The purified and dried tetanus toxin pre- 
pared by Brieger and Cohn was surely fatal to a 15-gram mouse in a 
dose of 0.000005 gram. The toxin is precipitated by saturating'the 
broth with ammonium sulphate and having been collected on the 
filter is compressed to eliminate the fluid clinging to it. The appalling 
strength of tetanus toxin may readily be appreciated when it is stated 
that it is twenty times as poisonous as dried cobra venom. 
The quantity of the toxin produced in nutrient media varies accord- 
ing to the age of the culture, the composition of the culture fluid, 
reaction, completeness of the exclusion of oxygen, etc. For some 
reason more toxin develops in broth inoculated with masses of tetanus 
spores in nutrient agar than with bacilli. The variation in strength is 
partly due to the extreme sensitiveness of the toxin, which deteriorates 
on keeping at blood-heat or on exposure to light. It is sensibly affected 
by most chemical reagents and is largely destroyed by heating to 55° 
to 60° C. for a few minutes. It retains its strength best when protected 
from heat, light, oxygen, and moisture. 
The tetanus cultures retain their ability to produce toxins unaltered 
when kept under suitable conditions; but when subjected to deleterious 
influences they may entirely lose it. 
Production of Toxin for Immunization of Horses.-The usual 
medium for the development of the toxin is a slightly alkaline veal 
bouillon containing 1 per cent, of peptone and 0.5 per cent. salt. The 
younger the calves from which the bouillon is prepared the better the 
results. The cultures are kept at 37° for ten days and then filtered. 
For other methods of preparing toxin and for further characteristics 
of toxin, see Part I. See also later in this chapter. 
Action of Tetanus Toxin in the Body.-After the absorption of the 
poison there is a lapse of time before any effects are noticed. With 
an enormous amount, such as 90,000 fatal doses, this is about nine 
hours; with 30,000, ten; with 3000, twelve; with 300, twenty hours; 
with ten fatal doses, thirty-six to forty-eight hours; with two fatal doses, 
two to three days; with one fatal dose, four to seven days. Less than 
a fatal dose will produce local symptoms. The parts first to be affected 
with tetanus are, in about one-third of the cases in man, and usually 
in animals, the muscles lying in the vicinity of the inoculation-for 
instance, the hind foot of a mouse inoculated on that leg is first affected, 
then the tail, the other foot, the back and chest muscles on both sides, 
and the forelegs, until finally there is a general tetanus of the entire 
body. In mild cases, or when a dose too small to be fatal has been 
received, the tetanic spasm may remain confined to the muscles adjacent 
to the point of inoculation or infection. The symptoms following a TECHNIQUE OE TESTING ANTITOXIN SERUM 
325 
fatal dose of toxin vary greatly with the method of injection. Intra- 
peritoneal injection is followed by symptoms which can hardly be 
distinguished from those due to many other poisons. Injection into 
the brain is followed by restlessness and epileptiform convulsions. 
In man the first symptoms are usually those of a contraction of the 
muscles of the lower jaw and then those of the neck. 
Presence of Tetanus Toxin in the Blood.-The blood during the first 
four days of the disease usually contains toxin. After that time antitoxin 
usually develops and soon makes the blood antitoxic. In St. Louis 
some years ago the serum of a horse dying of tetanus was given by acci- 
dent in doses of 5 to 10 c.c. to a number of children, with the develop- 
ment of fatal tetanus. In this connection Bolton and Fisch showed 
by a series of experiments that much toxin might accumulate in the 
serum before symptoms became marked. 
Tetanus Antitoxin.-Behring and Kitasato were the first to show 
the protective and curative effects of the blood-serum of immunized 
animals. It was found that animals could be protected from tetanus 
infection by the previous or simultaneous injection of tetanus anti- 
toxin, provided that such antitoxic serum was obtained from a thor- 
oughly immunized animal. From this it was assumed that the same 
result could be produced in natural tetanus in man. Unfortunately, 
however, the conditions in the natural disease are very much less 
favorable, inasmuch as treatment is usually commenced not shortly 
after the infection has taken place, but some hours after the appearance 
of tetanic symptoms, when the poison has already attacked the cells 
of the central nervous system. 
The Production of Tetanus Antitoxin for Therapeutic Purposes.-The 
tetanus antitoxin is developed in the same manner as the diphtheria 
antitoxin-by inoculating the tetanus toxin in increasing doses into 
horses. The horses receive 5 c.c. as the initial dose of a toxin of which 
1 c.c. kills 250,00.0 grams of guinea-pig, and along with this twice the 
amount of antitoxin required to neutralize it. In five days this dose 
is doubled. This overneutralized toxin stimulates the production of 
antitoxin. Recently we have preferred to inject the horses with 5000 
units of tetanus antitoxin and then give increasing doses of straight 
toxin. After some months of this treatment the blood of the horse 
contains the antitoxin in sufficient amount for therapeutic use. Some 
horses have produced as high as 600 units per c.c. 
Antitoxin Unit and Technique of Testing Antitoxin Serum.- 
Tetanus antitoxin is tested exactly in the same manner as diphtheria 
antitoxin, except that the unit is different. In April, 1907, the pro- 
ducers of serum in the United States agreed to a unit of antitoxin 
which is approximately ten times the size of the unit of diphtheria anti- 
toxin. A unit is defined as the amount of antitoxin required to just 
neutralize 1000 minimal fatal doses of tetanus toxin for a 350-gram 
guinea-pig. The United States government has adopted this unit and 
supplies the different producers with standardized toxin. 326 
PATHOGENIC MICROORGANISMS 
The amount of antitoxic serum which neutralizes an amount of test toxin 
which would destroy 40,000,000 grams of mouse contains 1 unit of antitoxin 
by the German standard. In the French method the amount of antitoxin 
which is required to protect a mouse from a dose of toxin sufficient to kill in 
four days is determined, and the strength of the antitoxin is stated by deter- 
mining the amount of serum required to protect 1 gram of animal. If 0.001 c.c. 
protected a 10-gram mouse the strength of that serum would be 1 : 10,000. 
The toxin used for testing is preserved by precipitating it with saturated 
ammonium sulphate and drying and preserving the precipitate in sealed tubes. 
As required, it is dissolved in 10 per cent, salt solution as above stated. For 
small testing stations the best way is to obtain some freshly standardized anti- 
toxin and compare serums with this. 
Persistence of Antitoxin in the Blood.-Ransom has clearly shown 
that the tetanus antitoxin, whether directly injected or whether pro- 
duced in the body, is eliminated equally rapidly from the blood of an 
animal, provided that the serum was from an animal of the same species. 
If from a different species it is much more quickly eliminated. 
The same author found some interesting facts in testing the anti- 
toxic values of the serum of an immunized mare, of its foal, and of 
the milk. The foal's serum was one-third the strength of the mare's 
and one hundred and fifty times that of the mare's milk. In two 
months the mare's serum lost two-thirds in antitoxic strength, the 
foal's five-sixths, and the milk one-half. 
Theories as to the Methods by which the Toxin Produces its Effects.- 
Gamprecht and Stintzing concluded from their experiments that the 
toxin from the wound passed to the central nervous system partly 
directly by the peri- and endoneural lymph spaces of the nerves which 
directly connected with subdural space and partly indirectly from the 
blood. The local tetanus they considered as due to the contact of the 
poison with the motor end plates. The experiments of Meyer and 
Ransom and of Marie and Morax have proved to them that the poison 
is transported to the central nervous system by the way of the motor 
nerves-and by no other channel. These authors thought that they 
had shown that the essential element for the absorption and transpor- 
tation of the toxin is not the lymph channels, but the axis-cylinder, the 
intramuscular endings of which the toxin penetrates. The poison is 
taken up quite rapidly. Marie and Morax were able to demonstrate 
the poison in the corresponding nerve trunk (sciatic) one and a half 
hours after the injection. Absorption, however, and conduction are 
dependent to a large extent on the nerves being intact. A nerve cut 
across takes very much longer to take up the poison (about twenty- 
four hours), and a degenerated nerve takes up no poison whatever. 
In other words, we see that section of the nerve prevents the absorption 
of the poison by way of the nerve channels. Similarly section of the 
spinal cord prevents the poison from ascending to the brain. The poison 
which passed through the general lymph channels to the blood was 
partly returned to the tissue fluids throughout the body and taken 
up by nerve endings and thus produced general tetanus. 
According to Meyer and Ransom, the reason why the sensory nerves UNION OF TOXIN WITH GRAY MATTER OF THE BRAIN 
327 
do not play any role in the conduction of the poison lies in the pres- 
ence of the spinal ganglion, which places a bar to the advance of the 
poison. Injections of toxin into the posterior root leads to a tetanus 
dolorosus, which is characterized by strictly localized sensitiveness to 
pain. 
Ascending centripetally along the motor paths, the poison reaches 
the motor spinal ganglia on the side of inoculation; then it affects the 
ganglia of the opposite side, making them hypersensitive. The visi- 
ble result of this is the highly increased muscle tonus-i. e., rigidity. 
If the supply continues, the toxin next affects the nearest sensory 
apparatus; there is an increase in the reflexes, but only when the af- 
fected portion is irritated. In the further course of the poisoning the 
toxin as it ascends continues to affect more and more motor centres, 
and also the neighboring sensory apparatus. This leads to spasm of 
all the striated muscles and general reflex tetanus. Zupink showed 
that local tetanus did not develop when toxin was injected where it 
did not come in contact with muscle. He believed, therefore, that the 
muscle spasm was due to direct action of the toxin on the muscle fibers. 
Field in our laboratory has shown that not only tetanus toxin, but 
diphtheria toxin and inert colloids can be demonstrated in the sciatic 
nerves after they have been injected subcutaneously or intramuscularly, 
and after varying periods may be found in the spinal cord. He believes 
that the toxins are absorbed by way of the lymphatics of the nerves, 
and not by way of the axis-cylinder. 
A later experiment of Cernovodeanu and Henni tends to confirm 
this contention. They ligated all the muscles and bloodvessels in a 
guinea-pig's leg, leaving intact only the sciatic nerve, skin and bone, 
and then injected a large amount of tetanus toxin below the point of 
ligation. The animals in which this was done never developed tetanus. 
In this case there was only a very slight flow of lymph into the ligated 
area, and so there could be only a slight flow up the nerve. 
If the toxin gets into the blood the only path of absorption to the 
central nervous system is still by way of the motor-nerve tracts. There 
seems to be no other direct path, as, for example, by means of the 
bloodvessels supplying the central nervous system. Even after intro- 
ducing the poison into the subarachnoid space, owing to the passage 
of the poison into the blood, there is a general poisoning and not a 
cerebral tetanus. This at least is the case if care has been taken dur- 
ing the operation to avoid injuring the brain mechanically. A very 
much smaller amount of toxin is required to produce fatal tetanus if 
it is injected into a nerve than if it is injected into the blood. 
The Union of Toxin with the Gray Matter of the Brain and Spinal 
Cord.-This union is a loose one and the toxin can be partially freed 
from its union by action of proteolytic ferments. A number of different 
elements of the cell substances seem to have this power of binding the 
toxins. Heating to 65° C. for ten minutes destroys the capacity to 
fix toxin. These brain substances which unite with toxin are certainly 
not of the nature of antitoxin and the brain cells if they produce anti- 328 
PATHOGENIC MICROORGANISMS 
toxin at all certainly share the power with other cells that have no power 
to bind toxin. Marie, in a recent communication, notes that adrenalin 
neutralizes tetanus toxin and that lecithin compounds are undoubtedly 
concerned in the mechanism of tetanus toxin on nerve cells. 
Rapidity of Absorption and Loss of Tetanus Antitoxin from Tissues.- 
The absorption of antitoxin administered subcutaneously takes place 
rather slowly. In his animal experiments Knorr found the maximum 
quantity in the blood only after twenty-four to forty-eight hours. From 
that time on the amount again steadily decreased, so that by the sixth 
day only one-third the optimum quantity was present. By the twelfth 
day only one-fiftieth and at the end of three weeks no antitoxin what- 
ever could be demonstrated. We injected one of our laboratory assis- 
tants with 10,000 units of tetanus antitoxin subcutaneously. The 
blood antitoxic strength was found to be as follows: 18 hours, 0.6 units; 
24 hours, 0.8; 48 hours, 1; 72 hours, 1; 148 hours, 0.7. The two 
important facts to be noted are the slow absorption of antitoxin from 
the subcutaneous tissues and its long retention in the blood. The two 
charts showing the absorption and disappearance of diphtheria anti- 
toxin apply equally to tetanus antitoxin (see pages 308 and 309). 
When injected intravenously the antitoxin very quickly passes into 
the lymph. Ransom, in 1901, was able to demonstrate it in the thoracic 
duct of a dog fifteen minutes after intravenous injection. Only after 
very massive intravenous doses and a considerable interval of time 
are small traces found in the cerebrospinal fluid. This is the reason 
that passively and actively immunized animals become tetanic if the 
poison is injected directly into the central nervous system or into a 
peripheral nerve. Antitoxin injected subdurally passes almost entirely 
over into the blood within twenty-four hours. 
So long as the toxin circulates in the blood it is neutralized by anti- 
toxin in about the same proportion as in test-tube experiments. By 
means of intravenous injections of antitoxin Ransom was able to 
render the lymph free from toxin in a very few minutes. According 
to Marie and Morax, toxin injected into the muscles is already demon- 
strable in the nerve tissue at the end of one and a half hours-i. e., 
it has already entered the channel, where it is reached with difficulty by 
the antitoxin. Donitz injected various rabbits intravenously, each 
with 1 c.c. of a toxin solution containing twelve fatal doses. There- 
upon he determined the dose of antitoxin which, when intravenously 
given, would neutralize this poison after various intervals of time. 
The antitoxin was of such a strength that in test-tube experiments 
1 c.c. of a 1 : 2000 solution just neutralized the amount of toxin em- 
ployed. He found that at the end of two minutes double the dose 
required in vitro would still neutralize the poison; at the end of four 
minutes about four times the dose was required, and at the end of 
eight minutes ten times. When one hour had been allowed to elapse 
forty times the original dose just sufficed to protect the animal from 
death, but not from sickness. 
From the foregoing it is not difficult to formulate the conditions INTRASPINAL ADMINISTRATION OF TETANUS ANTITOXIN 
329 
under which an antitoxin introduced into the organism can exert its 
neutralizing power on the toxin. We see that the poison deposited 
at any given place takes either of two paths to the central nervous 
system, one a direct path by way of the local peripheral nerves and 
the other an indirect path through the lymph channels and blood to 
the end plates of all other motor nerves. Only that portion can be 
surely neutralized which («) still lies unabsorbed at the site of inocula- 
tion, or (b) which, though it has passed into the blood and lymph, has 
noWyet combined with the motor-nerve cells. A curative effect can 
therefore result from antitoxin introduced intraspinally or intravenously 
only so long as a fatal dose of poison has not been taken up by the nerves, 
nerve cells, and possibly by the nerve lymphatics or fibers. The ana- 
tomical relations of the spinal cord and brain are such that fluids 
injected subdurally gain access to the surface of the brain and the 
spinal roots. This has led to experiments to discover whether the cells 
endangered by the tetanus toxin would be better protected by subdural 
or intravenous injections. 
Experiments on the Curative Value of the Intraspinal Administra- 
tion of Tetanus Antitoxin.-While tetanus antitoxin has proved most 
efficacious in the prevention of tetanus, its employment as a curative 
agent has been much less successful. This failure to produce more 
uniformly good results is chiefly due to its too late administration. 
Insufficient dosage and the use of the subcutaneous method have also 
been important factors. While the subcutaneous use of antitoxin, 
which at first was the usual method employed, may prove of value in 
large doses given within the first few hours after the onset of symptoms, 
nevertheless it frequently fails because of slow absorption and the time 
required to reach the tissues of the central nervous system. In the 
light of our present knowledge, this method can be justified only by 
an inability of the physician, for one reason or another, to give an 
intraspinal or an intravenous injection. The latter method is being 
more and more recommended, and when employed before the disease 
has made too much headway has given much better results than the 
subcutaneous. 
From time to time since 1903, single cases or small series of cases have 
been reported in which the antitoxin has been given intraspinally. 
Thus Neugebauer1 in 1905 reported forty-three cases from Continental 
and American sources with a mortality of twenty-two, or 51 per cent., 
and also three patients treated by him of whom two recovered. Many 
of these patients received also intravenous or subcutaneous injections 
or both. The results seemed to some observers to be better than when 
other methods were used without intraspinal dosage, but on the whole, 
this method of treatment has not made much headway. Experimental 
proof of the greater value of the intraspinal method has not until very 
recently been at all convincing. Permin of Copenhagen, working on 
rabbits and dogs, showed that local tetanus could be prevented by the 
1 Wiener klin. Woch., 1905, xviii, 450. 330 
PATHOGENIC MICROORGANISMS 
simultaneous injection of tetanus toxin intramuscularly and a dose of 
antitoxin given in the spine, whereas with the same dose of antitoxin 
given intravenously, local tetanus occurred. Four hours after the 
giving of the toxin neither method of preventing the occurrence of local 
tetanus was efficacious, and when nine hours were allowed to elapse 
between the giving of toxin and antitoxin, the animals could not be 
saved. 
The following series of experiments was undertaken with the object 
of determining to what extent tetanus antitoxin given in the spine has 
greater curative power, when the disease is actually established, than 
when given in the circulation. The comparative ineffectiveness of 
subcutaneous injections of tetanus antitoxin in developed cases is inci- 
dentally brought out. The use of guinea-pigs for this purpose has not 
been recorded so far as we know. They possess, however, a marked 
susceptibility to tetanus together with comparative freedom from 
intercurrent diseases. 
Administration and Result of Antitoxin in Experiment IV. 
No. 
Weight. 
Gm. 
Condition 
of leg. 
Method. 
Amount 
in units. 
Result. 
116 
290 
Fairly stiff 
Control 
D 3 days 
42 
310 
Fairly stiff 
Control 
D 3 days 
296 
250 
Slightly stiff 
H. 
100 
D 8 days 
227 
275 
Fairly stiff 
H. 
100 
D 4 days 
399 
300 
Fairly stiff 
H. 
100 
D 5 days 
216 
255 
Slightly stiff 
N. 
200 
D 4 days 
287 
255 
Fairly stiff 
N. 
200 
D 3 days 
289 
280 
Fairly stiff 
N. 
200 
D 3 days 
306 
285 
Slightly stiff 
N. 
200 
D 3 days 
59 
255 
Stiff 
Sp. 
10 
Discharged April 23; 
normal 
304 
275 
Fairly stiff 
Sp. 
10 
Discharged April 23; 
drags leg 
321 
320 
Fairly stiff 
Sp. 
10 
Discharged April 23; 
drags leg 
Six Given Antitoxin Twenty-two and a Half to Twenty-three Hours after 
Inoculation. 
102 
300 
Stiff 
H. 
100 
D 5 days 
10 
325 
Stiff 
H. 
200 
D 4 days 
272 
350 
Stiff 
H. 
200 
D 4 days 
263 
285 
Stiff 
Sp. 
50 
Discharged April 23; 
drags leg 
123 
325 
Stiff 
Sp. 
50 
D 5 days 
294 
350 
Stiff 
Sp. 
50 
Discharged April 23; 
drags leg 
This would seem absolutely conclusive of the superiority of the intra- 
spinal method of giving antitoxin over the intravenous method. It 
will be noted that only those animals receiving the antitoxin in the 
spine were able to survive. This result is the more striking since the 
amount of antitoxin given them was only a fractional part of that given 
in the circulation. An attempt was made to give the antitoxin to four 
guinea-pigs intraneurally. Under an anesthetic the sciatic nerve of the RESULTS OF USE OF ANTITOXIN FOR IMMUNIZATION 
331 
affected limb was cut down on and freed from the surrounding tissues, 
as much antitoxin as could be introduced into the nerve sheath injected, 
and the remainder intramuscularly directly along the course of the 
nerve. Owing to the small caliber of the nerve, most of the antitoxin 
passed to the deep intramuscular tissues. The animals lived no longer 
than those receiving subcutaneous injections. 
In all of these experiments no guinea-pigs were discharged from 
observation until there was absolutely satisfactory evidence that the 
disease had long ceased to make any progress. While a number of the 
animals, perhaps most, on being discharged exhibited more or less 
stiffness in the inoculated leg lasting for weeks or even months, they 
were nevertheless in the best of health in other respects. This condi- 
tion appears to be identical with that seen in human tetanus, in which 
the stiffness in different groups of .muscles is often very prolonged even 
when the patients are perfectly convalescent. 
Since beginning this work, we have obtained records of four consecu- 
tive clinical cases of tetanus in which an intraspinal injection of anti- 
toxin was given and all the patients recovered. Two of these occurred 
in private practice and two in hospitals. In all of them, in addition 
to the antitoxin used intraspinally, very much larger amounts were 
also given by other methods. 
On experimental and clinical grounds the following recommendation 
for the treatment of tetanus with antitoxin would seem to be amply 
justified: In every case strongly suspected of being tetanus, from three 
to five thousand units of tetanus antitoxin should be given at the first 
possible moment intraspinally, slowly by gravity, and always, if pos- 
sible, under an anesthetic. In order to insure its thorough dissemina- 
tion throughout the spinal meninges the antitoxin should be diluted 
if necessary, to a volume of from 3 to 10 c.c. or more, according to the 
patient's age. When fluid is drawn off previously to the giving of the 
antitoxin, an amount of the latter somewhat less than that of the fluid 
withdrawn should be given. A number of cases of "dry tap " have been 
observed in the disease by those so expert in spinal puncture as to leave 
no room for doubt that the canal was properly entered. In such cases 
only a small amount of tetanus antitoxin should be injected (from 3 to 
5 c.c.). In brief, tetanus antitoxin should be used in precisely the same 
way as antimeningitis serum. 
Results of the Use of Antitoxin for Immunization.-The striking 
results which have been obtained, both in human and in veterinary 
practice, with the prophylactic injection of tetanus antitoxin, would 
seem to warrant the treating of patients with immunizing doses of 
serum-at least, in neighborhoods where tetanus is not uncommon- 
when the lacerated and dirty condition of their wounds may indicate 
the possibility of a tetanus infection. 
Splended results have followed this practice in many places. It is 
the custom at many dispensaries in New York City and elsewhere to 
immunize all Fourth of July wounds by injecting 1000 units. None 
of these have ever developed tetanus. Even the few cases of human 332 
PATHOGENIC MICROORGANISMS 
tetanus reported as occurring after single injections of antitoxin prove 
the value of immunizing injections, for the mortality was low. They 
teach, however, that where tetanus infection is suspected the antitoxic 
serum should be given a second and even a third time at intervals of 
seven days. 
Results of Antitoxin in Treatment.-In cooperation with Dr. Cyrus 
W. Field, one of us (Park) tried a number of experiments upon guinea- 
pigs to test the importance of intravenous and of intraneural injections 
of antitoxin in animals in which tetanus had already developed. Forty 
guinea-pigs have been experimented upon. These were injected in the 
lower part of the hind leg with ten to twenty times the fatal dose of a 
mixture of tetanus toxin and bacilli. Within from one to two hours 
after the development of the first definite symptoms of tetanus the 
animals were operated upon and given antitoxin. The experiments 
show clearly that moderate doses of antitoxin given after the develop- 
ment of tetanus did not save the animals from death or even prolong 
life, while very large doses usually did both. Seventy-five per cent, of 
those receiving 500 units recovered. The surprising result developed 
that amputation of the infected leg at the hip-joint hastened the death 
of the animals in every case. Control animals which had not been 
infected stood the amputation perfectly well, and made good recoveries. 
Without antitoxin, excision of a piece of the nerve did not materially 
prolong life, nor did ligation of the nerve. In the guinea-pigs receiving 
antitoxin the ligation of the nerve seemed to be of benefit. The results 
of the experiments showed that large doses of antitoxin given shortly 
after the development of tetanus usually saved the animals, and that 
most of the toxin was absorbed by the blood and not by the nerves of 
the infected part. Every minute of delay after the appearance of tetanus 
was of importance. With Dr. Matthias Nicol, Jr., I have recently 
compared intravenous with intraspinal injections. The results with 
intraspinal injections were a little better than with intravenous. 
Repeated injections did not give any better results than a single large 
injection. In actual cases in which the treatment was given within 
six hours of the development of symptoms the results have been sur- 
prisingly good. The recoveries have been over 70 per cent. In some 
cases no beneficial results appeared. We have seen numerous cases of 
generalized tetanus that after a large intravenous injection have 
markedly improved and finally recovered, and these cases have certainly 
done better on the average than apparently similar ones receiving 
palliative treatment alone. Lambert, who some years ago made an 
exhaustive study of tetanus, states that in a total of 114 cases of this 
disease treated with antitoxin, according to published and unpublished 
reports, there was a mortality of 40.35 per cent. Of these, 47 were acute 
cases-that is, cases with an incubation period of eight days or less and 
with rapid onset, or cases with a longer period of incubation, but in- 
tensely rapid onset of symptoms; of these the mortality was 74.46 per 
cent. Of the chronic type-those with an incubation period of nine 
days or more, or those with shorter incubation with slow onset-there METHODS OF EXAMINATION IN A CASE OF TETANUS 
333 
were 61 cases, with a mortality of 16.39 per cent. With a still larger 
number of cases the results indicate that with tetanus antitoxin about 
20 per cent, better results are obtained than without. 
Differential Diagnosis between Tetanus and Tetanus-like Bacilli.- 
The differential diagnosis of the bacillus of tetanus is, generally speaking, 
not difficult, inasmuch as animal inoculation affords a sure test of the 
specific organism. No other microorganism known produces similar 
effects to the tetanus bacillus, nor is any other neutralized by tetanus 
antitoxin. The other characteristics also of this bacillus are usually 
distinctive, though microscopic examination alone cannot be depended 
on to make a differential diagnosis. Difficulty arises when other anae- 
robic, or aerobic bacilli, almost morphologically identical with the 
tetanus bacillus, are encountered which are non-pathogenic, such as 
the Bacillus pseudotetanicus anaerobius, already mentioned, and the 
Bacillus pseudotetanicus aerobius. It is possible, however, that both 
these bacilli, when characteristic in cultures, are only varieties of the 
tetanus bacillus, which, under unfavorable conditions of growth, have 
lost their virulence. These non-virulent types do not, as a rule, have 
spores absolutely at their ends, and the spores themselves are usually 
more ovoid than those in the true tetanus bacilli. 
Methods of Examination in a Case of Tetanus.-(a) Microscopic.- 
From every wound or point of suppuration film preparations should 
be made and stained with the usual dyes. The typical spore-bearing 
forms are looked for, but are usually not found; At the same time 
other bacteria are noted if present. 
(&) Cultures.-Bits of tissue, pus, cartridge wads, etc., are collected 
and dropped into glucose bouillon contained in small flasks or tubes. 
(c) Inocidation.-A salt solution emulsion of material from the 
wound is inoculated into mice or guinea-pigs subcutaneously. Successful 
results are usually obtained, if the bacilli or their spores are present. CHAPTER XXII. 
INTESTINAL BACTERIA. 
Significance of Bacteria in Intestines.-The constant presence of 
great numbers of bacteria in the intestinal tract has been the subject 
of much investigation which has given somewhat conflicting results. 
On the one hand, certain experiments seem to show that the presence 
of intestinal bacteria is not essential to life. For example, Nutall and 
Thierfelder experimenting with guinea-pigs succeeded in keeping 
the intestines free from bacteria for a limited time, during which the 
young pigs remained well. Furthermore, Levin makes the interesting 
statement that the intestinal tract of polar animals is for the most part 
sterile. 
On the other hand, the supporters of the opposite theory, namely, 
that certain intestinal bacteria are necessary for perfect physiologic 
action, state that in their experiments on feeding animals with sterile 
food they found that development was retarded; thus Schottelius 
claims this for chickens, and Mme. Metchnikoff obtained similar results 
with young frogs. However, whether or not the presence of bacteria 
in the intestinal canal is essential to the animal economy, it is, never- 
theless, evident that microorganisms play a certain role in aiding or 
inhibiting some of the alimentary processes dependent upon biological 
activity. Recently new interest has been added to the subject by the 
work of Metchnikoff who claims that old age is hastened by the in- 
creased growth and action of certain putrefactive bacteria normally 
found in small numbers in the intestines; and he, Herter, and others 
consider that the development of these harmful varieties may be 
checked by the growth of the obligate intestinal bacteria or by some 
substituted variety which has no harmful action upon the host. 
Conditions Influencing Development of Bacteria.-The intestinal 
canal presents such varying conditions dependent upon so many dif- 
ferent factors that of necessity its flora will reflect great diversity. As 
the organisms gain access to the tract chiefly through the air, food, and 
drink ingested, the character of these will influence the nature of the 
flora. The condition of the oral cavity and that of the respiratory 
passages on account of swallowing bacteria will also have an influence 
on the kind of bacteria found. Some few microorganisms, such as 
the colon group and the obligate anaerobes, have become established 
as regular inhabitants of the intestines and find in the different locali- 
ties of the canal their best environment. Together with these may be 
found those bacteria which having been ingested with various sub- 
stances have survived the action of the gastric and intestinal fluids. REGIONAL DISTRIBUTION OF BACTERIA IN DIGESTIVE TRACT 
335 
The length of time that the intestinal contents are retained at any 
one point of the tract will cause an increase or decrease of certain 
types, as well as the total number, since all portions of the canal are 
not equally adapted to the development of any one species nor to 
bacteria as a class. 
Under absolutely normal conditions organisms, which are not de- 
stroyed, pass through the intestinal tract without entering the body 
of the host, but if injury occurs to the intestinal wall or the normal 
resistance of the body's tissues is lowered for any reason they may pass 
into the circulation. Maklezow claims that after twenty-two hours of 
fecal impaction, intestinal organisms were found in the circulation. 
In chronic constipation intestinal bacteria are found in the urine. 
Escherich found that in utero and immediately after birth the 
meconium is sterile, unless, in exceptional cases where the mother 
has suffered from a severe bacterial infection and the invading organ- 
isms are found in the fetus. From three to seven hours after birth 
a few bacilli, cocci, and yeasts may be found, having presumably entered 
by the anus; after eighteen hours the number and kinds of bacteria 
increase, being taken in by the food or the swallowing of saliva. The 
stools of artificially-fed infants show a greater variety of organisms than 
those of the breast-fed child. 
Anaerobic Conditions in the Intestines.-Virchow first questioned 
the presence of free oxygen in the entire intestinal canal and con- 
cluded it was essentially anaerobic, the oxygen which is taken in being 
quickly absorbed or combined with hydrogen. 
The character of the flora also indicates an anaerobic condition 
of the small intestines with more or less aerobic conditions in the lower 
part of the colon and rectum. 
The anaerobes play the chief part in intestinal putrefaction, and 
certain varieties are thought to be at least the predisposing if not the 
chief cause of many cases of appendicitis. 
Regional Distribution of Bacteria in Digestive Tract.-Many 
different organisms may be found at times in all parts of the tract, 
but each species finds its best envorinment in some one location and 
is here found with greater frequency. In the stomach, very few 
bacteria develop, the sarcinge, B. gastricus, and cloacse group are 
rather constant, the larger number and variety taken in being destroyed 
to a great extent by the gastric juice. 
The fact that great numbers of bacteria are destroyed by the diges- 
tive juices, together with a rapid passage of the partly digested food 
and the strict anaerobic condition, accounts for the very few bacteria 
that are usually found in the upper part of the small intestines. It 
is in this location that the obligate anaerobes, which are usually spore 
bearers and often Gram-positive organisms, such as the B. putrificus of 
Bienstock, B. capsulatus aerogenes, and B. bifidus are usually found. 
The chief bacteria of the lower part of the small and the upper part of 
the large intestines are members of the B. coli group which reach their 
highest development in the cecum and upper colon. Here, too, other 336 
PA TH0GEN1C MICROORGA NISMS 
organisms which have been held in check by the above chemical and 
mechanical causes, finding a more suitable soil, develop, and a marked 
increase is found in many Gram-positive bacilli and cocci of various 
types. In the lower colon and rectum with the accumulation of specific 
antagonistic substances formed from the abundant growth higher up, 
many forms, especially of B. coli group, are more or less destroyed. 
The flora can be materially changed in dogs by the diet, as has been 
shown by Herter. 
The range of variation of the bacteria that appear normally from 
time to time in the intestinal tract is so very great that no one grouping, 
except in the most general sense, such as fermenters or non-fermenters 
of glucose, anaerobes, or aerobes, seems to apply to all cases. Ford 
isolated 50 distinct species from the human feces. 
Methods Used in Examination of Normal Feces.-The material should be 
taken from a perfectly fresh stool, preferably after a dose of castor oil has been 
given. This induces a quick and thorough emptying of the intestinal tract, 
with the least alteration of the chemical nature of the feces. The use of blind 
tubes or flushing are apt to give only the contents of the lower part of the colon 
and rectum and are useful only when the examination is to be limited to this 
area. 
To 1 gram of the material 100 c.c. of normal salt solution is gradually added, 
first rubbing up the feces in a small quantity of the diluent and then shaking 
thoroughly as more is added. Definite amounts of this emulsion can be used 
for plating. Two per cent, glucose agar with defibrinated blood added to it is 
very satisfactory for the isolation of anaerobes, while beerwort agar (10 per 
cent, sterile beerwort added to the usual stock agar) is used for the acidophile 
group. A very convenient method, according to Zinsser, of growing anaerobic 
cultures is to take crystallizing dishes of different sizes so that one dish fits 
within the other, leaving a space of three-fourths of an inch all around. The 
larger dish is placed over the smaller dish as a cover, they are then wrapped in 
filter paper and in this way can be easily sterilized. When ready for plating 
1 c.c. of defibrinated blood is placed in the bottom of the smaller dish and into 
this is poured 10 c.c. of glucose agar which has been inoculated with 1-10 c.c. of 
the above emulsion. By gently tipping the dish back and forth the blood and 
agar become very well mixed. This is covered with a petri dish or the com- 
panion crystallizing dish and allowed to stand until it is perfectly cold. In the 
larger dish are placed two pieces, about 1| inches to 2 inches long, of caustic 
soda and the dish is filled to about J its capacity with pyrogallic acid. The 
smaller dish is carefully inverted over this and sufficient sterile water poured 
into the larger dish to cover the acid. The whole is then sealed with paraffin 
or oil poured over the water that is collected outside the smaller dish; this pre- 
vents continual absorption of the oxygen from the air. Plates are made from 
beerwort agar and grown both anaerobically and aerobically. Fishings are 
made from these plates upon corresponding tube media and the cultures are 
further tested on such media as may be best adapted to each special organism. 
Substitution of One Variety of Bacteria for Others.-It is possible 
for the usual flora of the intestines to be almost entirely replaced 
temporarily by an invading organism. This occurs in disease when 
the microorganism producing a specific disease of the intestines is 
found in almost pure cultures, as in dysentery or cholera. 
As already stated, Metchnikoff claims that this possibility of substitu- 
tion can be used in cases where intestinal putrefaction is excessive and 
thus check the process by the introduction of a lactic acid bacterium. LACTIC ACID MILKS 
337 
This work of Metchnikoff and his followers has led to an extensive 
study of organisms causing lactic acid fermentation. 
Fig. 127 
B. bulgaricus; seventh day (44°) colony. Whey agar plate. X 50 diameters. 
(White and Avery.) 
Lactic-acid Milks.-For many years the people of western Asia and 
eastern Europe have looked upon sour milk as an essential part of daily 
diet. In western Europe and America buttermilk has been a favorite 
drink with many, but it never assumed as much importance as in the 
east. The term sour milk covers all milks or parts of milks in which 
lactic acid fermentation is pronounced. The ordinary buttermilk 
sours because of the growth of lactic acid bacteria in the raw milk 
which have been derived from the local surroundings. Sour milk 
from the dealers may be such milk, but it is more usually heated milk 
to which some special culture of bacteria (starter) has been added. 
Sour milk is usually nearly fat-free, but more or less of the cream may 
remain in it. Hansen, of Copenhagen, has for some time supplied a 
lactic acid bacillus which has been much used. Another starter now 
popular is one suppled by Metchnikoff, which he obtained from the 
east. There are a number of preparations of sour milk used at present, 
among these are: 
Kumyss, in which the fermentation is due to lactic acid bacteria and 
yeasts, and thus contains not only lactic acid, but carbon dioxide and 
about 1 per cent, of alcohol. 
22 338 
PATHOGENIC MICROORGANISMS 
Maadzoun and Yoghurt, the common sour milk of southeastern 
Europe, containing chiefly the B. bidgaricus and streptococci and 
diplococci, all producing lactic acid. 
Zoolak (matzoon) made by adding to heated milk the same bacteria 
as occur in maadzoun. 
Yohourd, blabberade, and other sour milks are made by the use of 
much the same organisms. 
Fig. 128 
B. bulgaricus. X 1000 diams. (Piffard.) 
The bacilli at present of most interest are those resembling the B. 
bulgaricus (B. of Massol) which are present in the eastern milks and 
are now through the advocacy of Metchnikoff used alone or in con- 
nection with a lactic acid streptococcus to produce much of the souring 
of milk of Europe and America. In 1906, Cohendy studied the action 
of this bacillus and found that it produced a large amount of lactic 
acid, 3.23 per cent, being found after ten days at 36° C. From other 
preparations slightly different bacilli were found which produce a firm 
clot, while the B. bulgaricus produces a soft curd. Some bacilli which 
resemble the B. bulgaricus in many respects produce gas as well as acid. 
The bacilli in all strains of B. bulgaricus show wide variations in 
length from 2g to 50g. Chains of bacilli occur in some strains to a 
more marked degree than in others. The bacilli are non-motile, non- 
sporulating, Gram-positive, except when in involution forms, when 
they are said to be Gram-negative. Difficult to cultivate in most 
media. When freshly isolated, growth obtained only on media con- 
taining whey or malt or milk. Grow equally well in aerobic and anae- 
robic conditions. Optimum temperature for growth is 44°, fair growth 
at 30°, slight at 25°, none at 20°. Gelatin is not liquefied. Colonies LACTIC ACID MILKS 
339 
on whey agar are round, grayish white, and measure 0.5 to 1.5 mm. 
Periphery of colonies mostly filamentous. The growth in whey pro- 
Fig. 129 
'Lactic acid" milk containing B. bulgaricus and a lactose fermenting streptococcus. 
Fig. 130 
Yeast cells and lactose fermenting bacilli in "fermented milk." X 1000 diams 
duces clouding, but this disappears in 5 to 14 days, leaving a sediment. 
Coagulates milk in 8 to 18 hours at 44°, and after longer time at lower 340 
PATHOGENIC MICROORGANISMS 
temperatures. The lactic acid formed is either inactive or levoro- 
tatory. A small quantity of volatile acid is also produced. No 
appreciable peptonization of the curd. 
The bacilli are non-pathogenic. These bacilli are probably widely 
distributed in nature, being frequently present in the intestines of man 
and animals. White and Avery, who have made an exhaustive study 
of this group of bacilli, consider that they all belong to one group 
which is identical with the group Bacterium caucasicum (Kern). There 
are apparently at least two distinct types which differ in the amount 
and kind of lactic acid formed. Baldwin showed that when milk plus 
a lactic acid bacillus was added to a mixed diet, the ethereal sulphates 
in the urine may be increased. 
Fig. 131 
B. bulgaricus from normal mouth; forty-eight hour colonies on acid whey agar at 
42° C. Small colony X 40 diameters. Large colony X 220 diameters. (Heinemann 
and Hofferan). 
Means of Supplying Lactic Acid Bacteria for Therapeutic Uses.-These 
are supplied in fermented milks, fluid cultures, and in tablets. Bendick 
has shown in work done in the research laboratory that most of the 
tablets are sterile and that the fluid cultures contain few living bacilli 
after storage for a few weeks. 
Prevalent Intestinal Bacteria.-B. Bifidus.-Tissier found the 
B. bifidus in the stools of breast-fed infants which at times forms 
nearly the entire flora. He found it, though less frequently, in arti- 
ficially fed infants. He also isolated it in the superficial ducts of the 
mammary gland of the mother. It is a strict anaerobe. In the feces 
and fresh cultures it presents the form of a slender bacillus with one end PREVALENT INTESTINAL BACTERIA 
341 
tapering and the other club-shaped. It varies in length from 2/x .to 
3a and even 4/x. 
It occurs mostly as a diplobacillus (see Fig. 132) with the pointed ends adja- 
cent and the swollen ends free, but at times this order is reversed and a fusi- 
form appearance results. As the line of separation is often obscure and as 
the two organisms come together at different angles these various arrangements 
give the impression of many different forms. 
The bacilli lie sometimes in parallel groups, but are seldom entangled. In 
old cultures, the swollen ends seen in the young cultures become bifurcated, 
others take the stain irregularly and Tissier designates this the vesicular form. 
In some instances several bacilli become grouped together at different angles, 
giving the appearance of multiple branching forms. As the medium becomes 
more acid, the bifurcated forms become more numerous. Vesicular forms 
bear a relation to vitality and bifucated forms a relation to media. It is non- 
motile, stains by Gram's method, old cultures staining irregularly. Does not 
seem to possess spores. Killed at 60° for 15 minutes. Does not die out 
quickly. Can be transplanted after three weeks. Grows best at 37° C., but 
also grows at 20° C. 
Fig. 132 
B. bifidus, representing the various forms described; the irregularly stained or vesicular 
forms being from old cultures. X about 1800 diameters. 
On glucose agar, after three days, fine regular colonies, oval in shape, appear. 
It is innocuous to guinea-pigs. It can be cultivated on beerwort agar and on 
glucose agar. In stab cultures made from the feces, in either medium the 
bacilli may be found in almost pure cultures at the bottom of the stab after two 
to fifteen days; the other organisms dying out unless the enterococcus which 
is a facultative anaerobe, is present, then B. bifidus, being less adaptable, is 
overgrown. Fermentation tubes of glucose broth inoculated with fecal material 
will show an abundant growth of B. bifidus in the sediment. 
Conradi and Kurpjuweit claim that in infants the B. bifidus plays 
the same role in inhibiting the growth of the more harmful organisms 
as the B. coli does in the adults. Cahn states that the B. bifidus 
is not found so constantly in artificially fed infants, but that the 
B. acidophilus of Mora takes its place. 342 
PA THOGENIC MICROORGA NISMS 
B. Acidophilus.-This organism belongs to the acidophile group and 
differs from the bifidus in several respects. It never shows the bifid 
forms. It is only found in the artificially fed infants and in milk 
from cows. Colonies arc irregular and send out filaments. It is a 
facultative anaerobe. Some consider it to be the same as the B. bifidus, 
Enterococcus.-Thiercelin in 1903 described the Enterococcus protei- 
formis (Fig. 133) as occurring as a coccus, diplococcus, streptococcus, 
staphylococcus, tetrad filaments and rods. It has a capside barely 
visible, sometimes forming a halo. 
Fig. 133 
Represents the gradation of the Enterococcus (Thiercelin) from the apparent bacillary 
forms to the coccus without a capsule.. X 1000 diameters. 
The arrangement depends upon the mode of divisions. When the 
organism assumes the form of a bacillus the division takes place in line 
of the short axis, the capsule being tough does not rupture but encloses 
2 or 4 more organisms. This form is observed in culture media con- 
taining alcohol, quinine, chromic acid, permanganate, and especially 
bichromate of potash. This bacillary form, before division takes place, 
is confusing as it is difficult to tell whether or not the culture is pure 
until transferred to a medium in which alcohol is present when the 
forms all become coccal. In strongly alkaline broth it grows in tetrads, 
on agar it resembles the staphylococcus, on gelatin the same. In 
broth containing a little methylene blue, picric or acetic acid and in 
hay infusions it is a distinct streptococcus. It is present in normal stools 
but may become pathogenic. It is found in the upper respiratory 
tract, skin, vagina, and is obtained in pure cultures from purulent 
discharges, being easily isolated by ordinary methods. 
It is sensitive to heat and direct sunlight, rather resistant to dis- 
infectants, does not grow in distilled water and sparingly in broth 
containing 2 per cent, sodium carbonate or nitrate. Grows in sterilized 
tap-water, does not ferment sugars, does not constantly coagulate milk, 
does not liquefy gelatin, grows well in the digestive fluids. It produces 
no gas, indol, or odor in the presence of sugar. E. AEROGENES CAPSULATUS 
343 
- It produces a toxin that kills mice in 24 to 48 hours. The organisms 
are found in all the organs after death. 
B. Putrificus.-Bienstock found in putrid mixtures an anaerobic, 
spore-bearing bacillus resembling tetanus in its morphology, which 
is capable of decomposing fibrin in the absence of oxygen, in this 
case the end products of putrefaction, such as indol, are not formed. 
When, however, B. putrificus is associated with some aerobes it acts 
upon fibrin in the presence of oxygen, forming the characteristic 
putrefactive products, which are further split up by the aerobes, 
forming indol. This action is not observed with all aerobes, for ex- 
ample, with B. coli and B. lactis aerogenes inoculated on fibrin with 
B. putrificus. 
B. putrificus is found commonly in the small intestines where it 
enters through the respiratory and digestive tract; that putrefaction 
does not occur, normally is supposed to be due to the presence of in- 
hibiting bacteria. It is isolated with difficulty from the feces. Is an 
obligate anaerobe with drum-stick spores. It is a slender rod with 
blunt ends, and sometimes forms threads, especially on liquid gelatin, 
is actively motile with flagella arranged on either side. Liquefies blood- 
serum with the production of a foul odor. Is Gram-positive. Is not 
pathogenic for animals. 
B. Aerogenes Capsulatus (B. welchii, B. perfringensfi--Found 
usually in small numbers in healthy adults. Increased in old age. 
It is considered by Herter to be the chief cause of intestinal putre- 
faction. For a full description of its pathogenicity and other char- 
acteristics, see p. 353. 
Klein found in the feces of patients during an outbreak of diarrhea 
at St. Bartholomew's Hospital, London, an organism which he named 
the B. aerogenes sporogenes, which may be a variety of B. welchii. 
Bibliography. 
Bienstock. Archiv. f. Hygiene. 1899, xxxvi. 
Escherich. Darmbakterien des Sauglings und ihre Beziehungen zur Physiologic der 
Verdauung, Stuttgart, 1886._ 
Herter. Bacterial Infections of the Intestinal Tract, New York, 1907. 
Klein. Ueber einen pathogenen anaeroben Darmbazillen B. enteritidis sporogenes, 
Centralbl. f. Bakt., 1895, Bd. Xviii. 
MacNeal, Latzer, and Kerr. Fecal Bacteria of Healthy Men, Jour. Infect. Dis., 1909, 
vi, 123 and 571. 
Metchnikoff, E. Sur les microbes de la putrification intestinale, C. R. Acad. Sci., 1908, 
cxlvii, 579. Etude sur la flore intestinale, Am. Inst. Past., 1908, xxii. 
Thiercelin. Formes d'involution de enterocoque enterobacteria, Comptes-rendus de 
la Soci6t6 de Biologie, 1902-1903. 
Tissier. Recherches sur La Flora Intestinale, Normale et Pathologique du Nourrison, 
1900. CHAPTER XXIII. 
THE COLON-TYPHOID GROUP OF BACILLI. 
THE COLON GROUP. 
There are a number of varieties of bacilli normally occupying the 
intestines of man and animals which, because they have similar charac- 
teristics and live in the colon, are generally grouped together as colon 
bacilli. Many of the varieties occurring in animals seem identical with 
those found in man. These bacilli are only pathogenic under unusual 
conditions. The specific pathogenic typhoid, paratyphoid, including 
the types responsible for meat poisoning, dysentery, and paradysentery 
bacilli, also have among themselves and between them and the colon 
bacilli resemblances and are often classed together in the group of the 
colon-typhoid bacilli. 
The chief common characteristics of this whole group are: (1) a 
similar morphology, i. e., short, rather plump rods with a tendency to 
thread formation; (2) a Gram-negative staining reaction; (3) similar 
growths on agar and gelatin; (4) non-liquefaction of gelatin (a few 
closely related organisms, such as B. cloacae, liquefy gelatin very 
slowly). 
In order to see more clearly the main points of difference between 
the subdivisions of this great group the following tabulations may be 
studied. 
Group of Colon-typhoid Bacilli. 
B. (coli) communis 
B. (coli) communior 
B. (acidi) lactici 
B. aerogenes types 
B. para typhosus, A 
B. paratyphosus, B 
B. paratyphosus, C 
B. enteritidis 
Colon group. 
Normal inhabitants of the intes- 
tines, under certain conditions, 
become pathogenic. 
Paratyphoid group 
(intermediate group) 
Usually pathogenic for man or ani- 
mals in varying degree. 
(Similar types, intermediates between colon and typhoid 
have been isolated from water and other sources, probably 
non-pathogenic.) 
B. typhosus Pathogenic for man. 
B. dysenteri® (mannite 
not fermented) 
B. paradysenteri® (man- 
nite fermented). Three 
varieties. 
Pathogenic for man, paradysen- 
teri® usually less so than dysen- 
tery type. 
Dysentery group. 
Alkaligenes group. 
B. alkaligenes. 
Occasionally pathogenic. MORPHOLOGY 
345 
Chief Differential Points between Members of the Typhoid-Colon Group. 
Organism. 
Dextrose. 
Lactose. 
Saccharose. 
Dulcite. 
Mannite. 
Indol. 
Milk. 
Colonies. 
Russell medium. 
Acid. 
Coag. 
Secondary 
alkalinity. 
Endo. 
Conradi. 
Slant. 
Butt. 
B. (ooli) communis 
++ 
H-F 
- 
++ 
♦ 
+ 
+ + 
- 
Red 
Red 
Red 
Red, gas 
B. (coli) communior 
++ 
++ 
++ 
+ + 
+ 
+ + 
- 
Red 
Red 
Red 
Red, gas 
B. (acidi) lactici . 
++ 
+ + 
+ 
+ + 
- 
Red 
Red 
Red 
Red,gas 
B. aerogenes . 
+ + 
~l- 
++ 
- 
* 
+ 
+ * 
- 
Red 
Red 
Red 
Red, gas 
B. paratyphoid, A 
++ 
- 
++ 
H-F 
- 
Slight 
Color- 
Blue 
Blue 
Red, gas 
B. paratyphoid, B 
++ 
- 
- 
++ 
++ 
- 
- 
Strong 
Color- 
less 
Blue 
Blue 
Red, gas 
B. enteritidis 
++ 
- 
- 
++ 
++ 
- 
=fc 
- 
Strong 
Color- 
less 
Blue 
Blue 
Red, gas 
B. typhosus . 
+ 
- 
- 
+ 
- 
=fe 
- 
Slight 
Color- 
less 
Blue 
Blue 
Red 
B. dysenteric 
+ 
- 
- 
- 
Slight 
Color- 
less 
Blue 
Blue 
Red 
B. paradysenteriae 
+ 
- 
* 
- 
+ 
+ 
=1= 
Slight 
Color- 
less 
Color- 
less 
Blue 
Blue 
Red 
B. alkaligenes 
- 
Strong 
Blue 
Blue 
Blue 
* Variable, used for further subdivision. 
+ acid positive; gas negative. 
+ + Usual, exceptions found. 
++ acid positive; gas postive. 
- acid negative; gas negative. 
± slight reaction. 
The Colon Group.-The first description of an organism of the 
colon type was by Emmerich (1885), who obtained it from the intes- 
tinal discharges of cholera patients. A similar organism was found 
by Escherich (1886) in the feces of healthy infants. He gave it the 
name of Bacterium coli commune. It has since been demonstrated 
that closely allied types of bacilli are normal inhabitants of the intes- 
tines of most of the lower animals. They are transferred through 
the feces as manure and sewage to cultivated land, surface waters, 
etc. During warm weather they may multiply outside of the animal 
body. Those strains having the chief cultural characteristics of the 
original strain are classed as colon bacilli, while those differing con- 
siderably from it are, while considered in the general group, given 
different names, such as paracolon, "atypical coli," etc. These types 
are especially common in water and are often due to the suppression 
of certain characteristics due to unfavorable environment. 
The colon group is divided into four main subgroups, B. communis, 
B. communior, B. aerogenes, and B. acidi lactici. These subgroups 
have numerous varieties. 
The colon group has interest not only because it excites disease at 
times in man and animals, but also because it is an index of fecal pollu- 
tion from man or animals. If from man it indicates the possibility of 
infection with the typhoid or dysentery bacilli. For the significance 
of the colon group in water see Bacteriology of Water. 
B. (Coli) Communis.-This type is described as an example of the 
group. 
Morphology.-B. communis varies in morphology. 
The typical form (Fig. 134) is that of short rods with rounded ends, 
from 0.4^ to 0.7m in diameter by 1^ to 3/z in length; sometimes, especially 
where the culture media are not suitable for their growth and in tissues. 346 
PATHOGENIC MICROORGANISMS 
the rods are so short as to be almost spherical, resembling micrococci 
in appearance, and, again, they are somewhat oval in form or are seen 
as threads of 6g or more in length. The various forms may often be 
associated in the same culture. The bacilli occur as single cells or 
as pairs joined end-to-end, rarely as short chains. There is nothing 
in the morphology of this bacillus sufficiently characteristic for its 
identification. 
Flagella.--Upon some varieties seven or eight peritrichic flagella 
have been demonstrated, the non-motile types show none. The flagella 
are shorter and more delicate than those characteristic of the typhoid 
bacilli. 
Staining.-The B. communis stains readily with the ordinary aniline 
colors; it is always decolorized by Gram's method. Under certain 
conditions the stained bacilli exhibit bipolar granules. 
Fig. 134 
Colon bacilli. Twenty-four-hour agar culture. X 1100 diameters. 
Biology.-It is an aerobic, facultative anaerobic, non-liquefying 
bacillus. It develops best at 37° C., but grows well at 20° C., and 
slowly at 10° C. It is usually motile, but the movements in some of 
the cultures are so sluggish that a positive opinion is often difficult. 
In fresh cultures, frequently, only one or two individuals out of many 
show motility. 
The B. coli does not form spores. 
Cultivation.--The B. communis develops well on all the usual culture 
media. Its growth on them is usually more abundant than that of the 
typhoid bacillus or the dysentery bacillus, but the difference is not 
sufficient for a differential diagnosis. 
Gelatin.-In gelatin plates, colonies are developed in from eighteen 
to thirty-six hours. They resemble greatly the colonies of the typhoid 
bacillus, except that many of them are somewhat larger and more 
opaque. (See Figs. 43-45, page 110.) When located in the depths (MS PRODUCTION 
347 
of the gelatin and examined by a low-power lens they are at first seen 
to be finely granular, almost homogeneous, and of a pale yellowish 
to brownish color; later they become larger, denser, darker, and more 
coarsely granular. In shape they may be round, oval, or whetstone- 
like. When the gelatin is not firm the margins of many colonies are 
broken by outgrowths, which are rather characteristic of colon bacilli. 
In stab cultures on gelatin the growth usually takes the form of a 
nail with a flattened head, the surface extension generally reaching 
out rapidly to the sides of the tube. 
Nutrient Agar.-In plate cultures: Surface colonies mostly circular, 
finely granular, and rather opaque. The deep colonies are apt to have 
protuberances. In streak cultures an abundant, soft, w'hite layer is 
quickly developed, but the growth is not characteristic. 
Bouillon.-In bouillon the B. communis produces diffuse clouding 
with sedimentation; in some cultures a tendency to pellicle formation 
on the surface is occasionally seen. 
Potato.-On potato the growth is rapid and abundant, appearing 
after twenty-four to thirty-six hours in the incubator as a yellowish- 
brown to dark cream-colored deposit covering the greater part of the 
surface. But there are considerable variations from the typical growth; 
there may be no visible growth at all, or it may be scanty and of a white 
color. These variations are often due to variations in the potato. 
Milk.-Milk is usually coagulated in from one to four days at 37° C. 
Coagulation is due principally to the production of lactic and acetic 
acid. Some strains produce acid but coagulation does not occur. It 
is possible that a lab ferment is partly responsible for the coagulation. 
Chemical Activities.-Behavior Toward Carbohydrates.-In cultures of 
B. communis many carbohydrates, especially sugars, become fermented 
with production of acid and gas. 
The important fermentation products, both qualitatively and quan- 
titatively, are produced from grape-sugar, probably, according to the 
following reaction: 
2CcHi2O6 + 
Grape-sugar. 
H2O 
Water. 
= 2C5H6O3 + 
Lactic acid. 
CHsCOOH 
Acetic acid. 
+ C2H5COOH 
Ethyl alcohol. 
+ 2CO2 
Carbonic 
acid. 
+ 2Hj 
Hydrogen. 
There are reasons to think that lactic acid is first produced and that 
from this other acids and products develop. Under aerobic conditions 
lactic is produced in excess of acetic acid, while in the absence of oxygen 
the reverse is apt to be true. 
Gas Production.-When B. communis is grown in a solution of glucose 
(dextrose), CO2 and H2 are produced, in the proportion of 1CO2 to 1H2 
upto lCO2to3H2. Anaerobic conditions aid gas formation. Very slight 
traces of gases other than H and CO2 are produced. The amount of gas 
varies in different varieties; the closed arm of the tube half-filled, and 
the II and CO2 in the proportion 2 to 1, is the characteristic type. 
The fermentation is not a simple hydrolytic action, but one in which 
combinations between the C and O atoms are sundered and formed. 348 
PATHOGENIC MICROORGANISMS 
This is not an oxidation process, but a change through breaking down 
-that is, a true decomposition. What oxidation takes place is chiefly 
due to the oxygen liberated from splitting the sugar molecules. For 
the carbohydrates fermented, see table, p. 345. 
Effect of B. Communis in Nitrogenous Compounds.-Indol Formation.- 
B. communis does not liquefy gelatin nor peptonize any albumins. They 
do, however, break down some of the higher nitrogenous compounds 
into smaller atom groups. The first noted of these compounds was 
indol, jjyCH. This is one of the most important products of 
colon activity, although some varieties lack the ability to produce it. 
The maximum amount of indol is present about the tenth day. In the 
intestinal canal in health very little indol appears to be produced by 
the bacilli. Sulphurated hydrogen is liberated from sugar-free proteid 
substances. 
Reduction Processes.-Nitrates are reduced to nitrites and from 
them ammonia and free nitrogen. Litmus and other dyes are also 
reduced. 
Toxins.-The bodies of dead bacilli contain pyogenic and other 
substances, which, injected into the circulation, produce paralysis 
of the striped muscle fibers, convulsions, coma, and death. Extracts 
from some cultures produce irritation of the mucous membranes of the 
large intestines with dysenteric symptoms. 
Growth with Other Bacteria.-The B. communis as well as other 
members of the colon group act antagonistically to many of the pro- 
teolytic bacteria in the intestinal tract, and so inhibit alkaline putre- 
faction otherwise caused by the latter. In milk the same antagonism 
exists, probably because of the acidity caused by the colon growth. 
Reaction to High and Low Temperatures.-B. communis is killed 
at 60° C. in from five to fifteen minutes. Frozen in ice a large propor- 
tion die, but some resist for six months. Frozen in liquid air 95 per 
cent, are killed in two hours. 
Resistance to Drying and Antiseptics.-Simple drying destroys the 
majority of organisms dried at any one time, but some bacilli of the 
number dried may remain alive, especially when held in the texture 
of threads, for five or six months, or all may die in forty-eight hours. 
To most antiseptics they are moderately resistant. They are killed 
in five to fifteen minutes in a 1 per cent, solution of carbolic acid. 
Effect of Acids.-B. communis grow in a wider range of acids and 
alkalies than most other bacteria. They develop in from 0.2 to 0.4 
per cent, of mineral acids, in from 0.3 to 0.45 per cent, of vegetable 
acids, and in from 0.1 to 0.2 per cent, of alkalies. 
Effect of Intestinal Juices.-Gastric juice kills unprotected B. communis 
bacilli unless it is too greatly diluted by food. All the members of 
the typhoid-colon group are more resistant to the gastric juices than 
most non-spore-bearing bacteria. With the food they readily pass 
from the stomach into the intestines. They grow in bile and in the 
intestinal juices. PATHOGENESIS IN MAN 
349 
B. '(Coli) Communior, this species differs from B. communis only 
in its sugar fermentations, fermenting saccharose with the production 
of gas. 
B. (Acidi) Lactici, this type differs from both of the preceding in 
failing to ferment dulcit. 
Most of the differential work has been done with strains from feces, 
similar work with strains from pathological lesions of various parts of 
the body has been, so far as we know, only limited in amount. For 
this reason the collective term B. coli is used in the following descrip- 
tions. 
Pathogenesis.-In Lower Animals.-Intraperitoneal and intravenous 
inoculation of guinea-pigs and rabbits may produce death, which, 
when it follows, usually takes place within the first forty-eight hours, 
accompanied by a decided fall of temperature, the symptoms of enter- 
itis, diarrhea, etc., and finally fibrinopurulent peritonitis. 
Subcutaneous inoculation in rabbits is followed usually by abscess 
formation at the point of inoculation. Dogs and cats are similarly 
affected. 
Cystitis and -pyelonephritis may be produced by direct injections 
into the bladder and ureters, if the urine is artificially suppressed. 
Angiocholitis and abscess are produced by direct injections into the 
liver. Osteomyelitis may follow the intravenous injections of cultures 
into young rabbits. 
From experiments on animals it would appear that the explanation 
of the pathogenesis of the colon bacillus is undoubtedly to be found in 
the toxic effects of the chemical substance and products of the cells. 
In Man.--In normal intestines with intact mucous membranes the 
toxic products formed by B. coli are absorbed but little or not at all, 
and the bacilli themselves are prevented from invading the tissues by 
the epithelial layer and the bactericidal properties of the body fluids. 
Possibly there is an acquired immunity to the colon varieties which 
have long inhabited the intestines. 
B. coli was at first regarded purely as a saprophyte. Later, because 
of the postmortem invasion and the great ease of growth of the colon 
bacillus on ordinary media, the other extreme was taken of attributing 
too much to it. 
The bacilli previously present in the intestines can, either by an 
increase in virulence or by a lowered resistence in the person, invade 
the tissues. Thus in the case of ulceration in typhoid fever B. coli may 
enter the blood, or in perforation produce peritonitis. In dying condi- 
tions they at times pass through the intact mucous lining. In the gall- 
bladder or urinary tract the spread of bacilli from the intestines may 
cause disease. The specific serum reaction in the body is a sign of infec- 
tion, but great care has to be observed in deciding that it is present, 
as group agglutinins also occur. Up to the present time it is very diffi- 
cult to state in any colon infection whether the bacilli were previously 
present in the intestines or were derived from outside sources through 
water, food, or direct contact with other cases. 350 
PATHOGENIC MICROORGANISMS 
Intestinal Lesions.-The lesions present in intestinal infection attri- 
buted to B. coli are those of enteritis; the duodenum and jejunum are 
found to contain fluid, the spleen is somewhat enlarged, and there are 
marked hyperemia and ecchymosis of the small intestines, together with 
swelling of Peyer's patches. 
Virulence of B. Coli from Normal and Diseased Intestines.-The virulence 
varies with the culture and the time since its recovery from the intestines. 
Other things being equal, it is usually more virulent from an intestinal 
inflammation. From severe diarrhea the colon bacilli in 0.25 c.c. 
bouillon culture may kill guinea-pigs if given intraperitoneally, while 
from the healthy bowel 2.5 c.c are usually required. 
B. Coli in Sepsis.-When lesions of the intestinal mucous mem- 
branes exist, or in colon cystitis, pyelitis, or cholecystitis, there is fre- 
quently just before death a terminal dissemination of the bacilli and 
consequent septicemia. Here special symptoms of intoxication may 
occur, such as diarrhea, changes in temperature, heart weakness, and 
hemorrhages. In most of these cases infection proceeds from the intes- 
tines, but in not a few from the wounded urethra or bladder. The colon 
septicemia is detected by blood-cultures. At times very few bacilli 
are found, and then the blood-infection may be less important than the 
local one. Cases occurring in typhoid and cholera have been observed, 
especially in relapses in typhoid. In very young infants a malignant 
septicemia with tendency to hemorrhages is due to B. coli. In a few 
cases in which B. coli but no typhoid bacilli were present the course of 
the disease has been similar to typhoid fever. An epidemic due to colon 
infection of water has been noted. Infection through food and water 
are usually brought about by other closely allied bacilli not belonging 
to the colon group. 
B. Coli in Diarrhea.-In diarrhea we find increased peristalsis, less 
absorption of foodstuff, increased and changed intestinal secretions. 
Tissier observed that under treatment with cathartics the colon varieties 
increased, while the anaerobic forms are inhibited. In diarrhea, there- 
fore, we should expect favorable conditions for multiplication and 
inhibiting causes to be lessened. This makes one question the signi- 
ficance of numerous epidemics which have been reported of acute 
diarrhea in children from one to five years of age in which almost pure 
cultures of colon bacilli have been found. The symptoms in such epi- 
demics begin with high fever which often rapidly falls, and frequent 
stools, only watery or containing mucus and streaks of blood. These 
symptoms may quickly abate or go on to a toxic state characterized 
by heart weakness and drowsiness. This may lead to lung complica- 
tions or death. 
B. Coli in Peritonitis.-Here the lesions must be considered as being 
due to mixed infection. Not only perforation of the intestines in man, 
but injury to the intestinal walls, allows colon infection of the peri- 
toneum to take place. At first most of these cases were believed to be 
a pure colon infection, but now it is known that this idea came largely 
from the overgrowth of colon bacilli in the cultures. More careful INFLAMMATION OF THE URINARY TRACT 
351 
investigations, through cultures and smears, have demonstrated the 
fact that streptococci, and less frequently staphylococci and pneu- 
mococci, are also usually present in peritonitis arising from intestinal 
sources. The colon bacilli found even in the same case commonly 
comprise many varieties. 
B. Coli in Inflammation of the Bile Tract.-The normal healthy 
gall-bladder is usually sterile. This is true in spite of the fact that 
bile is apparently a good culture medium for the colon group. Simple 
tying of the neck of the gall-bladder usually causes a colon infection 
to take place within twenty-four hours. Obstruction of the bile-duct 
through various causes is fairly common in man. The gall-bladder then 
becomes infected, and following the inflammation of the mucous mem- 
branes there is often the formation of gall-stones. Some cases of jaun- 
dice are believed to be due to colon inflammation of the gall-ducts. 
Atypical varieties of B. coli are frequently isolated from gall-bladder 
infections. 
Inflammation of the Pancreas.-Welch was the first to record a 
case of pancreatitis with multiple fat necroses due to B. coli infection, 
A few more cases have since been reported due to members of the colon 
group, either alone or in conjunction with the pyogenic cocci. 
Inflammation of the Urinary Tract.-As far back as 1879 Bouchard 
noted cystitis due to bacilli of the colon group. When cystitis is estab- 
lished the bacterial infection frequently spreads to the pelvis of the 
kidneys, causing a pyelitis or suppurative nephritis. The same process 
often occurs in man. In most cases of chronic cystitis the ureters and 
pelves of the kidneys become involved; any malformation of the ureters 
aids the process. From the pelvis the bacteria push up into the urinary 
tubules and excite inflammation and multiple abscesses. Colon infection 
of the different parts of the urinary tract may occur at any age, from 
infancy upward. Instead of starting in the bladder it may begin in the 
kidney itself, the colon bacilli coming from the blood or peritoneum. 
In many of these cases the bacilli isolated from the urine are clumped 
in high dilutions of the blood from the patient. 
Although other bacteria-the pyogenic cocci, the proteus, the typhoid 
bacillus, etc.-may excite cystitis, still in 90 per cent, of all cases some 
of the colon group are found, and this percentage is even higher in young 
children. The clinical picture of colon infection is very variable. The 
lightest cases progress under the guise of a bacteriuria. The urine is 
passed a little more frequently and shows a fine granular cloudiness. 
The reaction is acid. The cell elements are but little increased. There 
is an excess of mucus. Albumin is absent or present in only a trace. 
The condition may last for weeks or months and then spontaneously 
disappear or grow worse. With a somewhat more severe infection there 
is painful urination, perhaps tenesmus, increase of pus cells and slight 
fever. In a conical glass a sediment of pus cells forms at the bottom, 
and clear urine remains above. If the infection passes to the kidney 
colicky pain and tenderness over the region of the kidneys is usually 
present. The most important symptom of pyelitis is an irregular inter- 352 
PATHOGENIC MICROORGANISMS 
mittent fever resembling malaria. The albumin is increased in the urine 
and red-blood cells may be seen. If a general nephritis arises the symp- 
toms are all intensified and an anemic condition may develop. Septi- 
cemia may finally result. 
In most of these cases the microscopic examination is sufficient to 
make a probable diagnosis, since the bacteria are so abundant. The 
variety of colon bacillus present can, of course, only be told by cultures 
and other means. In the urine they appear as diplobacilli, or partly 
in short, almost coccus, forms, partly in long threads. As a rule, motility 
is absent. Not infrequently the cultures appear to be identical with 
those of the Bacillus aerogenes. 
The characteristics of the urine itself have much to do with the 
probability of infection; the more acid urines being less likely to afford 
a proper soil for growth. Some uriries are bactericidal even when they 
are neutral. The substances producing this condition are not known. 
The colon bacilli in the urine produce no appreciable effect on the reac- 
tion, but give up some of their toxins, which upon absorption cause the 
deleterious local and general effects. The serum of the patient usually 
agglutinates the cultures from the urine in 1: 20 or 1: 50 dilutions, but 
this property is sometimes absent, especially in light cases. 
In all cases in addition to the introduction of the colon bacillus a 
predisposing condition must be present, such as more or less marked 
retention of urine by an enlarged prostate or stricture, any unhealthy 
state of the mucous membrane or general depression of vitality. 
B. Coli as Pus Formers.-Members of this group are frequently the 
cause of abscesses in the region of the rectum, urethra, and kidney. 
They rarely produce pus in other locations. 
B. Coli in Inflammation Not Previously Mentioned.-Broncho- 
pneumonia, lobar pneumonia, and pleurisy have occasionally been 
caused by colon bacilli, probably from blood-sources. Not a few cases 
of meningitis and spinal meningitis in infants, follow localized B. coli 
infections. The symptoms are not well developed, as a rule. Some 
cases of endocarditis have also been noted. 
Immunity.-Natural infection in man, or inoculation into animals, 
is followed by the production of antibodies; agglutinins, precipitins, 
bactericidal substances, and opsonins being produced. The attempt to 
separate the members of this group by agglutination have shown the 
great dissimilarity of the different members in their immune reactions 
although group reactions are marked. Natural agglutinins for this 
group are commonly present in the serum of man and animals. 
Curative Vaccine Treatment.-Localized inflammations due to the 
B. coli have been treated quite successfully by injections of dead 
organisms. An injection of 25 millions can be made daily or 300 millions 
every few days. The dose should be increased at each injection depend- 
ing on the reaction, 1000 million being the usual maximum dose. 
Autogenous vaccines should always be prepared if possible. 
Methods of Isolation.-They may be isolated from lesions on ordi- 
nary media or bile media or Endo- or Conradi plates may be used. The B. AEROGENES 
353 
latter are used in isolation from feces or other mixed material. Blood 
cultures may be made in bile or broth. For the examination of water 
for members of the colon group, see Bacteriology of Water. 
Bacillus Aerogenes and Allied Encapsulated Bacilli.-The members 
of this group differ from the members of the colon group, already de- 
scribed, in producing a viscid or mucoid growth, and in smears a capsule is 
commonly demonstrable. For this reason the group is spoken of by some 
as the capsulatus group, and the aerogenes types described as allied 
varieties. There are wide variations in the group, varying from forms 
almost identical with the colon types already described, to forms in 
which the capsule production is marked, and the ability to ferment 
lactose is slight or absent. In the absence of biologic differentiation 
of types, the varieties are best considered according to their source. 
For this reason the term B. aerogenes is collectively used for the types 
normally found in feces, milk, etc. 
B. Aerogenes.-Normally present in feces and also found in sewage 
and water where it has the same significance as other types of the colon 
group. It is constantly present in milk and is one of the chief causes 
of the souring of milk and cream. 
Morphologically it is variable in length and capsule production. In 
cultures the growth is abundant and viscid or mucoid in variable degree. 
It usually ferments the various carbohydrates more vigorously than 
other colon types, the closed arm of the fermentation tube being usually 
full or nearly full of gas. Different strains differ in the carbyhodrates 
attacked. Indol production is variable. B. aerogenes is much more 
resistant to acids and other deleterious substances than the other colon 
types. 
Pathogenesis.-Probably slight, although it has been isolated from 
infections of the urinary tract, peritoneum and liver and gall-bladder. 
In some of the cases reported there is a possibility that the infection 
was really due to other of the colon types. In the absence of reliable 
methods of differentiation there is always a question whether the more 
serious infections are not due to the more virulent bacillus of Friedlander. 
Bacillus Pneumoniae, Friedlander (B. Mucosus Capsulatus).-This 
bacillus was first described by Friedlander in 1882. He confused this 
bacillus with the pneumococcus, then undescribed, and regarded it as 
the causative agent in lobar pneumonia. 
Morphology.-It varies from coccoid forms to longer bacilli; they 
may be single, in pairs, or in short chains. They are surrounded by 
a wide capsule which is easily demonstrable not only in the material 
directly from the lesions but also in smears from cultures. It is Gram- 
negative and non-motile. 
Biology.-It grows freely on ordinary media. On agar the colonies 
are characteristically mucoid, with a tendency to confluence. Indol 
is not produced. The fermentative properies of different strains is 
very varied not only in the sugars fermented, but also in the amount 
of acid or gas or both produced. Milk-sugar is usually fermented, but 
milk is not coagulated. 
23 354 
PATHOGENIC MICROORGANISMS 
Pathogenesis.-It is pathogenic for mice and guinea-pigs in variable 
degree. Injection intraperitoneally or into the lung is followed by peri- 
tonitis or local hepatization with septicema. Rabbits are killed by some 
strains after intraperitoneal or intravenous injection; subcutaneous 
injection, however, commonly causes only a local lesion. 
In man it causes both broncho- and lobar pneumonia. This type of 
pneumonia is relatively infrequent and is characterized by its very high 
mortality. The bacilli are present in the sputum as a rule in almost 
pure culture. It is commonly present in the upper respiratory tract 
and the accessory sinuses, and may cause inflammation. By extension 
infection of the ear occurs, which may be complicated by meningitis. 
The pleura or the pericardium may be infected as a complication of 
pneumonia. A septicemia may develop as a complication of pneumonia 
or infection elsewhere. Inflammations of the eye are occasionally due 
to this organism. 
B. Ozaena.-This organism is culturally indistinguishable from the 
preceding. It receives its name from its constant presence in ozena or 
fetid rhinitis. Abel and others consider it the causative agent in this 
disease, basing their opinion on its constant presence. 
Bacillus of Rhinoscleroma.-This bacillus differs from the Bacillus 
pneumoniae only in its weaker fermentative properies. Its name comes 
from its frequent presence in rhinoscleroma. Its etiological relationship 
to this disease is not established. 
Immunity and Serum Reactions of the Capsulatus Types.-Active im- 
munity cannot as a rule be produced in animals by the injection of 
killed cultures or the products. Careful injections of animals, however, 
lead to the production of agglutinins, precipitins, and complement 
fixing antibodies. The attempt to differentiate the various types by 
means of serum reactions has as yet led to no uniform results. 
In common with other encapsulated bacteria, they are not easily 
affected by sera. Forges found that the absence of agglutination could 
be attributed to the presence of the capsule and devised a method of 
destroying the capsule by first treating the bacterial suspension with 
acids and heat and then neutralizing with alkali. 
Vaccine Therapy.-Only a limited number of cases have been treated, 
some with reported improvement, others not. 
Other Bacilli Allied to the Colon Group.-There are various types of 
which B. cloacae is an example, which resemble the colon bacillus in 
many respects but liquefy gelatin in varying degree, some very slightly 
and only after prolonged growth. CHAPTER XXIV. 
THE TYPHOID BACILLUS. 
This organism was first observed by Eberth, and independently by 
Koch, in 1880, in the spleen and diseased areas of the intestine in 
typhoid cadavers, but was not obtained in pure culture or its prin- 
cipal biologic features described until the researches of Gaffky in 1884. 
The methods of identification employed by Gaffky were found insuffi- 
cient to separate the typhoid bacillus from other bacilli of the colon- 
typhoid group. The absolute identification of the bacillus only became 
possible with the increase of our knowledge concerning the specific 
immune substances developed in the bodies of immunized animals. 
Its etiologic relationship to typhoid fever has been particularly difficult 
of demonstration, for although pathogenic for many animals when 
subcutaneously or intravenously inoculated, it has been impossible to 
produce infection in the natural way or produce gross lesions corre- 
sponding closely to those occurring generally in man. Nevertheless the 
specific reactions of the blood-serum of typhoid patients, the constant 
presence of the Bacillus typhosus in the intestines and some of the organs 
of the typhoid cadavers, the very frequent isolation of this bacillus from 
the roseola, spleen, blood, and excretions of the sick during life, the 
absence of the bacilli in healthy persons, unless they have at some time 
been directly exposed to, or are convalescent from, typhoid infection, all 
these have demonstrated scientifically that this bacillus is the chief 
etiologic factor in the production of the great majority of cases desig- 
nated as typhoid fever. 
Morphology and Staining.-Typhoid bacilli are short, rather plump 
rods of about Im to 3m in length by 0.5m to 0.8m in diameter, having 
rounded ends, and often growing into long threads. They are longer 
and somewhat more slender in form than most of the members of the 
colon group of bacilli (Figs. 135 and 136). 
The typhoid bacilli stain with the ordinary aniline colors, but a 
little less intensely than do most other bacteria. Like the bacilli of 
the colon and paratyphoid groups, they are decolorized by Gram's 
method. Bipolar staining is sometimes marked. 
Biology.-The typhoid bacillus is a motile, aerobic, facultative, 
anaerobic, non-liquefying bacillus. It develops best at 37° C.; above 
40° and below 30° growth is retarded; at 20° it is still moderate; below 
10° it almost ceases. It grows slightly more abundantly in the presence 
of oxygen. It does not form spores. 
Resistance.-When a number of typhoid bacilli are dried most of 
them die within a few hours and a few frequently remain alive for 356 
PA THOGENIC MICROORGA NI SMS 
months, but sometimes all the bacilli die very quickly. In their 
resistance to heat and cold they behave like the average non-spore- 
bearing bacilli. With rare exceptions they are killed by heating to 
60° C. for one minute. 
Fig. 135 
Fig. 136 
Typhoid bacilli from nutrient agar. 
X 1100 diameters. 
Typhoid bacilli from nutrient gelatin. 
X 1100 diameters. 
Motility.-Typhoid bacilli, when living under favorable conditions, 
are very actively motile, the smaller ones having often an undulat- 
ing motion, while the larger rods move about rapidly. In different 
cultures, however, the degree of motility varies. 
Fig. 137 
Fig. 138 
Flagella, heavily stained, attached to bacilli. 
(Van Ermengen's method.) 
Typhoid bacillus with faintly stained flagella. 
(Loffler's method.) 
Flagella.-These are often numerous and spring from the sides as 
well as the ends of the bacilli, but many short rods have but a single 
terminal flagellum (Figs. 137 and 138; see also Plate III). 
Cultivation.-Its growth on most sugar-free culture media is quite 
similar to that of the Bacillus coli, but it is somewhat slower and not 
quite so luxuriant. INDOL REACTION 
357 
Growth on Gelatin Plates (Fig. 139).-The colonies growing deep 
down in this plate medium have nothing in their appearance to distin- 
guish them from submerged colonies of the colon group; they appear 
as finely granular round points with a sharp margin and a yellowish- 
brown color. The superficial colonies, however, particularly when 
young, are often quite characteristic; they are transparent, bluish- 
white in color, with an irregular outline, not unlike a grape-leaf in shape. 
Slightly magnified they appear homogeneous in structure, but marked 
by a delicate network of furrows. Surface colonies from some varieties 
of colon bacilli give a similar picture. 
In stick cultures in gelatin the growth is mostly on the surface, appear- 
ing as a thin, scalloped extension, which gradually reaches out to the 
sides of the tube. In the track 
of the needle there is but a limited 
growth, which may be granular 
or uniform in structure, and of a 
yellowish-brown color. There 
is no liquefaction. 
Growth in Bouillon.-This me- 
dium is uniformly clouded by 
the typhoid bacillus, but the 
clouding is not so intense as by 
the colon bacillus. When the bou- 
illon is somewhat alkaline a deli- 
cate pellicle is sometimes formed 
on the surface after eighteen to 
twenty-four hours' growth. 
Growth on Agar.-The streak 
cultures on agar are not dis- 
tinctive; a transparent, filiform, 
grayish streak is formed. 
Growth on Potato.-The growth 
on this medium was formerly of 
great importance in identification, but now other media, giving more 
specific characteristics, have been discovered. When characteristic, 
the growth is almost invisible but luxuriant, usually covering the 
surface of the medium, and when scraped with the needle offers a 
certain resistance. In some cases, however, the growth is restricted 
to the immediate vicinity of the point of inoculation. Again, the 
growth may be quite heavy and colored yellowish brown, and with 
a greenish halo, when it is very similar to the growth of the colon 
bacillus. These differences of growth on potato appear to be chiefly 
due to variations in the substance of the potato, especially in its re- 
action. For the characteristic growth the potato should be slightly 
acid. A new lot of potato should always be tested with a typical 
typhoid bacillus as a control. 
Indol Reaction.-It does not produce even a trace of indol in peptone- 
water solution. This test was proposedfby Kitasato for differentiating 
Fig. 139 
A superficial colony (1) and a deep colony (2) 
of typhoid bacilli in gelatin. X 20 diameters. 358 
PATHOGENIC MICROORGANISMS 
the typhoid bacillus from other similar bacilli such as those of the 
colon group, which, as a rule, give the indol reaction. 
The typhoid bacillus, like the colon bacillus, produces alkaline 
substances from peptone. 
Neutral Red.-In stick cultures in glucose agar the typhoid bacillus 
produces no change, while the colon bacillus decolorizes the medium 
and produces gas. 
Effect of Inhibiting Substances in Culture Fluids.-The typhoid bacil- 
lus is inhibited by weaker solutions of formaldehyde, carbolic acid, 
and other disinfectants than is the colon bacillus. Most typhoid-like 
bacilli resemble the typhoid bacillus in this respect. Some sub- 
stances, such as brilliant green, inhibit the colon bacillus more. 
Action on Different Sugars.-The determination of the action upon 
sugars of any bacillus belonging to the typhoid or colon group is one 
of the most important of all the cultural differential tests. It has 
been given in detail in the table, p. 345. 
While the typhoid bacillus does not produce gas from dextrose 
galactose, and levulose, it does produce acid from these substances. 
Milk.-The typhoid bacillus does not cause coagulation when grown 
in milk. A very slight acidity is evidenced in litmus milk from the 
action of the typhoid on traces of fermentable substances other than 
lactose. 
Production of Disease in Animals.-It is impossible experimentally 
to produce the characteristic lesions usually met with in human 
typhoid fever in animals. Sickness or fatal results without the appear- 
ance of the typical pathologic changes may be produced by animal 
inoculations, but in most cases they could easily be traced to the 
toxemia produced by the substances in the bodies of the bacilli 
injected. Typhoid bacilli, freshly obtained from typhoid cases and 
introduced subcutaneously in animals, rapidly die. In the peritoneal 
cavity they may increase, causing a fatal peritonitis with toxic poison- 
ing. By accustoming bacilli to the animal body a certain degree of 
increased virulence for the animal can be obtained, so that smaller 
amounts of culture may prove fatal. Among the most successful 
efforts in this direction are the experiments of Cygnaeus and Seitz, 
who, by the inoculation of typhoid bacilli into dogs, rabbits, and mice, 
produced in the small intestines conditions that were histologically, 
and to the naked eye, analogous to those found in the human subject. 
Their results, however, were not constant. Very similar results followed 
inoculation of virulent strains of colon bacilli. In rabbits a septicemia 
can be produced by intravenous inoculation. Localization in the gall- 
bladder follows and a "carrier" state similar to that found in man 
results. 
Distribution of Bacilli in the Human Subject. Toxic Effects.- 
Typhoid fever during its early stages, at least, is accompanied by a 
bacteremia. The bacilli thus pass to all parts of the body and become 
localized in certain tissues, such as the bone marrow, lymphatic tissues 
and spleen, liver and kidneys. Wherever found in the tissues the ELIMINATION OF TYPHOID BACILLI FROM BODY 
359 
typhoid bacilli are usually observed to be arranged in groups or foci; 
only occasionally are they found singly. These foci are formed during 
life, as is proved by the degenerative changes often seen about them; 
but it is possible that the bacilli may also multiply somewhat after 
death. 
Important Primary Characteristic Lesions in Man.-The lesions of the 
intestines which are most pronounced in the lower part of the ileum consist 
of an inflammatory enlargement of the solitary and agminated lymph nodules. 
Necrosis with ulceration frequently follows the hyperplasia in the more severe 
cases. In the severest cases the ulceration and sloughing may involve the 
muscular and peritoneal coats and perforation may occur. Peritonitis and 
death usually follow. In rare cases the perforation is closed by adhesions. 
The minute changes are an hyperplasia of normal elements of the lym- 
phatic tissue, namely, the lymph cells and the endothelium of the trabecula; and 
sinuses. In severer forms necrotic changes are apt to intervene. These 
changes are attributed to the toxic substances formed by the typhoid bacilli, 
but may be directly brought about by the occlusion of the nutritive bloodvessels, 
as pointed out by Mallory. 
The mesenteric lymph nodes undergo changes similar to those in the ileum. 
The spleen is enlarged because of congestion and hyperplasia. The liver and, 
to a less extent, the kidneys are apt to show foci of cell proliferation. 
In typhoid fever, as in other infectious diseases, toxic poisoning may be mani- 
fested by disturbances in the circulatory, respiratory, and heat-regulating 
mechanism as well as by manifest lesions. In a few cases the intestinal lesions 
are absent. Some of the inflammatory complications which occur in typhoid 
fever are due to the growth of the bacillus in excessive numbers in unusual places 
in the body; but many of them are due to a secondary infection with other 
bacteria, expecially the pyogenic cocci and bacilli of the colon group. 
Unusual Location of Typhoid Lesions Occurring as Complications 
of Typhoid Fever.-Cases of sacculated and general peritonitis, ab- 
scess of the liver and spleen, subphrenic abscess, osteomyelitis, peri- 
ostitis, and inflammatory processes of other kinds have been reported 
as being due to the typhoid bacillus. In certain cases of typhoid 
pneumonia, serous pleurisy, empyema, and inflammations of the brain 
and spinal cord or their membranes, typhoid bacilli exclusively have 
occurred. The inflammation produced may or may not be accom- 
panied by the formation of pus. There are indeed a number of cases 
now on record in which the typhoid bacillus has played the part of 
pus producer. 
The Importance of Mixed Infection.-Frequently when complications 
occur in typhoid fever they are due to secondary or mixed infection 
with the staphylococcus, pneumococcus, streptococcus, pyocyaneus, 
and colon bacillus. Frequently these bacteria are found side by side 
with typhoid bacilli; in such cases it is difficult to say which was the 
primary and which was the secondary infection. 
Elimination of Typhoid Bacilli from Body.-Not infrequently 
typhoid bacilli are found in the secretions. They are present in the 
urine in about 25 to 50 per cent, of the cases during some part of the 
disease. Slight pathological lesions in the kidneys almost always 
occur in typhoid fever, but severe lesions also sometimes occurs. In 
some cases the urine is crowded with typhoid bacilli. 360 
PATHOGENIC MICROORGANISMS 
In cases of pneumonia due to the typhoid bacillus it is abundantly 
present in the sputa, and care should be taken to disinfect the expec- 
toration of typhoid patients. In typhoid fever the bacilli are almost 
always present in the gall-bladder. The bacilli are usually eliminated 
by the feces, being derived from the ulcerated portions of the intestines; 
their growth within the intestinal contents is, with few exceptions, 
not extensive. They may be excreted, however, very early in the 
disease, and have been found in the stools even during the incubation 
period. 
Not only do the very great majority of cases examined bacterio- 
logically and pathologically, but the epidemiological history of the 
disease, proves that the chief mode of invasion of the typhoid bacillus 
is by way of the mouth and stomach. The infective material is dis- 
charged principally by means of the excretions and secretions of the 
sick-namely, by the feces, the urine, and occasionally by the sputum. 
Occurrence in Healthy Persons.-The bacilli usually disappear 
from the body in the fourth or fifth week, but may remain for months 
or exceptionally years in the urine and throughout life in the gall- 
bladder. They have been found in deep abscesses one year after 
recovery from typhoid fever. 
Typhoid Carriers.-Examinations of convalescent typhoid cases 
show that about 1 to 5 per cent, continue to pass typhoid bacilli for 
years, perhaps for life. Petruschky in 1898 reported that typhoid 
bacilli sometimes remained in the urine of typhoid convalescents 
for months. Cushing soon after observed a case who had had ty- 
phoid fever five years before. In 1902 Frosch, and a little later 
Conradi and Drigalski, reported persons who passed typhoid-infected 
feces months after recovery from typhoid fever. Some bacilli carriers 
did not know either that they had had typhoid fever or been in contact 
with it, and others knew only that they had been in contact with it. 
In Washington, typhoid bacilli were isolated 0.3 per cent, of persons 
with no known exposure, but who did not or had not had typhoid. 
These cases may be called "healthy or normal carriers" in contra- 
distinction to "convalescent carriers." Lentz in 1905 found out of a 
large number of examinations that about 4 per cent, of persons convales- 
cent from typhoid fever were typhoid carriers. In our laboratory we 
have found six in one hundred and forty institution convalescents. The 
focus of infection is either the gall-bladder or gall-ducts. The majority 
are women. 
A remarkable case of a cook was under our care for three years. 
A visitor of the family in which this woman was cook developed 
typhoid fever some ten days after entering the household. This was in 
1901. The cook had been with the family three years and it is difficult 
to judge which infected the other. The cook went to another family. 
One month later the laundress in this family was taken ill. 
In 1902 the cook obtained a new place. Two weeks after arrival 
the laundress was taken ill with typhoid fever; in a week a second case 
developed and soon seven members of the household were sick. COMMUNICABILITY 
361 
In 1904 the cook went to a home in Long Island. There were 4 
in the family as well as 7 servants. Within 3 weeks after arrival, 4 
servants were attacked. 
In 1906 the cook went to another family. Between August 27 
and September 3, 6 out of its 11 inmates were attacked with typhoid. 
At this time the cook was first suspected. She entered another family 
on September 21. On October 5, the laundress developed typhoid fever. 
In 1907 she entered a family in New York City, and two months 
after her arrival two cases developed, one of which proved fatal. 
Altogether during five years this cook is known to have been the cause 
of 26 cases of typhoid fever. 
The cook was removed to the hospital March 19, 1907. Cultures 
taken every few days showed bacilli off and on for three years. Some- 
times the stools contained enormous numbers of typhoid bacilli and 
again for days none would be found. We recently traced some hundreds 
of cases of typhoid fever to a milk supply produced at a farm, looked 
after by a typhoid carrier who had typhoid fever forty-seven years ago. 
Treatment of Typhoid Carriers.-Medicinal treatment or surgery 
seems so far to have yielded only slight results. Urotropin in 
very large amounts is reported to have cured one case, in which 
operation alone had failed. Removal of the gall-bladder cannot be 
relied upon, as the bacilli may be present in the gall-ducts. Meader 
reports some success with long-continued treatment with killed cultures. 
Duration of Life Outside of the Body.-It is of importance to 
know for whgt length of time the typhoid bacillus is capable of liv- 
ing outside of the body; but, unfortunately, owing to the great diffi- 
culties in proving the presence of this organism in natural conditions, 
our knowledge on this point is still incomplete. In feces the length 
of life of the typhoid bacilli is very variable, depending on the composi- 
tion of the feces and on the varieties of bacteria present; sometimes 
they live but a few hours, usually a day, exceptionally for very long 
periods. Thus, according to Levy and Kayser, in winter typhoid 
bacilli may remain alive in feces for five months. Foote says that 
they can be found in living oysters for a month at a time, but in nu- 
merous experiments we have not been able to find them after five days. 
Their life in privies and in water, however, is usually very much 
shorter. As a rule, they can be detected in river water no longer than 
seven days after introduction, and often not after forty-eight hours. 
The less the general contamination of the water, the longer the bacilli 
are apt to live. The life of the typhoid bacillus varies according to the 
abundance and varieties of the bacteria associated with it, and accord- 
ing to the presence or absence of such injurious influences as deleterious 
chemicals, high temperature, light, desiccation, etc., to which it is 
known to be sensitive. In ice typhoid bacilli rapidly die, none prob- 
ably ever live as long as six months (see p. 372). 
Communicability.-The bacilli may reach the mouth by means of 
infected fingers or articles of various kinds, or by the ingestion of 
infected food, milk, water, etc., or by more obscure ways, such as the 362 
PATHOGENIC MICROORGANISMS 
eating of raw oysters and clams or the contamination of food by 
Hies. Of the greatest importance, however, is the production of infection 
by contaminated drinking-water or milk. In a very large number of 
cases indirect proof of this mode of infection has been afforded by find- 
ing that the water had been contaminated with urine or feces from 
a case of typhoid. In a few instances the proof has been direct- 
namely, by finding typhoid bacilli in the water. Examples of in- 
fection from water and milk have frequently come under our direct 
observation.- The following instances may be cited: A large force 
of workmen obtained their drinking-water from a well near where 
they were working. Typhoid fever broke out and continued to spread 
until the well was filled up. Investigation showed that some of the 
sick, in the early stages of their disease, repeatedly infected the soil 
surrounding the well with their urine and feces. Another example 
occurred in which typhoid fever broke out along the course of a creek 
after a spring freshet. It was found that, far up near the source of the 
creek, typhoid feces had been thrown on one of its banks and had 
then been washed into the stream. 
In the late epidemic at Ithaca some 1500 cases developed among 
those using the infected water supply of the town. The students 
and townspeople not drinking the infected supply escaped. The epi- 
demic at Scranton, Pa., during the winter of 1907 was most interesting. 
A little over 1 per cent, of the inhabitants were attacked. No pollu- 
tion of the water with typhoid infected feces or urine could be discov- 
ered, although typhoid bacilli were isolated from the water of a small 
intercepting reservoir by Dr. Fox. This was only accomplished by 
using large quantities of water. The bacillus isolated was identical 
by all known tests with the typhoid cultures from cases of typhoid 
fever. 
An instance of milk infection secondary to water infection was in 
the case of a milk dealer whose son came home suffering from ty- 
phoid fever. The feces were thrown into a small stream which ran 
into a pond in which the milk cans were washed. A very alarming 
epidemic of typhoid developed, which was confined to the houses and 
asylums supplied with this milk. During the Spanish-American war 
not only water infection, but food infection was noticed, as in the 
case of a regiment where certain companies were badly infected, while 
others nearly escaped. Each company had its separate kitchen and 
food supply, and much of the infection could be traced to the food, 
the contamination coming partly through the flies. Several epidemics 
have been traced to oysters. Contact infection is probably responsible 
for as many cases as are due to the causes, already given. Even during 
outbreaks due to these causes, contact infection plays a large role in 
the development of secondary cases. These cases develop either by 
contact with known typhoid cases or through convalescent or healthy 
carriers in the households. The early undiagnosed cases may have 
typhoid bacilli in their stools and act as a source of contagion; like- 
wise short, mild cases develop which are never surely diagnosed. VACCINATION AGAINST TYPHOID FEVER 
363 
For these reasons "typhoid precautions" should not await positive 
diagnosis, but should be instituted on the slightest suspicion. 
Individual Susceptibility.-In this, as in all infectious diseases, 
individual susceptibility plays an important role in the production 
of infection. Without a suitable soil upon which to grow, the seed 
cannot thrive. There must in many be some disturbance of the di- 
gestion, excesses in drinking, etc., or a general weakening of the power 
of resistance of the individual, caused by bad food, exposure to heat, 
overexertion, etc., as occurs with soldiers and prisoners, for example, to 
bring about the conditions suitable for the production of typhoid fever. 
Immunization.-After recovery from typhoid fever a considerable 
immunity is present which lasts for years. This is not absolute, as 
about 2 per cent, of those having typhoid fever have a second attack, 
which is usually a mild one. Specific immunization against experi- 
mental typhoid infection has been produced in animals by the usual 
method of injecting at first small quantities of the living or dead typhoid 
bacilli and gradually increasing the dose. The blood-serum of animals 
thus immunized has been found to be highly bactericidal and to possess 
some protective power against toxic extracts of the bacilli. It is also 
rich in agglutinins, precipitins, and opsonins. These characteristics 
have also been observed in the blood-serum of persons who are con- 
valescent from typhoid fever. The attempt has been made to employ 
the typhoid serum for the cure of typhoid fever in man, but, although 
a number of individual observers have reported good results with one 
or another of the sera, most consider that little or no good is derived 
from serum. 
Vaccination During Typhoid Fever.-The value of vaccines in the 
treatment of typhoid fever has not as yet been definitely determined, 
although used by Richardson and later by Watters and Eaton1 and 
Callison.2 The use of vaccines early in the disease in doses of 
500,000,000 every second or third day would seem to shorten the course 
of the disease in a certain number of cases and to produce a somewhat 
lower death rate. Very recently Boinet3 has reported favorable results 
in twenty-five cases treated with sensitized vaccines prepared according 
to the method of Besredka. 
Vaccination Against Typhoid Fever.-The vaccination against 
typhoid fever is now a procedure of established worth. Its use began 
in 1896, when Pfeiffer and Kolle and, independently, Wright, demon- 
strated that persons injected with killed typhoid bacilli developed 
the same antibodies in their blood as are found in recovered cases of 
typhoid fever. Wright then introduced its use in the English army, 
and the results led to its introduction in other countries. Vaccination 
was started in the American army in 1909 under the supervision of 
Russell. 
The rate per 1000 in the whole American army has dropped from 
3.2 in 1908 to 0.03 in 1913. During the recent maneuvers in Texas 
1 Med. Rec., 1911, Ixxix, 797. 
3 Ann. de 1'Inst. Pasteur, 1914, xxviii, 540. 
2 Ibid., 1911, Ixxix, 1129. 364 
PATHOGENIC MICROORGANISMS 
there were 20,000 men in the encampments with only two cases of 
typhoid during a period of four months. This remarkable improve- 
ment over the rate in encampments of previous years is probably due, 
in large part, to improvements in camp sanitation, but not altogether. 
The soldiers visited ate and drank at Galveston and San Antonio, 
and in the former city there were 192 cases and in the latter 49 cases 
of typhoid fever during the same period of time. The civil population 
in this way acts as a control 
Similar results have been obtained in the English army. To cite 
one instance only, Firth reports that in India for 1911 the rates per 
thousand were: vaccinated 1.7, deaths 0.17; unvaccinated 6.7; deaths 
1.15. These figures are interesting not only because of the reduction 
in cases, but it gives a fair estimate of the reduction in the death rate 
of typhoid among those vaccinated. 
Similar results have followed its use in civil life. It has given good 
results even in the presence of an epidemic. Cullinan, for instance, 
vaccinated 500 persons in an asylum during an epidemic. Of the vac- 
cinated only 1.36 per cent, contracted typhoid, and these were nearly 
all in the incubation period of the disease; of the un vaccinated, 114 
nurses, 14.9 per cent., contracted typhoid. The use of vaccine during 
exposure has not led to any results, therefore that would warrant 
the fear of the production of a "negative phase," that is, reduction of 
resistance to infection. 
Since January, 1913, the Department of Health of New York City 
has detailed inspectors to vaccinate on request. The members of 
families where typhoid exists are especially urged to be vaccinated. 
The inspectors have vaccinated 2794 persons, of which about 80 per 
cent, have received full doses. Of these, 1014 were not directly exposed 
to infection and none have developed typhoid. The exposed persons 
numbered 1780, among these 31 cases of typhoid developed, the great 
majority receiving only one injection, and were already in the incuba- 
tion period of the disease. Two cases receiving three doses developed 
typhoid fever within ten days of the last dose. Of the remaining cases 
two nurses exposed to infection developed typhoid, one four months, 
the other eight months after injection. One died of hemorrhage. 
Another case died six months after vaccination of typhoid complicated 
by pregnancy with miscarriage. Her mother was a typhoid carrier, 
and four of her children had typhoid about the same time. Of about 
8000 persons exposed to typhoid during the above period of time there 
have been 190 cases of typhoid. During the same time vaccine for over 
2000 persons has been given to private physicians and institutions. No 
cases of typhoid have been reported among the persons injected. We 
know of two other cases' of typhoid after vaccination, one a nurse who 
died of hemmorrhage, the other a physician who recovered. 
These cases show that the protection conferred is only relative, and 
with an undue amount of exposure infection may result. 
The duration of the immunity is shown by the experience of Firth 
in the Indian army. He found that the immunity diminished after USE OF DRIED BLOOD 
365 
two and one-half years, but even after four to five years some im- 
munity persists, the rate among the vaccinated being only one-fourth 
of that among the unvaccinated troops. 
Three doses of vaccine are administered, the site of injection being 
the subcutaneous tissues over the insertion of the deltoid. The first 
dose is 500,000,000, the second and third 1,000,000,000 typhoid bacilli. 
The period between injections is seven to ten days, the injection being 
given in the late afternoon, so that if a reaction occur the person will 
be in bed. 
The reaction usually consists of a reddening and swelling at the 
point of injection, which is tender. In some instances this area is large 
and the axillary lymph nodes become swollen and tender. A general 
reaction is usually absent, or if present consists of slight malaise. In 
a few cases, less than 1 per cent., a more marked reaction occurs with 
prostration and considerable rise of temperature. The reaction is of 
no importance further than the discomfort caused. 
The vaccine usually employed is a suspension of the bacilli grown 
on agar, standardized by the method of Wright and killed by heating 
to 56° C. Sensitized vaccines of either killed or live bacilli have also 
been used, but their use has been too limited to warrant their general 
adoption as superior to the vaccine in general use. 
Diagnosis by Means of the Widal or Agglutination Reaction.-The 
chief practical application of our knowledge of the specific substances 
developed in the blood of persons sick with typhoid fever has been as 
an aid to diagnosis. 
Gruber-Widal Test.-The first application of the use of serum for 
the early diagnosis of typhoid fever on an extensive scale was made 
by Widal, and reported with great fulness and detail in a communication 
published in June, 1896. For history and theory of test, see p. 201. 
Since then the serum test for the diagnosis of typhoid fever has come 
into general use in bacteriologic laboratories in all parts of the world, 
and though the extravagant expectations raised at the time when 
Widal first announced his method of applying this test have not been 
entirely fulfilled, it has, nevertheless, proved to be of great assistance 
in the diagnosis of obscure cases of the disease, and is now one of the 
recognized tests for the differentiation of the typhoid bacillus. 
It should also be mentioned that to Wyatt Johnson, of Montreal, 
belongs the credit of introducing its use into municipal laboratories, 
suggesting that dried blood should be employed in place of blood-serum, 
and in October, 1896, the serum test was regularly introduced in the 
New York Department of Health Laboratory for the routine examina- 
tion of the blood-serum of suspected cases of typhoid fever. Since 
then all health departments have followed the example set by those of 
Montreal and New York. 
Use of Dried Blood.-Directions for Preparing Specimens of Blood. 
-The skin covering the tip of the finger or the ear is thoroughly 
cleansed, and is then pricked with a needle deeply enough to cause 
several drops of blood to exude. Two fair-sized drops are then placed 366 
PATHOGENIC MICROORGANISMS 
on a glass slide, one near either end, and allowed to dry. Glazed 
paper may also be employed, but it is not as good, for the blood soaks 
more or less into it, and later, when it is dissolved, some of the paper 
fibre is apt to be rubbed off with it. The slide is placed in a box for 
protection. 
Preparation of Specimen of Blood for Examination.-In preparing the 
specimens for examination the dried blood, if accuracy is desired, is 
first weighed and then brought into solution by adding to it the quantity 
of normal salt solution to make the desired dilution, remembering of 
course to allow for the loss in water through drying. A drop of each 
dilution is placed on individual cover-slips and to each is added a drop 
of an eighteen-hour broth culture of typhoid. These are then mounted 
on hollow slides in the ordinary way. (See p. 72.) The drops, after 
being mixed, have in a 1 : 10 dilution a distinct reddish color and in 
1 : 20 a faint pink tinge. The cover-glass with the mixture on the 
surface is inverted over a hollow slide (the edges about the concavity 
having been carefully smeared with vaselin, so as to make a closed 
chamber). Ordinarily the dried blood is not weighed, but the measure 
of dilution is estimated by the color of the drop. To judge this the 
beginner must carefully make dilutions of fluid blood and notice the 
depth of color in 1 : 10 and 1 : 20 dilutions. Besides the faulty judg- 
ment of the dilution color by the examiner, the variation in depth 
of color of different specimens of blood makes the estimation of dilu- 
tions more or less inaccurate, but fortunately this does not greatly 
interfere with the value of the test. 
Use of serum or fluid blood. (See Agglutination for methods.) 
Advantages and Disadvantages of Serum, Dried Blood, and Fluid Blood 
for the Serum Test.-The dried blood is easily and quickly obtained, 
and does not deteriorate or become contaminated by bacterial growth. 
It is readily transported, and seems to be of nearly equal strength with 
the serum in its agglutinating properties. It must in use, however, 
be diluted with at least five times its bulk of water, otherwise it is 
too viscid to be properly employed. The amount of dilution can 
only be determined roughly by the color of the resulting mixture, 
for it is impossible to estimate accurately the amount of dried blood 
from the size of the drop, and it is generally considered too much 
trouble to weigh it accurately. Serum, on the other hand, can be 
used in any dilution desired, from a mixture which contains equal 
parts of serum and broth culture to any dilution desired; and this can 
be exactly measured by a graduated pipette, or, roughly, by a measured 
platinum loop. The disadvantages in the use of serum are entirely due 
to the slight difficulty in collecting and transporting it. If the serum 
is obtained from blood after clotting has occurred a greater quantity 
of blood must be drawn than is necessary when the dried-blood method 
is used. For scientific investigations and for accurate results, par- 
ticularly in obscure cases, the use of serum is to be preferred to dried 
blood. Practically, however, the results are nearly as good for diag- 
nostic purposes from the dried blood as from the serum. DILUTION OF THE BLOOD-SERUM 
367 
The Culture to be Employed.-It is important that the culture em- 
ployed for serum tests should be a suitable one, for although all cul- 
tures show the reaction, yet some respond much better and in higher 
dilutions than others. Cultures freshly obtained from typhoid cases 
are not as sensitive as those grown for some time on nutrient media. 
Those kept for a long time on artificial media sometimes show a decided 
tendency to' spontaneous agglutination. Artificial cultivation is usually 
accompanied by increase of capacity for agglutination. At present a 
strain known as Mt. Sinai is used at the laboratory. A broth culture 
of the typhoid bacillus developed at 25° to 35° C., not over twenty- 
four hours old, in which the bacilli are isolated and actively motile, 
has been found to give us the most satisfactory results. If the broth 
culture is heavy it should be diluted. Cultures grown at temperatures 
over 38° C. are not apt to agglutinate so well as those grown at lower 
temperatures. Stock cultures of typhoid bacilli can be preserved on 
nutrient agar in sealed tubes and kept in the ice-box. These remain 
alive for months. From time to time one of these is taken out and used 
to inoculate the broth. 
Dilution of the Blood serum to be Empolyed and Time Required for 
the Development of Reaction.-The serum test, as has been pointed out, 
is quantitative and not qualitative, that is, the diagnostic reaction must 
be in dilutions higher than occur because of the presence of normal 
or group agglutinins. (See p. 195.) It is most important to remember 
that it is purely a matter of experience to determine in any type of 
infection what agglutinating strength of a serum is of diagnostic 
value. 
The results obtained in the Health Department Laboratories, as 
well as elsewhere, have shown that in a certain proportion of cases 
not typhoid fever a slow reaction occurs in a 1 : 10 dilution of serum 
or blood; but very rarely does a complete reaction occur in this dilution 
within fifteen minutes. When dried blood is used the slight tendency 
of non-typhoid blood in 1:10 dilution to produce agglutination is 
increased by the presence of the fibrinous clumps, and perhaps by 
other substances derived from the disintegrated blood cells. 
From many cases examined it has been found that in dilutions of 
1:20 a quick reaction is almost never produced in any febrile dis- 
ease other than due to typhoid or paratyphoid bacillus infection, while 
in typhoid fever such a distinct reaction often occurs with dilutions of 
1:100 or more. It is possible that some cases of paratyphoid infection 
give a prompt reaction in 1: 20 dilutions, but if this is so, it is not a serious 
drawback. The rare cases of persons who, though never having had 
typhoid fever, yet are typhoid-bacillus carriers usually have specific 
agglutinins in their blood. 
The mode of procedure as now employed is as follows: With serum, 
one part of a 1: 10 dilution is added to one of the bouillon culture. 
With dried blood, a solution of the blood is first made, and the dilution 
guessed from the color. To obtain an idea of the dilution by the color, 
known amounts of blood are dried and then mixed with definite amounts 368 
PATHOGENIC MICROORGANISMS 
of water; the colors resulting are fixed in the memory as guides for future 
tests. If there is no reaction-that is to say, if within five minutes no 
marked change is noted in the motility of the bacilli, and no clumping 
occurs-nothing more is needed; the result is negative. If marked 
clumping and immobilization of the bacilli immediately begin and 
become complete within five minutes, this is termed a marked immediate 
typhoid reaction, and no further test is considered necessary, though it is 
always advisable to confirm the reaction with higher dilutions up to 1: 50 
or more, so as to measure the exact strength of the reaction. If in the 
1: 20 dilution a complete reaction takes place within thirty minutes, the 
blood is considered to have come from a case of typhoid infection, while 
if a less complete reaction occurs it is considered that only a probability 
of typhoid infection has been established. By many the time allowed 
for the development of the reaction with the high dilutions is from 
one to two hours, but to us twenty minutes with the comparatively low 
dilution of 1: 20 seems safer and more convenient. Positive results 
obtained in this way may be considered conclusive, unless there be 
grounds for suspecting that the reaction may be due to a previous fairly 
recent attack or to recent vaccination. The failure of the reaction in 
one examination by no means excludes the presence of typhoid infection. 
If the case clinically remains doubtful, the examination should be 
repeated every few days. 
Use of Dead Cultures.-Properly killed typhoid bacilli respond well 
to the agglutination test. For the physician at his'office the dead 
bacilli offer many advantages. The reaction is slower than with the 
living cultures and is observed either macroscopically or microscopically. 
A number of firms now supply outfits for the serum test. These outfits 
consist of a number of small tubes containing an emulsion of dead 
typhoid bacilli. Directions accompany the outfit. 
Proportion of Cases of Typhoid Fever in which a Definite Reaction 
Occurs, and the Time of its Appearance.-As the result of a large num- 
ber of cases examined in the Health Department Laboratories, it 
was found that about 20 per cent, give positive results in the first 
week, about 60 per cent, in the second week, about 80 per cent, in the 
third week, about 90 per cent, in the fourth week, and about 75 per 
cent, in the second month of the disease. In 98 per cent, of the cases 
in which repeated examinations were made (hospital cases) a definite 
typhoid reaction was present at some time during the illness. 
Persistence of the Reaction.-A definite typhoid reaction has been 
observed from three months to a year after convalescence, and a slight 
reaction, though much less than sufficient to establish a diagnosis of 
typhoid infection, from one to fifteen years after the disease. In persons 
in whom the typhoid bacilli persist the serum reaction may last as 
long as the bacilli remain in the body. 
Typhoid Bacilli in the Blood.-A blood-culture is usually positive during 
the first week of typhoid fever, and is the best method for early diag- 
nosis. They appear in the blood even in the first few days of the disease. 
In the first week, nearly 100 per cent, positive results are obtained, TYPHOID BACILLI IN FECES 
369 
in the second week 50 per cent, and then progressively less till the 
end of the disease. In relapses they reappear. 
Method.-The blood is drawn from the median basilic vein by syringe 
and is inoculated into either broth or bile medium. If broth is used, 
several flasks are used with sufficient broth to dilute the blood 50 
times or more, so that if there are any bactericidal substances in the 
blood they will be too diluted to act 
Bile media (see p. 105) are more convenient, as the bulk need not 
be so large. The bile inhibits coagulation. Three parts of bile medium 
are used to one part of blood. The bile is placed on Endo, or Conradi, 
after eighteen hours' incubation. It should also be examined after two 
to three days, as growth is sometimes delayed. 
Another method, not as satisfactory as the preceding, but valuable in 
emergencies, is to inoculate the blood into ten times its volume of distilled 
water. Hemolysis results and the proteins supply the nutrient substances 
for growth. 
Typhoid Bacilli in Feces.-^The following table by Schroder gives the 
number and time of positive results: 
No. examined. 
Positive. 
Per cent. 
First week 
115 
11 
9.6 
Second week 
160 
45 
28.1 
Third week 
71 
16 
22.5 
Fourth week 
41 
9 
21.9 
Fifth week 
26 
4 
15.3 
Sixth week 
12 
2 
16.6 
Convalescent 
190 
10 
7.9 
These results were obtained, using several media in combination. 
The more promptly the stools are plated after passage, the better 
is the chance of obtaining typhoid bacilli. Hiss, examining the stools 
very quickly after passage, obtained a very high percentage of positive 
results, in 43 consecutive cases, 37 of which were in the febrile stage 
and 6 convalescent. In a number of instances only one stool was 
examined, but even under these adverse conditions the average of 
positive results in the febrile stage was 66.6 per cent. Out of 26 cases 
of typhoid fever examined in hospitals, 21 were in the febrile stage 
and 5 convalescent. In the febrile cases in 17 the presence of typhoid 
bacilli, often in great numbers, was demonstrated. Thus in these care- 
fully followed cases the statistics show over 80 per cent, of the febrile 
cases positive. The bacilli were isolated from these cases as early as 
the sixth day, and as late as the thirtieth day, and in a case of relapse 
on the forty-seventh day of the disease. These observations, with 
regard to the appearance of the bacilli in the stools during the febrile 
stage and their usually quick disappearance, except in the permanent 
typhoid carriers, after the defervescence, have been confirmed by others. 
We have, however, a carrier under observation who was discharged 
from the hospital after three negative examinations. The bacilli are 
isolated in some cases in which no Widal reaction was demonstrated, 
24 370 
PATHOGENIC MICROORGANISMS 
or before its appearances. Between the seventh and twenty-first day 
of the disease, experience seems to indicate that the bacilli may be 
obtained from about 25 per cent, of all cases on the first examination 
and from about 75 per cent, after repeated examinations. In some 
samples of feces typhoid bacilli die out or are overgrown within twenty- 
four hours; in others they remain alive for days or even weeks. This 
seems to depend on the bacteria present in the feces and upon its chemical 
character. Probably the presence of typhoid bacilli in some stools 
and their absence in others must be explained largely by the character- 
istics of the intestinal contents. The short life of the typhoid bacillus 
in many specimens of feces suggests that stools be examined as quickly 
as possible. In fact, unless the physician wishes to take the trouble to 
have the sample of feces sent immediately to the laboratory, it is hardly 
worth while for the bacteriologist to take the trouble to make the 
test. 
Method.-The feces, if solid, are rubbed up with broth, otherwise 
they can be used without further preparation. The fluidified feces 
should not consist of merely a loopful of the outer portion, but a mixture 
of a generous amount. 
The density of the suspension to employ is a matter of experience. 
Plates are then streaked (see p. 108) and the colonies of typhoid can 
then be agglutinated directly either microscopically or by using the 
slide method. In the latter a drop of saline for control and a drop of 
high-titre serum are placed on a slide and the colony rubbed up in the 
saline and then in the serum. With appropriate dilutions of the serum 
agglutination will take place almost immediately. 
For fishing, Russell's medium is used, which gives a tentative indica- 
tion of the nature of the organism, which can be confirmed by agglu- 
tination and further cultural tests. 
Typhoid Bacilli in the Urine. - Of great interest is the frequent 
occurrence of typhoid bacilli in large numbers in the urine. The 
results of the examinations of others as well as our own indicate that 
the typhoid bacilli are not apt to be found in the urine until the end of 
the second week of the fever, and may not appear until much later. 
From this on to convalescence they appear in about 25 to 50 per cent, 
of the cases, usually in pure culture and in enormous numbers, even as 
high as 100 million per c.c. They are found until several weeks or 
months after convalescence; in exceptional cases they persist for years. 
When we think of the chances such cases have to spread infection 
as they pass from place to place, we begin to realize how epidemics 
can start without apparent cause. The more we investigate the 
persistence of bacteria in convalescent cases of disease, the more diffi- 
cult the prevention of their dissemination is seen to be. The disinfection 
of the urine should always be looked after in typhoid fever, and 
convalescents should not be allowed to go to places where contamina- 
tion of the water supply is possible, without at least warning them of 
the necessity of great care in disinfecting their urine and feces for 
some weeks. IMPORTANCE OF ICE IN PRODUCTION OF TYPHOID FEVER 
371 
Method.-If the bacilli are numerous, as evidenced by examination 
in the hanging drop, they are easily obtained by direct plating, other- 
wise the urine should be centrifuged and the sediment plated, and 
larger amounts inoculated into bile medium for enrichment. 
Typhoid Bacilli in Rose Spots and Spleen.-Although the bacilli 
have been frequently isolated from rose spots, it is a less convenient 
method than blood-cultures. Spleen puncture has been employed and 
although cultures are frequently positive the operation is dangerous, 
and has been abandoned. 
Detection of Typhoid Bacilli in Water.-There is absolutely no 
doubt that the contamination of streams and reservoirs is a frequent 
cause of the outbreak of epidemics of typhoid fever, but the actual 
finding and isolation of the bacilli is a very rare occurrence. This is 
often due to the fact that the contamination has passed away before 
the bacteriologic examination is undertaken, and also to the great 
difficulties met with in detecting a few typhoid bacilli when they are 
associated with large numbers of other bacteria. The greater the 
amount of contamination entering the water, and the shorter the 
time which elapses between this and the drinking of the water, the 
greater is the danger. A recent isolation of the typhoid bacillus is 
that from the small storage reservoir supplying Scranton. The bacillus 
was isolated by Fox from about 1 liter of water. Tested alongside of 
a culture from one of the Scranton cases it seemed identical 
The Importance of Ice in the Production of Typhoid Fever.- 
The total number of instances of typhoid fever which have been 
directly traced to ice infection are remarkably few. One was in 
France, where a group of officers placed ice made from water polluted 
by a sewer in their wine and afterward a large percentage developed 
typhoid fever, while those of the same company not using ice escaped. 
A second case was a small epidemic which occurred in those who used 
ice from a pond. It was found that water directly infected with typhoid 
feces had flowed over its frozen surface and been congealed there. 
The fact that freezing kills a large percentage of typhoid bacilli 
makes it indeed possible to conceive that ice from moderately infected 
water contains so few living typhoid bacilli that only the exceptional 
person here and there becomes infected, and so the source of the 
infection remains undetected. 
If this be true and scattered cases occur, there should be at least 
some increase on some or every year in March, April, and May in 
such a city as New York, where four-fifths of all the ice consumed is 
from the Hudson River, which is known to be contaminated with 
typhoid bacilli. 
When we examine the records for the past ten years we find no in- 
crease of typhoid fever in Greater New York during those months, 
with the one exception of 1907, when we had in the borough of Man- 
hattan a sharp outbreak lasting four weeks. This outbreak did not 
occur at all in Brooklyn. As the people of Brooklyn drank different 
water, but received ice from the same places of the Hudson River as 372 
PATHOGENIC MICROORGANISMS 
those of Manhattan, this directed attention to the water or milk rather 
than the ice. Examination of the Croton watershed at the time showed 
that a small epidemic of typhoid existed there and that pollution of 
the water was probable. This suggested still more strongly that the 
water and not the ice was the cause of the typhoid infection. 
It happened that most of the cases occurred in those living in the 
section of the upper West Side, where only well-to-do people live. An 
investigation showed that the majority of the infected had used only 
artificial ice and several had used no ice in their water at all. 
Life of the Typhoid Bacillus in Ice in Laboratory Experiments.-The 
first important investigation was that of Prudden, who showed that 
typhoid bacilli might live for three months or longer in ice. This experi- 
ment is frequently wrongly interpreted, as when a recent writer states: 
"It has been amply demonstrated that the germs of typhoid fever 
are not killed by freezing and that they have been known to live in 
ice for long periods of time." 
It is true that in Prudden's experiment a few typhoid bacilli re- 
mained alive for three months, when the experiment was terminated, 
but those were but a small fraction of 1 per cent, of the original num- 
ber. Following Prudden's experiment Sedgwick and Winslow in Boston 
and Park in New York City carried on independently a series of experi- 
ments. These led to the same conclusions. A table summarizing a 
final experiment of ours in which twenty-one different strains, mostly 
of recent isolation, were subjected to the test is given below: 
Average number 
of bacilli in ice. 
Percentage typhoid 
bacilli living. 
Before freezing 
2,560,410 
100.0 
Frozen three days 
1,089,470 
42.0 
Frozen seven days 
361,136 
14.0 
Frozen fourteen days 
203,300 
8.0 
Frozen twenty-one days .... 
10,280 
0.4 
Frozen five weeks 
2,950 
0.1 
Frozen nine weeks 
127 
0.005 
Frozen sixteen weeks 
107 
0.004 
Frozen twenty-two weeks .... 
0 
0 
Life of Twenty-one Strains of Typhoid Bacilli in Ice. 
In these experiments twenty-one different flasks of Croton water 
were inoculated each with a different strain of typhoid bacilli. In 
one a little of the feces rich in typhoid was directly added. The in- 
fected water in each flask was then pipetted into thirty tubes. These 
tubes were placed in a cold-storage room in which the temperature 
varied from 20 to 28° F. At first tubes were removed and tested twice 
a week, later once a week. The object of using so many different 
strains was because it has become evident that some cultures live 
longer than others. 
At the end of five weeks the water infected with six cultures was 
sterile, at the end of sixteen weeks only four strains remained alive. 
Interesting investigations of Hudson River ice were carried out in 
1907 by North. DIFFERENTIAL DIAGNOSIS 
373 
There was noticed a considerable difference between the number of 
bacteria in the top, middle, and bottom layers of ice. This is natural, 
since while water in freezing from above downward markedly purifies 
itself, 75 per cent, of the solids and a fair proportion of bacteria being 
eliminated, yet this cannot happen in the case of the snow blanket 
which becomes flooded by rain or by cutting holes through the ice. 
Here all impurities, such as dust and leaves which have fallen on the 
surface and dirt which may come from the water, remain with the 
bacteria which they carry, since all are retained in the porous snow. 
The bacteria in freshly cut bottom ice generally show the least destruc- 
tion by freezing. 
The river water in the forty specimens averaged 1800 bacteria per c.c., 
the top ice 306, the bottom ice 36, and the middle ice 14. Only four 
specimens of top ice had over 500 bacteria per c.c.; none of the specimens 
of middle or bottom ice. 
The great destruction by freezing is noticeable in these figures. 
Even the top ice soiled by the horses and men gathering it contained 
but 16 per cent, as many bacteria as the water from which it was 
obtained. The bottom ice, the last to be frozen, had but 2 per cent, 
of those in the water. 
Conclusions in Regard to Ice Pollution.-The danger from the use 
of ice produced from polluted water is always much less than the use 
of the water itself. Every week that the ice is stored the danger be- 
comes less, so that at four weeks it has become as much purified from 
typhoid bacilli as if subjected to sand filtration. At the end of four 
months the danger becomes almost negligible, and at the end of six 
months quite so. 
Differential Diagnosis.-The typhoid bacillus is easily separated 
from the other members of the group, and for practical purposes it is 
sufficient identification if the colony on the special plating media and 
the growth on Russel's medium is characteristic and is agglutinated in 
relatively high dilutions of serum. If a strain does not agglutinate 
freely it should be transferred daily on plain agar and again tested. 
The range of group agglutinins in the serum used should be known or 
error will result. 
In important examinations or strains from unusual sources as water, 
etc., a more extensive cultural study should be done. Likewise, absorp- 
tion of agglutinins and possibly the production of agglutinins for a 
known strain should be tested. 
B. (fecalis) Alkaligenes.-This group resembles the typhoid bacillus 
but produces acid from no sugars. It is frequently present in the 
intestines. It is usually only a saprophyte, but has been found in a few 
cases of disease in man. 
Other typhoid-like bacilli have been isolated from cholera infected 
swine, cow's feces, and water. CHAPTER XXV. 
PARATYPHOID GROUP. 
Gartner, in 1888, found the Bacillus enteritidis in association with 
a meat-poisoning epidemic. A cow sick for two days with profuse 
diarrhea had been slaughtered and the meat sold for food. Of the 
persons eating the meat fifty-seven became ill and one died. 
Similar bacilli were isolated by Smith from swine suffering from hog 
cholera, from mice by Loffler, and the bacilli became known as the hog 
cholera group. 
Similar bacilli were isolated by others from cases resembling typhoid 
fever. Schottmuller reported in 1900 that he was able to differentiate 
the strains from 5 cases into two groups, one resembling typhoid very 
closely, the other corresponding to the enteritidis types. The work 
of Durham and Buxton gave the basis for classification into subgroups. 
Numerous types have been isolated from infections in man and 
animals, and most of the terms in use refer to their source; but many 
cannot be differentiated one from the other, so fall into distinct groups 
according to their bacteriological and immune reactions. 
Paratyphoid A.-Disease Produced.-First isolated by Gwyn, later by 
Schottmuller,1 Brown, and Kayser, and others. It produces a typhoid- 
like disease in man, but is of relatively infrequent occurrence. The 
bacillus may be present in the feces, urine, blood, and bile. The post- 
mortem findings have been variable. In some the typical intestinal 
lesions of typhoid infections were present, in others they were absent. 
A diffuse inflammation of the intestines may be found. 
Morphology and Biology.-Similar to typhoid in many respects, but 
resembles Paratyphoid B. in its sugar fermentations (see table, p. 345). 
Communicability.-Communicability is the same as for typhoid. It 
has been isolated in a few instances from animals, and perhaps in water 
and foodstuffs, but its distribution can in no way be compared with 
that of the other members of this group. 
Diagnosis.-See below. 
Paratyphoid B.-This group includes many types, isolated not only 
in disease of man but also in the disease of many animals. 
Disease Produced in Man.-This varies and may be subdivided into 
several clinical types, depending on whether the disease is primarily 
an infection or an intoxication. 
Typhoid-like Type.-A variable period of incubation is usually present. 
The onset is commonly more acute than in typhoid fever, and is usually 
accompanied by chills. As a rule it runs a shorter and milder course. 
1 Muller strain. COMMUNICABILITY 
375 
In some epidemics, abortive attacks occur with no symptoms except 
fever for a short time. The bacili may be present in the feces, blood, 
urine, and gall-bladder; in the first two, early in the disease and com- 
monly during the whole course. Complications similar to those in 
typhoid may occur. 
Castro-enteric Type.-The onset is acute and the symptoms come on 
promptly after eating the infected food or after twenty-four to forty- 
eight hours. The symptoms are those of an acute enteritis ushered 
in by chills and rise of temperature. Nervous symptoms are common 
and may be severe. The bacillus can be isolated from the blood as 
early as the first day of the disease. In the mild cases cultures are nega- 
tive. They are present in the stools during the acute stage but quickly 
disappear in the acute cases. 
Cholera-like Type.-This differs from the preceding only in the degree 
of the toxic and intestinal symptoms. The bacillus may be present in 
the blood. 
Localized infections occur as complications, occasionally as primary 
infections without any other symptoms of paratyphoid infection 
elsewhere, such as pyelitis (sometimes associated with septicemia), 
cystitis, arthritis, periostitis, phlebitis, cholecystitis, etc. It occasion- 
ally occurs as a complicating infection of other diseases. 
Occurrence in Healthy Persons; Bacillus Carriers.-The bacillus may be 
found in the urine after convalescence for shorter or longer periods of 
time. Its localization in the gall-bladder and its excretion in the feces 
by chronic carriers is fairly frequent. As a rule the acute cases do not 
develop into carriers. Careful bacteriologic examination has revealed 
bacilli of the paratyphoid type in the feces of a considerable per- 
centage of healthy persons. 
Morphology and Biology.-They closely resembe the other members of 
this group. For differential characteristics, see table, p. 345. 
Frequency of the Disease.-In this country the disease is relatively 
infrequent, though statistics are not available. It is frequent in Europe, 
especially in certain districts. 
Communicability.-In contrast to typhoid the individual case and the 
carrier either through contact or by the contamination of water and milk, 
etc., are not the only sources of infection. Another important source 
is the wide distribution of the bacillus in domestic animals. Food 
infection, however contaminated, plays the important role. Infection 
has followed the consumption of the following foods: milk and milk 
products; fish; potato salad; variously prepared string beans. In water 
it is more capable of a saprophytic existence than the typhoid bacillus, 
and has been isolated more often. 
Meats or edible organs from cattle, swine, horse, sheep, geese, as well 
as various products containing these, are the main sources of infection. 
Although contamination of meat may occur in other ways the important 
source in epidemics is meat from infected animals. Such meat may by 
contact infect meat from healthy animals. Pickled, salted, and smoked 
meat may be a source of infection. Cooking cannot be relied upon to 376 
PATHOGENIC MICROORGANISMS 
kill the bacilli because of the slow penetration of the heat. It is occa- 
sionally found in the feces, meat, and organs of some of the domestic 
animals, although apparently healthy. It has also been isolated from 
meat products and market milk. 
B. Enteritidis.-In human pathology this bacillus plays the same 
role as the B. paratyphosus B. Meat is the usual source of infection, 
although other foodstuffs have been the cause. The disease is almost 
always of the acute type (see above). It is found in the feces, urine, 
and blood. Chronic carriers have not been found, although the bacilli 
have been found in gall-stones, and probably do occur. They may be 
present in the stools of healthy persons exposed to infection. Contact 
infection occurs, but infrequently. The pathological lesions are the 
same as those caused by the B. Paratyphosus B. Infection of domestic 
animals occurs and the dissemination of the bacillus is the same as for 
paratyphoid B. It has not been found in water, probably not in milk, 
and infrequently in meat products. In cultures it is identical with 
B. paratyphosus B. For methods of diagnosis see below. 
Virulence.-Although varying in degree, the various members of the 
group are highly virulent for white mice, guinea-pigs, rabbits, and rats. 
Death usually results from a septicemia. Toxic substances are freely 
produced in fluid media, which are heat resistant. The paratyphoid A. 
is probably the least virulent of the group. 
Immunity.-In experimental animals immunity is produced by feed- 
ing and by injection. Immunity is produced not only against the homo- 
logous strain but, as a rule, also against related strains. It has been 
suggested that because of the cross-protection of the members of these 
groups and the production of group antibodies active against typhoid 
or paratyphoid when one or the other is injected that a polyvalent 
vaccine of the typhoid-paratyphoid group be used. 
Differential Diagnosis of Members of the Groups.-The cultural 
differentiation only separates the A type from the B and enteritidis 
type. The members of the latter two groups, whether from man 
or animals, show no constant differences. 
Immunity reactions, such as agglutination, fails to separate the 
members of the different groups, although the different groups are 
easily separated. As with other allied bacteria, however, group agglu- 
tinins are present. Other immune reactions give the same results. 
Variation in Types.-The related enteritidis-paratyphoid strains show 
a wide ability for variation, not only culturally but in their patho- 
genicity and virulence. The above-described differences hold good 
for strains isolated from natural infections. There is every reason to 
believe that a strain from man can adapt itself and develop a full degree 
of virulence for an animal host, and vice versa. 
Even its original agglutination reactions may vary in degree, so that 
there is an apparent shifting from the paratyphoid to the enteritidis 
type; the antigenic properties do not change, however, in a parallel 
manner. Because of adaptability of the members of the group the pres- 
ence.of any organism belonging to this group should be looked upon as 
a potential source of infection for man. ENTERITIDIS TYPES 
377 
Diagnostic Methods."-The diagnostic methods are the Widal reac- 
tion, blood-culture, isolation from the stool or urine or other sites of 
infection. The use of the Widal reaction for a differential diagnosis, 
from typhoid or between infection with different members of the group, 
may occasionally lead to errors because of the presence of group agglu- 
tinins. For surety of diagnosis the other methods should be employed. 
Their application is the same as in typhoid fever. For final diagnosis 
immune sera against the various types is necessary. The range of 
group agglutination of the sera used must be known before they are 
used for identification. 
Paratyphoid-like Bacilli.-B. Paratyphosus C.-Bacilli having all char- 
acteristics of the paratyphoid group, but not agglutinated by paratyphoid 
or enteritidis serum, have been isolated from swine suffering from hog 
cholera, feces of man, meat, etc. Other similar types have been isolated, 
showing cultural variation from the paratyphoid group, as indol pro- 
duction, absence of gas in glucose media, etc. Bacilli of these types 
have been found in infections in man. 
Types of the Paratyphoid-Enteritidis Group found in Animal Dis- 
eases.-B. Paratyphosus B. Types.--B. suipestifer, hog-cholera bacillus, 
commonly present as a secondary invader. The disease is caused by a 
filtrable virus. 
B. typhi murium, mouse typhoid bacillus, used for the destruction 
of mice, successfully when the technical details for the maintenance 
of virulence are carefully carried out. Instances of infection in man 
have been reported, and in Prussia its use is restricted by law. 
B. psittacosis, the cause of enteritis in parrots, infectious for man, 
the disease being spoken of as psittacosis. 
Pseudotuberculosis of guinea-pigs. A paratyphoid bacilli is found in 
the pseudotubercular lesions in the spleen and liver and lymph nodes. 
Sporadic cases or epidemics are common. Recently the condition 
developed in a series of guinea-pigs which we had injected with milk 
to test for the presence of tubercle bacilli. 
Diarrhea of calves, an acute contagious disease of the first few' days 
of life, characterized by diarrhea and the development of a septicemia, 
or by the development of a septic pneumonia or of septicemia alone. 
Colon-like bacilli as well as paratyphoid and enteritidis types have been 
isolated. 
Enteritidis Types.- Rat Viruses- Danyz isolated an organism from 
an epidemic of mice which had a high degree of virulence for rats and 
mice. It has been widely used for the extermination of rodents. Similar 
strains have been isolated by others. They are obtainable in the market 
as Ratin, Danyz-Virus, Liverpool Virus, etc. Recently infection in 
man has been reported. Domestic animals, meat and meat products, 
should be protected from contact with the virus. 
B. Paratyphosus C.-Infectious Abortion in Mares.-Various inves- 
tigators have isolated paratyphoid types from infected mares. The 
recent studies of Meyer and Boerner revealed an organism belonging 
to the above group. It is possible that other members of the group 
may cause this disease. CHAPTER XXVI. 
DYSENTERY GROUP. 
Dysentery may be divided into acute and chronic. Amebre appear 
to be the chief exciting factor in most cases of chronic dysentery, though 
bacilli of the colon group also play a part. 
In temperate climates acute dysentery is but very rarely due to 
amebae, but usually to the bacilli identified by Shiga or to allied strains 
identified by Kruse, Flexner, and Park. The usual summer diarrheas 
are not excited by the dysentery bacilli. 
Historical Note.-In 1897 Shiga found in the stools of cases of 
dysentery a bacillus which had not been before identified. In 1900 
Flexner and Strong isolated bacilli which they at that time considered 
the same as those isolated by Shiga. In the same year Kruse in Germany 
isolated bacilli from cases of asylum dysentery which differed, however, 
in their agglutinative properties. Park and Dunham, 1902, isolated a 
bacillus from a severe case of dysentery during an epidemic at Seal 
Harbor, Mt. Desert, Maine, which they showed differed from the 
Shiga dysentery bacillus in that it produced indol and differed in agglu- 
tinating characteristics. At first they considered it the same as the 
Flexner strain, but it was shown later by Park to be a distinct variety, 
and later found by him in widely separated epidemics. 
Martini and Lentz in December, 1902, found that the Shiga type 
was present in separate epidemics in Europe, but also that other types 
similar to those isolated by Flexner, Park, Kruse, and others were found. 
These types differed from the Shiga type in fermenting mannite and 
in agglutination. In January, 1903, Hiss and Russell showed that a 
strain isolated by them differed from the Shiga type in same 
characteristics. 
German observers at first were inclined to consider the Shiga type 
as the only one producing dysentery, while the American observers con- 
sidered both types of equal importance. Park investigated several 
epidemics and isolated not only the Shiga type but two mannite fer- 
menting types, thus definitely proving the importance of the mannite 
fermenting types. The results obtained by others were the same, so 
that no doubt exists that all of the types produce true dysentery. 
Morphological and Cultural Characteristics of Dysentery Bacilli.- 
Microscopic.-Similar to bacilli of the colon group. 
Staining.-Similar to bacilli of the colon group. 
Motility.-No definite motility has been observed. The molecular 
movement is very active. Flagella are absent. 
Appearance of Cultures.-On gelatin the colonies appear more like 
the typhoid than the colon bacilli. Gelatin is not liquefied. On agar PATHOGENESIS 
379 
growth is somewhat more delicate than that of the average colon 
cultures. 
On Potato.-A delicate growth just visible or distinctly brownish. 
In Bouillon.-Diffuse cloudiness with slight deposit and sometimes 
a pellicle. 
See table (p. 345) for comparison with other members of colon- 
typhoid group. 
The fermentation reactions vary, and on this bases the dysentery 
group is divided into subgroups and types. 
Glu- 
cose. 
Man- 
nite. 
Mal- 
tose. 
Saccha- 
rose. 
Indol. 
B. dysenterise . . 
B. paradysenterise: 
. . + 
- 
- 
- 
- 
Type 1 (Mt. Desert, Y) 
. . + 
+ 
- 
- 
+ 
Type 2 (Flexner) 
. . + 
+ 
+ 
- 
+ ' 
Type 3 (Strong) 
. . + 
+ 
- 
+ 
+ 
In differential tests 2 per cent, mannite and 2.5 per cent, maltose give 
surer and more prompt reactions than the usual 1 per cent, 
Fig. 140 
Fig. 141 
Dysentery bacilli. X 1000 diameters. 
Colony of dysentry bacilli in gelatin. 
X 40 diameters. 
Pathogensis.-Animal Tests.-No characteristic lesions with one ex- 
ception have followed the feeding of large quantities of bacilli. Dogs 
at times have had diarrhea with slimy stools, but section showed merely 
a hyperemia of the small intestine. The disease can be produced and 
occurs spontaneously in monkeys. 
Many animals are very sensitive to bacilli injected into vein or 
peritoneum; 0.1 mg. of agar culture injected intravenously produced 
diarrhea, paralysis, and death; 0.2 mg. under the skin have killed, 
and the same amount in the peritoneum has caused bloody peritonitis, 
with lowered temperature and diarrhea. Both small and large animals 
are very sensitive to killed cultures. 
The autopsy of animals dying quickly from injection into the peri- 380 
PATHOGENIC MICROORGANISMS 
toneum of living or dead bacilli shows the peritoneum to be hyper- 
emic, the cavity more or less filled with serous or bloody serous exudate. 
The liver is frequently covered with fibrinous masses. 'The spleen is 
moderately or not at all swollen. The small intestine is filled with fluid, 
the large intestine is usually empty. The mucous membrane of both 
is hyperemic and sometimes contains hemorrhages. Conradi found 
ulcer formation in one case. 
Subcutaneous injections of dead or living cultures are followed by 
infiltration of tissues and frequently by abscess formation. The dysen- 
tery bacilli are not found in the blood or organs of animals. 
Toxins.--A highly poisonous endotoxin is found in autolysates and in 
filtered broth cultures. When injected into animals death results and on 
autopsy the same lesions are found as following the injection of live or 
dead bacilli. This endotoxin can be neutralized by large doses of im- 
mune serum. Kraus and others claim to have demonstrated the presence 
of an extracellular toxin against which an antitoxin can be produced. 
The Shiga type is the more toxic, the others less so or irregularly so. 
In Man. - The etiological significance of the dysentery bacillus in 
man is not only shown by its constant presence and by the presence 
of immune bodies in the serum of infected persons, but also by experi- 
mental infection of man. Thus Strong infected two condemned criminals 
with pure cultures. Jehle infected himself. Kruse reported two acci- 
dental laboratory infections, and others have occurred. 
As a general rule infection with the Shiga type is more severe and the 
mortality higher than infection from other types. But the severity 
of individual cases vary widely during an epidemic. 
Prevalence of the Disease.-The disease is distributed over the whole 
world. The Shiga type and Type I of the paradysentery varieties are 
most commonly found. 
Character of Disease in Man.-In the onset, acute dysentery is sudden 
and ushered in by cramps, diarrhea, and tenesmus. The stools, at first 
feculent, then seromucous, become bloody or composed of coffee- 
ground sediment. At the height of the disease there are ten to fifty 
stools in the twenty-four hours. After two to seven days the blood 
usually disappears. In temperate climates the mortality varies from 
5 to 20 per cent. Bacillary dysentery is a disease especially of the mucous 
membrane of the large intestines. The epithelium is chiefly involved. 
In the lightest cases a catarrhal inflammation is alone present, in the 
more severe the lymph follicles are swollen and some necrosis of epithe- 
lium takes place. 
In severe cases in adults the lesions are of a diphtheritic character 
and may be very marked. The entire lumen of the intestines may be 
filled with a fibrinous mass of pseudomembrane. In young children, 
even in fatal cases, the lesions may be more superficial. The following 
macroscopic and microscopic report of the intestinal findings on a fatal 
case of bacillary dysentery in an infant is a typical picture: 
Small Intestines.--Slightly distended. Mesenteric glands large and red. 
Peyer's patches are swollen slightly without ulceration. PROTECTIVE INOCULATION 
381 
Large Intestines.-Outer 'surface vessels congested and prominent, on section, 
covered with a yellowish mucus. Mucous membrane seems to be absent in 
places. Solitary follicles are elevated and enlarged, especially in the region of 
sigmoid flexure. In some instances the centres of the follicles are depressed 
and appear to be necrotic. 
Large Intestines.-Mucous glands are for the most part normal, but over the 
solitary follicles they have broken down somewhat and contain polynuclear 
leukocytes. The interglandular stroma in these places has undergone 
necrosis. The necrotic area extends down into the submucosa in the region 
of the solitary follicles. The capillaries of the solitary follicles are much dilated 
and congested. The submucosa is thickened and slightly edematous. The 
connective-tissue cells appear to have undergone a slight hyaline degeneration. 
The musculature is not affected, neither is the peritoneal coat. 
Small Intestines.-Normal. 
Distribution of the Bacilli.-The bacilli are only found in the intes- 
tines. They do not invade the rest of the body. The feces, therefore, 
are the only excretions containing them. 
Duration of Life Outside of the Body.-The resistance to the adverse 
conditions outside of the body is relatively small. The Shiga type is 
the least resistant. They die in stools in one to two days. In water 
they die out in several days to a week, exceptionally after a longer time. 
In general it resembles the typhoid bacillus in its resistance. 
Communicability.-Contact infection is mainly responsible for its 
spread. Especially dangerous are the mild or subacute cases, or carriers. 
Such infection may be direct or indirectly by contamination of food, 
dishes, linen, and clothes; water may also be infected and be the 
cause of epidemics. The bacillus has been isolated from water in 
such epidemics. 
Bacillus Carriers.-Both healthy and convalescent carriers are found. 
The former may be equal to one-fourth or even one-half of the number 
of cases. The duration of the carrier state is usually short, some become 
chronic carriers. The Widal reaction is present in many of these carriers. 
Convalescents commonly excrete the bacilli for weeks and some become 
chronic carriers. The excretion is irregular, and slight relapses occur, 
when the bacilli are most numerous. The importance of finding and 
isolating the carriers during an epidemic is obvious. 
Susceptibility.-The frequent occurrence of healthy carriers and mild 
cases along with severe and fatal cases shows the varying resistance to 
infection. Disturbances of digestion, alcohol, or other conditions lower- 
ing the general resistance are factors in infection. 
Immunity.-Immune bodies appear in the blood some time after 
infection. In animals immune bodies are also produced by injection. 
Agglutinins, bactericidal substances, precipitins, and opsonins are found. 
Neutralization of the toxic products is also possible with immune sera. 
True antitoxin is also probably produced. 
Active immunity in animals is produced with difficulty against the 
Shiga type because of its high toxicity. Against the other types, 
however, immunity is easily produced. 
Protective Inoculation.-Some encouraging reports have appeared. 
With the Shiga type, however, the reactions are very severe. For this 382 
PATHOGENIC MICROORGANISMS 
reason Shiga has attempted the simultaneous injection of immune 
serum and vaccines. Oral immunization has also been attempted. 
Serum Therapy.-The use of immune sera for treatment have been 
very good. Employed early in the disease in large doses, it not only 
limits the severity and duration of the disease, but also reduces the 
mortality very decidedly. Where various types of bacilli are prevalent 
a polyvalent serum is necessary. 
Diagnosis.-The use of the Widal reaction is limited, because the 
reaction does not appear during the acute stage, but later it may be 
used. Group or normal agglutinins are commonly present and interfere 
greatly with the value of the reaction in the paradysentery types. The 
action on the infecting subgroup is usually sharp, but infection by the 
types of the paradysentery group can frequently not be differentiated. 
A Widal reaction is commonly present in carriers. For diagnosis the 
serum should agglutinate in 1:50 in infections with B. dysenteries and 
in 1:100 in other types. 
Isolation of the bacillus is the only method of diagnosis applicable 
during the acute stage. The mucus flakes in the stool should be selected 
for plating. The methods of isolation and identification are the same 
as for typhoid or paratyphoid, except that the crystal violet should be 
omitted from the Conradi medium. The bacilli are so abundant in the 
mucus of most cases that they can be readily isolated from nutrient 
agar plates. If Endo is used the plates should be fresh. When the color 
returns the growth of the Shiga types is commonly inhibited. 
Differential Diagnosis of Type.-The cultural differences have been 
given. The actual nature of the bacillus should be verified by agglu- 
tination, although the cultural characters will suffice for a tentative 
diagnosis from a case clinically typical. 
Agglutination.-Sera from rabbits or goats are used, as horses develop 
normal and group agglutinin^ to a high degree. 
For practical purposes two sera, one for B. dysenteries and a poly- 
valent serum for the paradysentery types may be used, further differ- 
entiation being determined culturally. In the use of sera for differen- 
tiation of the latter groups univalent sera for each of the types must 
be on hand and all the precautions against error from group agglutinins 
should be observed. Variations in agglutinability of freshly isolated 
strains adds to the difficulty. 
Variability.-Cultures, after prolonged artificial cultivation, may show 
variations in their sugar fermentations. By passage through animals 
(monkey, etc.) the original type is returned. CHAPTER XXVII. 
BACILLUS PYOCYANEUS (BACILLUS OF GREEN AND 
OF BLUE PUS). BACILLUS PROTEUS (VULGARIS). 
BACILLUS PYOCYANEUS. 
The blue and green coloration which is occasionally found to ac- 
company the purulent discharges from open wounds is usually due 
to the action of the Bacillus pyocyaneus. According to recent in- 
vestigations, this bacillus appears to be very widely distributed and 
not infrequently the cause of infection. It was first obtained in pure 
culture and its significance noted by Gessard. 
Morphology.-Slender rods from 0.3/z to 1/z broad and from 2/x 
to 6m long; frequently united in pairs or in chains of four to six ele- 
ments; occasionally growing out into long filaments and twisted 
spirals. The bacillus is actively motile, a single flagellum being at- 
tached to one end. Does not form 
spores. Stains with the ordinary 
aniline colors; does not stain with 
Gram's solution. 
Biology. - Aerobic, liquefying, 
motile bacillus. Capable also of 
an anaerobic existence, but then 
produces no pigment. Grows 
readily on all artificial culture 
media at the room temperature, 
though best at 37° C., and gives 
to some of them a bright green 
color in the presence of oxygen. 
In gelatin-plate cultures the col- 
onies are rapidly developed, im- 
parting to the medium a fluores- 
cent green color; liquefaction begins 
at the end of two or three days, 
and by the fifth day the gelatin 
is usually all liquefied. The deep colonies, before liquefaction sets in, 
appear as round, granular masses with scalloped margins, having a 
yellowish-green color; the surface colonies have a darker green centre, 
surrounded by a delicate, radiating zone. In stick cultures in gelatin 
liquefaction occurs at first near the surface, in the form of a small funnel 
and gradually extends downward; later the liquefied gelatin is separated 
from the solid part of the medium by a horizontal plane, a greenish- 
Fig. 142 
Bacillus pyocyaneus. (From Kolle and 
Wassermann.) 384 
PATHOGENIC MICROORGANISMS 
yellow color being imparted to that portion which is in contact with 
the air. On agar a wrinkled, moist, greenish-white layer is developed, 
while the surrounding medium is bright green; this subsequently becomes 
darker in color, changing to blue-green or almost black. In bouillon 
the green color is produced, and the growth appears as a delicate, 
flocculent sediment. Milk is coagulated and assumes a yellowish-green 
color. 
Pigment.-Two pigments are produced by this bacillus-one of a 
fluorescent green which is common to many bacteria. This is soluble 
in water but not in chloroform. The other (pyocyanin) of a blue color 
is soluble in chloroform, and may be obtained from pure solution in 
long, blue needles. This pigment distinguishes the Bacillus pyocyaneus 
from other fluorescing bacteria. 
Ferment.-Besides the ferment causing liquefaction of gelatin 
there is one which acts on albumin. It resists heat. This ferment 
called pyocyanase has the power to dissolve bacteria, and it has been 
stated to have some protective power when injected into animals. It 
has been used locally in diphtheria in a number of cases. We do not 
think it has any advantage over the cleansing preparations. 
Distribution.-This bacillus is very widely distributed in nature; 
it is found on the healthy skin of man, in the feces of many animals, 
in water contaminated by animal or human material, in purulent 
discharges, and in serous wound secretions. 
Pathogensis.-Its pathogenic effects on animals have been care- 
fully studied. It is pathogenic for guinea-pigs and rabbits. Sub- 
cutaneous or intraperitoneal injections of 1 c.c. or more of a bouillon 
culture usually cause the death of the animal in from twenty-four 
to thirty-six hours. Subcutaneous inoculations produce an extensive 
inflammatory edema and purulent infiltration of the tissues; a sero- 
fibrinous or purulent peritonitis is induced by the introduction of the 
bacillus into the peritoneal cavity. The bacilli multiply in the body 
and may be found in the serous or purulent fluid in the subcutaneous 
tissues or abdominal cavity, as well as in the blood and various organs. 
When smaller quantities are injected subcutaneously the animal 
usually recovers, only a local inflammatory reaction being set up 
(abscess), and it is subsequently immune against a second inoculation 
with doses which would prove fatal to an unprotected animal. It 
is interesting to note that Bouchard, Charrin, and Guignard have 
shown that in rabbits, which have been inoculated with a culture of 
the bacillus anthracis a fatal result may be prevented by inoculating 
the same animal soon after with a pure culture of the bacillus pyo- 
cyaneus. Loew and Emmerich have shown that the enzymes produced 
in the pyocyaneus cultures are capable of destroying many forms 
of bacteria in the test-tube, and have a slight protecting value in the 
body. The pyocyaneus bacillus produces these effects not only through 
ferments, but by intracellular toxins. 
Our knowledge of the pathogenic importance of the Bacillus pyo- 
cyaneus in human diseases has been much increased by recent inves- BACILLUS PROTEUS 
385 
tigations. Its presence in" wounds greatly delays the process of re- 
pair, and may give rise to a general depression of the vital powers 
from the absorption of its toxic products. This bacillus has been 
obtained in pure culture from pus derived from the tympanic cavity 
in disease of the middle ear, from cases of ophthalmia, and broncho- 
pneumonia. Kruse and Pasquale have found the organism in three 
cases of idiopathic abscess of the liver, in two of them in immense 
numbers and in pure culture. Ernst and Schiirmayer report the 
presence of the bacillus pyocyaneous in serous inflammations of the 
pericardial sac and of the knee-joint. Ehlers gives the history of a 
disease in two sisters who were attacked simultaneously with fever, 
albuminuria, and paralysis. It was thought that they would prove 
to be typhoid fever or meningitis, but on the twelfth day there was 
an eruption of blisters, from the contents of which the bacillus pyo- 
cyaneus was isolated. Krambals refers to seven cases in which a 
general pyocyaneus infection occurred, and adds an eighth from his 
own experience. In this the bacillus pyocyaneus was obtained post- 
mortem from green pus in the pleural cavity, from serum in the 
pericardial sac, and from the spleen in pure culture. Schimmel- 
busch states that a physician injected 0.5 c.c. of sterilized (by heat) 
culture into his forearm. As a result of this injection, after a few 
hours he had a slight chill, followed by fever, which at the end of 
twelve hours reached 38.8° C.; an erysipelatous-like swelling of the 
forearm occurred, and the glands in the axilla were swollen and pain- 
ful. Wassermann reports an epidemic of septic infection of the new- 
born, starting in the umbilicus. In all there were eleven deaths. 
Lartigau found it in well-water, and in great abundance in the intesti- 
nal discharges of a number of cases made ill by drinking the water. 
It has also been found in a certain number of cases of gastro-enteritis 
where no special cause of infection could be noted. 
We may therefore conclude from these facts that the Bacillus pyo- 
cyaneus, although ordinarily but slightly pathogenic for man, may 
under certain conditions, as in general debility, become a dangerous 
source of infection. Children would seem to be particularly susceptible. 
The differential diagnosis of the pyocyaneus from other fluorescing 
bacteria is easy enough as long as it retains its pigment-producing 
property. When an agar culture is agitated with chloroform a blue 
coloration demonstrates the presence of this bacillus. When the 
pyocyanin is no longer formed, however, the diagnosis is by no means 
easy, particularly when the pathogenic properties are also gone. 
Immunity.-Animal infection is followed by the production of anti- 
toxic and bactericidal substances. No practical use has been made of 
this knowledge. 
BACILLUS PROTEUS (VULGARIS). 
This bacillus, which is one of the most common and widely dis- 
tributed putrefactive bacteria, was discovered by Hauser (1885) 
along with other species of proteus in putrefying substances. These 
25 386 
PATHOGENIC MICROORGANISMS 
bacteria were formerly included under the name " Bacterium ter mo" 
by previous observers, who applied this name to any minute motile 
bacilli found in putrefying infusions. 
Morphology.-Bacilli varying greatly in size; most commonly occur- 
ring 0.6/z broad and 1.2/z long, but shorter and longer forms may also 
be seen, even growing out into flexible filaments which are sometimes 
more or less wavy or twisted like braids of hair. 
The bacillus does not form spores, and stains readily with fuchsin 
or gentian violet. 
Biology. - An aerobic, facultative anaerobic, liquefying, motile 
bacillus. Grows rapidly in the usual culture media at the room 
temperature. 
Growth on Gelatin.-The growth upon gelatin plates containing 5 
per cent, of gelatin is very characteristic. At the end of ten or twelve 
hours at room temperature small, round depressions in the gelatin 
are observed, which contain liquefied gelatin and a whitish mass con- 
sisting of bacilli in the centre. Under a low-power lens these depres- 
sions are seen to be surrounded by a radiating zone composed of two 
or more layers, outside of which is a zone of a single layer, from which 
ameba-like processes extend upon the surface of the gelatin. These 
processes are constantly undergoing changes in their form and posi- 
tion. The young colonies deep down in the gelatin are somewhat 
more compact, and rounded or humpbacked; later they are covered 
with soft down; then they form irregular, radiating masses, and simu- 
late the superficial colonies. But it is difficult to describe all the forms 
which the proteus vulgaris takes on in all the stages of its growth on 
gelatin plates. When the consistency of the medium is more solid, 
as in 10 per cent, gelatin the liquefaction and migration of surface 
colonies are more or less retarded. In gelatin-stick cultures the growth 
is less characteristic-liquefaction takes place rapidly along the line of 
puncture, and soon the entire contents of the tube are liquefied. 
Upon Nutrient agar a rapidly spreading, moist, thin, grayish-white 
layer appears, and migration of the colonies also occurs. Milk is 
coagulated, with the production of acid. 
Cultures in media containing albumin or gelatin have a disagree- 
able, putrefactive odor, and become alkaline in reaction. Growth 
is most luxuriant at a temperature of 24° C., but is plentiful also at 
37° C. It is a facultative anaerobe and grows also in the absence of 
oxygen, but the proteus then loses its power of liquefying gelatin. 
It produces indol and phenol from peptone solutions. The proteus 
develops fairly well in urine, and decomposes urea into carbonate of 
ammonia. 
Pathogensis.-This bacillus is pathogenic for rabbits and guinea- 
pigs when injected in large quantities into the circulation, the ab- 
dominal cavity, or subcutaneously, producing death of the animals 
with symptoms of poisoning. Hauser has obtained the Bacillus 
proteus (vulgaris) from a case of purulent peritonitis, from purulent 
puerperal endometritis, and from a phlegmonous inflammation of the BACILLUS PROTEUS 
387 
hand. Brunner also reports similar infections in which this organism 
was found associated with pus cocci, and Charrin describes a case 
of pleuritis during pregnancy, in which the proteus was present and a 
foul-smelling secretion was produced. Death in this case, which ensued 
without further complication, is said to have been due probably to the 
poisonous products of the proteus. 
An interesting example of pure toxemia resulting from the toxin 
of the proteus is reported by Levy: While conducting some experi- 
ments on this organism he had an opportunity of making a bacterio- 
logical examination in the case of a man who died after a short attack 
of cholera morbus. From the vomited material and the stools he 
obtained a pure culture of the proteus; but the blood, collected at 
the autopsy, was sterile. In the meantime seventeen other persons 
who had eaten at the same restaurant were taken sick in the same 
way. Upon examination at the restaurant it was found that the 
bottom of the ice-chest in which the meat was kept was covered with 
a slimy, brown layer, which gave off a disagreeable odor. Cultures 
from this gave the proteus as the principal organism present. In- 
jections into animals of the pure cultures produced similar symp- 
toms as occurred in the human subjects. 
Levy concludes that in so-called "flesh poisoning" bacteria of this 
group are commonly concerned, and the pathogenic effects are due to 
toxic products evolved during their development. Next to the para- 
typhoid-enteriditis types they are probably the most frequent cause of 
meat poisoning. 
Booker, from his extended researches into this subject, concludes 
that the proteus plays an important part in the production of the 
morbid symptoms which characterize cholera infantum. Proteus 
vulgaris was found in the alvine discharge in a large proportion of 
the cases examined by him, but was not found in the feces of healthy 
infants. "The prominent symptoms in the cases of cholera infantum 
in which the proteus bacteria were found were drowsiness, stupor, 
and great reduction in flesh, more or less collapse, frequent vomiting 
and purging, with watery and generally offensive stools." 
Next to the Bacillus coli communis the Proteus vulgaris appears 
to be the microorganism most frequently concerned in the etiology 
of pyelonephritis. In cases of cystitis and of pyelonephritis this 
bacillus is often found in pure cultures or associated with other bac- 
teria. It probably gets into the bladder chiefly through catheteri- 
zation. From the animal experiments of the authors above men- 
tioned, simple injection of pure cultures of proteus into the bladder, 
without artificial suppression of urine, invariably produces severe 
cystitis. The fact that this organism grows in urine is sufficient to 
account for the extension of the purulent process finally to the kidneys. 
The Proteus vulgaris is usually a harmless parasite when located 
in the mucous membrane of the nasal cavities. Here it only decom- 
poses the secretions, with the production of a putrefactive odor. It is 
found occasionally in the discharge from cases of otitis media in con- 
nection with other bacteria. CHAPTER XXVIII. 
THE BACILLUS AND THE BACTERIOLOGY OF TUBER- 
CULOSIS. 
A knowledge of phthisis was certainly present among men at the 
time from which our earliest medical descriptions come. For over 
two thousand years many of the clearest-thinking physicians have 
considered it a communicable disease; but it is only within compara- 
tively recent times that the infectiousness of tuberculosis has become 
an established fact in scientific medicine. Villemin, in 1865, by 
infecting a series of animals through inoculations with tuberculous 
tissue, showed that tuberculosis might be induced, and that such 
tissue carried the exciting agent of the disease. He also noticed the 
difference in virulence between tuberculous material of human and 
bovine sources, and says that not one of the rabbits inoculated with 
human material showed such a rapidly progressive and widespread 
generalization as those receiving material from the cow. Baumgarten 
demonstrated early in 1882 bacilli in tissue sections which are now 
known to have been tubercle bacilli. But these investigations and 
those of others at the same time, though paving the way to a better 
knowledge of the disease, proved to be unsatisfactory and incom- 
plete. The announcement of the discovery of the tubercle bacillus 
was made by Koch in March, 1882. Along with the announcement 
satisfactory experimental evidence was presented as to its etiological 
relation to tuberculosis in man and in susceptible animals, and its 
principal biological characters were given. He submitted his full re- 
port in 1884. Innumerable investigators now followed Koch into this 
field, but their observations served only to confirm his discovery. 
Distribution of Bacilli.-They are found in the sputum of per- 
sons and animals suffering from pulmonary or laryngeal tubercu- 
losis, either free or in the interior of pus cells; in miliary tubercles 
and fresh caseous masses in the lungs and elsewhere; in recent tuber- 
culous cavities in the lungs; in tuberculous glands, joints, bones, 
serous effusions, mucous membranes, and skin affections. They are 
also found in the feces of those suffering from tuberculous disease of 
the intestines or of those swallowing tuberculous sputum. They are 
frequently present in the blood in very small numbers, in large number 
only in acute miliary tuberculosis. 
Morphology.-The tubercle bacilli are slender, non-motile rods of 
about 0.3^ in diameter by 1.5m to 4m in length. (Plate VI, Figs. 1 and 2.) 
The morphology is extremely variable, especially on culture media, and 
varies with the type of medium used. Commonly they occur singly or PLATE VI 
FIG. 1 
FIG. 2 
Tubercle bacilli in red. 
Streptobacilli in blue. 
Tubercle bacilli in red. 
Tissue in blue. 
X 1OOO diameters. 
X UOO diameters. 
FIG. 3 
FIG. 4 
Leprosy bacilli in nasal secre- 
tion of person suffering from 
nasal lesions. (Hansen.) 
Short smegma bacilli in red, 
rest of material in blue. 
X 500 diameters. 
X 11OO diameters. RESISTANCE 
389 
in pairs, and are then usually slightly curved; frequently they are ob- 
served in smaller or larger bunches. Under exceptional conditions 
branching and club-shaped forms are observed. The tubercle bacillus, 
therefore, is closely allied to nocardia. In stained preparations there 
are often seen unstained portions. In old cultures irregular forms may 
develop, the rods being occasionally swollen at one end or presenting 
lateral projections. Here also spherical granules appear which stain 
with more difficulty than the rest of the bacillus and also retain the 
stain with greater tenacity. The bacilli, however, containing these 
bodies are not appreciably more resistant than those not having them; 
therefore they cannot be considered true spores. The bacilli have a thin 
capsule, shown in one way by the fact that they appear thicker when 
stained with fuchsin than with methylene blue. 
Chemical Constituents of the Tubercle Bacilli.-Water 86 per cent., 
dry substance 14 per cent., 25 per cent, of which is soluble in alcohol 
and ether, consisting of free fatty acids and fatty acids combined with 
the higher alcohol "mykol" to form a wax; lecithin proteins, largely 
nucleo-albumins, nuclein, protamin, nucleic acid and inorganic bases 
constitute the remainder. 
Staining Peculiarities.-These are very important, for by them its 
recognition in microscopic preparations of sputum, etc., is rendered 
possible. Owing to content of waxy substance it does not readily take 
up the ordinary aniline colors, but when once stained it is very difficult 
to decolorize, even by the use of strong acids. The more recently 
formed bacilli are much more easily stained and decolorized than the 
older forms. 
Methods of Staining.-Carbol-fuchsin.-Steam three minutes, decolorize 
with 3 per cent, hydrochloric acid in alcohol, counter-stain with methylene 
blue. Herman Stain.-A, crystal violet, 3 per cent, solution in alcohol; B, 
ammonium carbonate, 1 per cent, solution in water; mix one part of A with 
three parts of B just before using; steam as above, decolorize with 10 per cent, 
nitric acid, wash in alcohol, and counter-stain in Bismarck brown. 
Biology.-The bacillus of tuberculosis is a parasitic, aerobic, non- 
motile bacillus, and grows only at a temperature of about 37° C., 
limits 30° to 42° C. It does not form true spores. 
Resistance.-The bacilli, because of the protection given by their 
waxy substances, it has been assumed, have a somewhat greater resisting 
power than most other pathogenic bacteria. Frequently a few out of 
a great number of bacilli resist desiccation at ordinary temperatures for 
months; most bacilli die, however, soon after drying. There is a greater 
resistance shown by the tubercle bacillus than by most other non- 
sporebearing bacilli to the action of the products of bacterial growth 
as in souring milk, in water, and in sewage. In water and sewage they 
may remain viable for weeks. They frequently retain their vitality 
for several weeks, or even months, in putrefying material, such as 
sputum. Upon cultures the bacilli do not live longer than three months, 
unless the media be favorable, such as egg or serum; transplants after 
this time may fail to grow. A few bacilli, sufficient to infect guinea-pigs, 390 
PATHOGENIC MICROORGANISMS 
may persist much longer. Cold has little effect upon them. When dry, 
some of the organisms stand dry heat at 100° C. for twenty minutes 
but are dead in forty-five minutes; but when in fluids and separated 
as in milk, they are quickly killed-viz., at 60° C. in twenty minutes, 
at 65° C. in fifteen minutes, at 70° C. the great majority in one minute, 
all in five minutes, at 80° C. the great majority in one-half minute, all 
in one minute, and at 95° C. in one-half minute. In some experiments 
they appear to withstand a higher temperature. As pointed out by 
Theobald Smith, when milk is heated in a test-tube in the usual way 
the cream which rises on heating is exposed on its surface to a lower 
temperature than the rest of the milk, and as this contains the greatest 
percentage of the bacteria some of them are exposed to less heat than 
those in the rest of the fluid receive. Rosenau points out another source 
of error: If a moderate number of 
killed bacilli are injected, limited 
lesions will arise and caseation may 
follow. On killing and autopsying 
the animals, tubercle bacilli can 
then be demonstrated in smears 
from the lesions, and the inocula- 
tion is considered positive. If, 
however, this material is reinjected 
into a second pig, the latter will 
show nothing on autopsy. This 
capacity of dead bacilli to cause 
macroscopic lesions has long been 
shown by Prudden and Hodenpyl. 
Its importance, however, is not 
sufficiently considered. 
The resisting power of this bacil- 
lus to chemical disinfectants, dry- 
ing, and light is considerable, but not as great as it is apt to appear, 
for, as in sputum, the bacillus is usually protected by mucus or cell 
protoplasm from penetration by the germicidal agent. It is not al- 
ways destroyed by the gastric juice in the stomach, as is shown by 
successful infection experiments in susceptible animals by feeding 
them with tubercle bacilli. They are destroyed in sputum in six 
hours or less by the addition of an equal quantity of a 5 per cent, so- 
lution of carbolic acid. Bichloride of mercury is less suitable for 
the disinfection of sputum as it combines with the mucus and forms 
a more or less protecting envelope. Iodoform has no effect upon cul- 
tures until 5 per cent, is added. The fumes from four pounds of 
burning sulphur to each 1000 cubic feet of air space will kill tubercle 
bacilli in eight hours when fully exposed to the action of the gas, 
providing they are moist, or abundant moisture is present in the air. 
Formaldehyde gas is quicker in its action, but not much more effi- 
cient. Ten ounces of formalin should be employed for each 1000 cubic 
feet of air space. The tubercle bacillus resists the action of alkaline 
Fig. 143 
Tubercle bacilli. Impression prepara- 
tion from small colony on coagulated blood- 
serum. X 1000. CULTIVATION OF THE TUBERCLE BACILLUS 
391 
hypochlorite solution ("antiformin") in dilutions which quickly dis- 
solve non-acid fast bacteria. 
The tubercle bacillus in sputum when exposed to direct sunlight is 
killed in from a few minutes to several hours, according to the thick- 
ness of the layer and the season of the year; it is also usually destroyed 
by diffuse daylight in from five to ten days when placed near a window 
in fine powder. Protected in cloth the bacilli survive exposure to 
light for longer periods. Tuberculous sputum expectorated upon 
sidewalks, etc., when left undisturbed in the shade may be infectious 
for weeks, but when exposed to the action of direct sunlight, will in 
many cases, especially in summer, be disinfected by the time it is in 
condition to be carried into the air as dust, but not before children 
and flies get into it. This action of sunlight and other more impor- 
tant hygienic reasons, suggest that the consumptive patients should 
occupy light, sunny rooms. 
Fig. 144 
Fig. 145 
Tubercle bacilli, bovine. X 1000 
diameters. 
Tubercle bacilli, human. X 1000 
diameters. 
Dried sputum in rooms protected from abundant light has occa- 
sionally been found to contain virulent tubercle bacilli for as long as 
ten months. For a year at least it should be considered dangerous. 
The Rontgen rays have a deleterious effect on tubercle bacilli in cul- 
tures, but practically none upon those in tissues. 
Multiplication of Tubercle Bacilli in Nature Takes Place Only in 
the Living Animal.-The tubercle bacillus is a strict parasite-that is 
to say, its biological characters are such that it could scarcely find 
natural conditions outside of the bodies of living animals favorable for 
its multiplication. Under exceptional conditions, such as in freshly 
expectorated sputum, tubercle bacilli may increase for a limited time. 
Cultivation of the Tubercle Bacillus.-On account of their slow 
growth and the special conditions which they require, tubercle ba- 
cilli cannot be grown in pure culture by the usual plate method on 
ordinary culture media. Koch first succeeded in cultivating and 
isolating this bacillus on coagulated beef serum, which he inoculated 
by carefully rubbing the surface with sections of tuberculous tissue ami 392 
PATHOGENIC MICROORGANISMS 
then leaving the culture, protected from evaporation, for several weeks 
in the incubator. Cultures are more readily obtained of human or 
avian than of bovine bacilli. 
Growth on Coagulated Dog or Bovine Serum or on Egg.-On these, one of 
which is generally used to obtain the first culture, the growth is usually visible 
at the end of ten days at 37° C., and at the end of three or four weeks a dis- 
tinct and characteristic development has occurred. On serum small, grayish- 
white points and scales first appear on the surface of the medium. As develop- 
ment progresses there is formed an irregular, membranous-looking layer. On 
egg the growth is in the form of more or less elevated colonies which may become 
confluent. 
Growth on Nutrient 3-5 Per Cent. Glycerin Agar.-Owing to the greater 
facility of preparing and sterilizing glycerin agar, it is now usually employed 
in preference to blood-serum for continuing to produce later cultures. When 
numerous bacilli have been distributed over the surface of the culture medium, 
a rather uniform, thick, white layer, which subsequently acquires a slight 
yellowish tint, is developed; when the bacilli sown are few in number, or are 
associated in scattered groups, separate colonies are developed, which acquire 
considerable thickness and have more or less irregular outlines. The growth 
appears similar to that shown upon bouillon as seen in Fig. 146. 
Fig. 146 
Growth of tubercle bacilli upon glycerin bouillon. (Kolle and Wassermann.) 
Growth on Nutrient Veal or Beef Broth Containing 5 per cent, of Glycerin. 
-This is of importance, because in this way tuberculin is produced. 
On these media the tubercle bacillus also grows readily if a very 
fresh thin film of growth from the glycerin agar is floated on the 
surface. Glycerin broth is used for the development of tuberculin 
and must be neutral to litmus, viz., between 1.5 per cent, to 2 per 
cent, acid to phenolphthalein. The small piece of pellicle removed 
from the previous culture continues to enlarge while it floats on the 
surface of the liquid, and in the course of three to six weeks covers it 
wholly as a single film, which on agitation is easily broken up and then 
settles to the bottom of the flask, where it ceases to develop further. 
The liquid remains clear. A practical point of importance, if a quick 
growth is desired, is to remove for the new cultures a portion of the 
pellicle of a growing bouillon culture, which is very thin and actively 
increasing. PURE CULTURES OF TUBERCLE BACILLUS FROM SPUTUM 
393 
Growth on Potato. - A good growth from cultures and sometimes even 
from tissue takes place on potato, and this forms the most uniform medium 
for stock cultures. 
Obtaining of Pure Cultures of the Tubercle Bacillus from Sputum, 
Infected Tissue, and other Materials.-On account of the time re- 
quired and the difficulties to be overcome, this is never desirable ex- 
cept when careful investigations of importance are to be undertaken. 
Pure cultures can be obtained directly from tuberculous material if 
the tubercle bacilli are present in sufficient number and mixed infection 
is not present using the proper blood-serum or egg culture medium; 
but it is difficult to get material free from other bacteria which grow 
much more rapidly and take possession of the medium before the 
tubercle bacillus has had time to form visible colonies. It is usually 
necessary first to inoculate guinea-pigs, subcutaneously or intramus- 
cularly, preferably in the thigh, and then obtain cultures from the 
animals as soon as the tuberculous infection has developed. In this 
way, due to the susceptibility of the guinea-pig to tuberculosis, cultures 
may be obtained from material containing very few tubercle bacilli, 
although contaminating bacteria may be very numerous. 
Animals inoculated usually die at the end of three weeks to four 
months. It is better, however, not to wait until the death of the ani- 
mals, but at the end of four to six weeks to kill a guinea-pig without vio- 
lence, using illuminating gas, chloroform, or ether in a closed tin or jar. 
(Animals which develop tuberculosis acutely are apt to have abundant 
tubercle bacilli and give successful cultures, while the chronic cases 
usually have few bacilli and may give unsuccessful cultures.) The 
animal after being killed is tied out in trays, and after washing with 
a 5 per cent, solution of carbolic acid, immediately autopsied. The 
skin over the anterior portion of the body having been carefully turned 
back, the inguinal nodes are removed with fresh instruments. The 
nodes on the side of injection are especially favorable for cultures. 
The abdomen is then opened and the spleen and retroperitoneal nodes 
removed. As the organs are removed they should be placed in Petri 
dishes and thoroughly minced with knife and forceps. Fresh instru- 
ments should be used for each operation. The sternal nodes may be 
used for cultures, but the lungs are almost useless as the majority of 
cultures will be contaminated. The minced tissue is then placed on 
the surface of the culture media, both egg and glycerin egg being used, 
and evenly and thoroughly smeared over its surface, then the cotton 
plug is dipped in hot paraffin and the tube corked with a tightly fitting 
charred cork, to keep the media from drying. The tubes are incubated 
in the inclined position. On egg, growth is visible in from seven to ten 
days, and well marked at the end of three weeks. Many tubes should 
be inoculated as it is only with the dexterity acquired by practice that 
contaminations are avoided. As will be noted further on, the growth 
of the bovine type will be very sparse and on glycerin egg probably 
negative. 394 
PATHOGENIC MICROORGANISMS 
Pathogenesis.--The tubercle bacillus is pathogenic not only for man, 
but for a large number of animals, such as the cow, monkey, pig, 
cat, etc. Young guinea-pigs are very susceptible, and are used for 
the detection of tubercle bacilli in suspected material. When inocu- 
lated with the minutest dose of the living bacilli they usually succumb 
to the disease. Infection is most rapidly produced by intraperitoneal 
injection. If a large dose is given, death follows in from ten to twenty 
days. The omentum is found to be clumped together in sausage-like 
masses and converted into hard knots, which contain many bacilli. 
There is no serous fluid in the peritoneal cavity, but generally in both 
pleural sacs. The spleen is enlarged, and it, as well as the liver and 
peritoneum, contains large numbers of tubercle bacilli. If smaller 
doses are given the disease is prolonged. The peritoneum and inte- 
rior organs-spleen, liver, etc., and often the lungs-are then filled 
with tubercles. On subcutaneous injection, for instance, into the 
thigh, there is a thickening of the tissues about the point of inocu- 
lation, which may break down in one to three weeks and leave a 
sluggish ulcer covered with cheesy material. The neighboring lymph 
nodes are swollen, and at the end of two or three weeks may attain 
the size of hazel-nuts. Soon an irregular fever is set up, and the animal 
becomes emaciated, usually dying within four to eight weeks. If 
the injected material contained only a small number of bacilli the 
wound at the point of inoculation may heal up and death be postponed 
for a long time. On autopsy the lymphatic nodes are found to have 
undergone cheesy degeneration; the spleen may be very much enlarged, 
and throughout its substance, which is colored dark red, are distributed 
masses of nodules. The liver is also commonly increased in size, 
streaked brown and yellow, and the lungs are filled with grayish-white 
tubercles; but, as a rule, the kidneys contain no nodules. Occasionally 
the lesions are limited to the inguinal and retroperitoneal nodes. 
Tubercle bacilli are found in the affected tissues, but the more chronic 
the process the fewer the bacilli present. 
Injection into the thigh is to be preferred for diagnostic purposes, 
the swelling of the local lymph nodes being then palpable. As soon 
as this is appreciable the node may be removed with or without killing 
the pig, the presence or absence of tuberculous lesions noted, and smears 
made for the detection of tubercle bacilli, thus saving time. It must 
be remembered that the pig may not show the usual picture of gener- 
alized tuberculosis, but only a swelling of the local lymph nodes. 
Fortunately tubercle bacilli are usually easily demonstrable in smears 
made from the crushed nodes. If there is any doubt the remaining 
tissue should be emulsified and reinjected into a second set of pigs. 
Another point to be considered is that other organisms may, rarely, 
give a picture difficult to distinguish macroscopically from tuberculosis, 
as, for instance, streptothrix. To safeguard against error smears 
should be stained and tubercle bacilli demonstrated. Chronic guinea- 
pig septicemia may be accompanied by lesions in the spleen which 
might be taken for tuberculous lesions. POINT OF ENTRANCE OF INFECTION 
395 
Rabbits are very susceptible to tuberculosis of the bovine type, 
less so to that of the human type. This will be given more in detail 
under the differences between human and bovine tuberculosis. 
Monkeys are very susceptible to infection with both types of bacilli. 
Cats, dogs, rats, and mice are susceptible, the last two usually show 
no tuberculous lesions, but there is great multiplication of the bacilli 
in the tissues. 
Tubercle Toxins.-The tubercle bacillus produces no true toxins. 
The bodies of the bacteria contains substances which cause necrosis 
of tissue with subsequent, caseation or abscess. In broth cultures, 
after filtration, are present substances which produce fever and inflam- 
matory reactions of tissues. These substances as well as extracts 
from tubercle bacilli are highly toxic for tuberculous animals, little 
if at all for normal animals, and cause fever only in the former and the 
tissue inflammation spoken of is marked about tuberculous lesions. 
These poisons will be considered in detail later in connection with 
tuberculins. 
Action upon the Tissues of the Poisons Produced by the Tubercle Bacillus. 
-Soon after the introduction into the tissues of tubercle bacilli, 
either living or dead, the cells surrounding them begin to show that 
some irritant is acting upon them. The connective-tissue cells be- 
come swollen and undergo mitotic division, the resultant cells being 
distinguished by their large size and pale nuclei. A small focus of 
proliferated epithelioid cells is thus formed about the bacilli, and 
according to the intensity of the inflammation these cells are sur- 
rounded by a larger or smaller number of the lymphoid cells. When 
living bacilli are present and multiplying the lesions progress, the 
central cells degenerate and die, and a cheesy mass results, which 
later may lead to the formation of cavities. Dead bacilli, on the other 
hand, unless bunched together, give off sufficient poison to cause the 
less marked changes only (Prudden and Hodenpyl). Of the gross 
pathological lesions produced in man by the tubercle bacilli the most 
characteristic are small nodules, called miliary tubercles. When 
young, and before they have undergone degeneration, these tubercles 
are gray and translucent in color, somewhat smaller than a millet 
seed in size, and hard in consistence. But miliary tubercles are not the 
sole tuberculous products. The tubercle bacilli may cause diffuse 
growth of tissue identical in structure with that of miliary tubercles, 
that is, composed of a basement substance, containing epithelioid, 
giant, and lymphoid cells. This diffuse tuberculous tissue also tends to 
undergo cheesy degeneration. 
Point of Entrance of Infection.-Infection by the tubercle bacillus 
takes place usually through the respiratory tract or the digestive tract, 
including the pharynx and tonsils, more rarely through wounds of the 
skin. 
Tuberculosis may be considered to be caused chiefly by the direct 
transmission of tubercle bacilli to the mouth through soiled hands, 
lips, handkerchiefs, milk, etc., or by the inhalation of fine particles of 396 
PATHOGENIC MICROORGANISMS 
mucus thrown off by coughing or loud speaking, or of tuberculous dust 
contaminated by sputum or feces. 
Tuberculosis of Skin and Mucous Membranes.-When the skin or 
mucous membranes are superficially infected through wounds there 
may develop lupus, ulceration, or a nodular growth. The latter two 
forms of infection are apt after an interval to cause the involvement 
of the nearest lymphatic nodes. 
Tuberculosis of Respiratory Tract.-The lungs are the most frequent 
location of clinically recognizable tuberculous inflammation. On 
account of their location they are greatly protected from external 
infection. Most of the bacilli are caught upon the nasal or pharyngeal 
mucous membranes. Only a small percentage can find their way to 
the larynx and trachea, and still less to the smaller bronchioles. From 
the examination of the lungs of miners as well as from experimental 
tests there is no doubt but that some of the bacilli may find their way 
into the deeper bronchi. The deeper the bacilli penetrate the more 
unlikely that they can be cast out. On the other hand the lungs are 
the most likely point of localization of tubercle bacilli which find their 
way into the blood-stream. It is in this way that the lungs frequently 
become infected. It is now well established that infection taking place 
through the intestine may find its way by the blood to the lungs and 
excite there the most extensive lesions with or without leaving any 
trace of its point of entrance. Even if infection of the lung is slight or 
even absent the tubercle bacilli then find their way to the bronchial 
nodes where lesions may develop. Lesions either active or latent, any 
where in the body may be the source of a subsequent pulmonary infec- 
tion. Should bacilli find their way into the blood infection of the 
lung tissue will result if the resistance is lowered for any reason. The 
nasal cavities are rarely affected with tuberculosis, but more often the 
retropharyngeal tissue. Tuberculosis of this tissue as well as that of 
the tonsils is apt to give rise to infection of the lymph nodes of the 
neck. It is believed that just as bacilli may pass through the intestinal 
walls to infect the mesenteric nodes, so bacilli may, without leaving 
any trace, pass through the tonsils to the nodes of the neck. 
Primary infection of the larynx is rare. Secondary infection is 
fairly common. The region of the vocal cords and the interarytenoid 
space are the special sites attacked. 
Infection by Inhalation of Dried and Moist Bacilli.-A common mode 
of infection is by means of tuberculous sputum, which, being coughed 
up by consumptives, is either disseminated as a fine spray and so 
inhaled, or, carelessly expectorated, dries and, broken up by tramping 
over it, sweeping, etc., distributes numerous virulent bacilli in the 
dust. As long as the sputum remains moist there is no danger of dust 
infection, but only of direct contact; it is when it becomes dry, as on 
handkerchiefs, bedclothes, and the floor, etc., that the dust is a source 
of danger. 
A great number of the expectorated and dried tubercle bacilli 
undoubtedly die, especially when exposed to the action of direct sun- INFECTION BY INHALATION OF DRIED AND MOIST BACILLI 
397 
light; but when it is considered that as many as five billion virulent 
tubercle bacilli may be expectorated by a single tuberculous individual 
in twenty-four hours, it is evident that even a much smaller pro- 
portion than are known to stay alive will suffice in the immediate 
vicinity of consumptives to produce infection unless precautions are 
taken to prevent it. The danger of infection is greatest, of course, 
in the close neighborhood of tuberculous patients who expectorate 
profusely and indiscriminately, that is, without taking the necessary 
means for preventing infection. We found that of 100 tuberculous 
men admitted to one of the consumption hospitals, only 20 claimed 
to have taken any care to prevent the contamination of their surround- 
ings by their sputum. There is much less danger of infection at a 
distance, as in the streets for instance, where the tubercle bacilli have 
become so diluted that they are less to be feared. In rooms the sputum 
is not only protected from the direct sunlight, but it is constantly broken 
up and blown about by the walking, closing of doors, etc. In crowded 
streets on windy days infected dust must sometimes be in the air unless 
the expectoration of consumptives is controlled. 
Exhaustive experiments made by many observers have shown that 
particles of dust collected from the immediate neighborhood of con- 
sumptives, when inoculated into guinea-pigs, produce tuberculosis 
in a considerable percentage of them; whereas, the dust from rooms 
inhabited by healthy persons or dust of the streets does so only in 
an extremely small percentage. Fliigge is probably right in think- 
ing that the dust which is fine enough to remain for a long time in 
suspension in the air is usually free from living bacilli. It is in the 
coarser though still minute particles, those in which the bacilli are 
protected by an envelope of mucus, that the germs resist drying 
for considerable periods. These are carried only short distances 
by air currents. Such results as those obtained by Straus, who, on 
examining the nasal secretions of twenty-nine healthy persons living 
in a hospital with consumptive patients, found tubercle bacilli in 
nine of them, must be accepted with some reserve, since we know that 
in the air there are bacilli which look and stain like tubercle bacilli 
and yet are totally different. It may be said that the danger of in- 
fection from slight contact with the tuberculous is not so great as it is 
considered by many, but that on this account it is all the more to be 
guarded against in the immediate neighborhood of consumptives. 
Those who are most liable to infection from this source are especially 
young children; the adult members of the family, the nurses, the fellow- 
workmen, and fellow-prisoners of persons suffering from the disease 
are relatively very less likely to become infected. In this connection, 
also, attention may be drawn to the fact that rooms which have been 
recently occupied by consumptives are not infrequently the means of 
producing infection (as has been clinically and experimentally demon- 
strated) from the deposition of tuberculous dust on furniture, walls, 
floors, etc. The danger is not apt to last beyond three months. Fliigge 
has drawn attention to the fact that in coughing, sneezing, etc., very 398 
PATHOGENIC MICROORGANISMS 
fine particles of throat secretion containing bacilli are thrown out 
and carried by air currents many feet from the patient and remain 
suspended in the air for a considerable time. To encourage us we now 
have a mass of facts which go to show that when the sputum is care- 
fully looked after there is very little danger of infecting others except 
by close personal contact. 
Tuberculosis of Digestive Tract.-Tuberculosis of the gums, cheeks, 
and tongue is rare. The tonsils and pharynx are somewhat more 
often involved. The stomach and esophagus are almost never at- 
tacked. The small intestines are rather frequently the seat of infection 
from bacilli swallowed with the food or dust-infected mucus. In a 
striking case four previously healthy children died within a short period 
of one another. Their nurse was found to have tuberculosis of the 
antrum of Highmore, with a fistulous opening into the mouth. She 
had the habit of putting the spoon with which she fed the children 
into her mouth so as to taste the food before it was given to them. 
As already noted, the bacilli frequently pass through the mucous 
membrane to the lymph glands without leaving any lesions. 
Infection by Ingestion of Milk and Milk Products.-Milk serves as 
a conveyor of infection, whether it be the milk of nursing mothers or 
the milk of tuberculous cows. In this case evidence of infection is 
usually shown in the mesenteric and cervical lymph nodes or general- 
ized tuberculosis may be caused, while the intestinal walls are fre- 
quently not affected. Bacilli accompanied by fat pass much more 
readily through the intestinal mucous membrane or that of the tonsils 
and pharynx. The transmission of tubercle bacilli by the milk of 
tuberculous cows has been abundantly proved. 
Formerly it was thought that in order to produce infection by milk 
there must be a local tuberculous affection of the udder; but it is now 
known that tubercle bacilli may be found in the milk in small numbers, 
when adjacent tissue is infected and when careful search fails to detect 
any udder disease. Schroder has shown that the feces are a very 
dangerous factor in the dissemination of tubercle bacilli. He compares 
feces in cattle to sputum in man, since the tubercle bacilli are swal- 
lowed by cattle and are to a great extent passed through the intestinal 
tract without destruction. He found that when milk from phthisical 
cows having healthy udders was obtained so as not to become infected 
by feces it was free from bacilli, but when obtained without special 
precautions it was frequently infected. The milk of every cow which 
has any well-developed internal tuberculous infection must there- 
for be considered as possibly containing tubercle bacilli. Rabino- 
witsch, Kempner, and Mohler also proved beyond question that not 
only the milk of tuberculous cattle, which showed no appreciable udder 
disease, but also those in which tuberculosis was only detected through 
tuberculin, frequently contained tubercle bacilli. Different observers 
have found tubercle bacilli in 10 to 30 per cent, of the samples of un- 
heated city milk. Butter may contain tubercle bacilli in higher per- 
centages of samples examined. When we consider the prevalence of BOVINE INFECTION IN MAN 
399 
tuberculosis among cattle we can readily realize that, even if the 
bovine bacillus infects human beings with difficulty, there is danger to 
children when they are exposed to this source of infection. The milk 
from cattle suffering from udder tuberculosis usually contains a few 
hundred bacilli per c.c., but may contain many millions. It is also 
important to mention the fact that mixed milk from a herd, though 
tending to dilute the milk of cows excreting tubercle bacilli, may be 
badly infected from one cow, especially if this cow has udder disease. 
Taking the abattoir statistics of various countries, we find that 
about 10 per cent, of the cattle slaughtered were tuberculous. A less 
probable source of infection by way of the intestines is the flesh of 
tuberculous cattle. Here the danger is considerably less, from the 
fact that meat is usually cooked, and also because the muscular tissues 
are seldom attacked. In view of the finding of the bovine type of bacilli 
in a considerable percentage of the cases of tuberculous children 
tested, the legislative control and inspection of cattle and milk is an 
absolute necessity. As a practical and simple method of preventing 
infection from suspected milk, sufficient heating of the milk used as 
food must commend itself to all. Human tubercle bacilli may be 
found in milk as instanced by one sample of city milk examined in the 
Research Laboratory by Hess. 
Method of Examining Milk for Tubercle Bacilli.-Thirty c»c. of milk 
are centrifuged at high speed and 10 c.c. of the lower milk and sedi- 
ment collected. Four cubic centimeters of the cream is thinned with 
a little sterile water and injected into two guinea-pigs. The sediment 
is injected in amounts of 3 to 5 c.c. into other pigs. Larger amounts 
than this are apt to kill too many pigs from the associated bacteria. 
Subcutaneous injection is to be preferred. There are certain precau- 
tions that must be taking in drawing conclusions as the different types 
of acid-fast "butter bacilli" may cause lesions, and their presence will 
be noted in smears made from these lesions. To avoid this source of 
error, two methods are resorted to. If cultures are made from the sus- 
pected lesions on glycerin agar, these bacilli develop in a few days, 
whereas tubercle bacilli would not. When one is ready to kill the pigs, 
2 c.c. of old tuberculin should be injected into each pig late in the 
day. The following morning the tuberculous pigs will be dead or dying. 
Autopsies should be done on all to confirm the test. The milk should 
be as fresh as possible to prevent the growth of other bacteria. 
Bovine Infection in Man.-Numerous investigations have been made 
on this point. To Ravenel probably belongs the credit of isolating the 
first bovine bacillus from a child. The following tables summa- 
rizing the results of a large series of cases give a fair idea of inci- 
dence of such infection. As will be seen, children are especially the 
ones infected, and usually the point of entry is clearly alimentary as 
shown by the lesions. Cervical adenitis and abdominal tuberculosis 
are the most frequent types of infection. Generalized tuberculosis 
due to bovine infection is less frequent. Bone and joint tuberculosis 
is most commonly of the human type. The meninges are less 400 
PATHOGENIC MICROORGANISMS 
Table I.-Tabulation of cases reported.1 
Diagnosis. 
Adults sixteen 
years and over. 
Children five to 
sixteen years. 
Children under 
five years. 
Human. 
Bovine. 
Human. 
Bovine. 
Human. 
Bovine. 
Pulmonary tuberculosis 
Tuberculous adenitis axil- 
497 
3 
6 
- 
28 
1 
lary 
Tuberculous adenitis cer- 
2 
- 
- 
2 
• - 
vical 
27 
1 
17 
14 
9 
11 
Abdominal tuberculosis 
Generalized tuberculosis, 
15 
4 
-7 
8 
9 
11 
alimentary origin 
6 
1 
3 
4 
16 
13 
Generalized tuberculosis . 
Generalized tuberculosis, 
including meninges, ali- 
27 
- 
4 
1 
56 
3 
mentary origin . 
Generalized tuberculosis, 
- 
- 
1 
- 
5 
-10 
including meninges . 
4 
- 
10 
- 
51 
- 
Tubercular meningitis 
Tuberculosis of bones and 
- 
- 
1 
- 
2 
2 
joints 
31 
1 
31 
3 
20 
- 
Genito-urinary tuberculosis 
16 
1 
- 
- 
- 
Tuberculosis of skin . 
Miscellaneous cases: 
9 
3 
4 
6 
2 
- 
Tuberculosis of tonsils 
Tuberculosis of mouth and 
- 
• - 
- 
1 
- 
- 
cervical nodes 
Tuberculous sinus or ab- 
- 
1 
- 
- 
- 
- 
scess 
1 
- 
- 
- 
- 
- 
Sepsis, latent bacilli 
- 
- 
- 
- 
1 
- 
Totals .... 
635 
14 
85 
37 
201 
51 
Mixed or double infections: 
Pulmonary tuberculosis. 
10 cases. 
20 years. 
27 years. 
Sputum, human and bovine types. 
Sputum, human and bovine types. 
Abdominal tuberculosis. 
70 years. 
Mesenteric nodes, human type. 
Retroperitoneal, human and bovine type. 
Generalized tuberculosis 
(Alimentary origin). 
5 J years. 
Spleen, human type. 
Mesenteric node, bovine type. 
Lung, culture not obtained. 
Mesenteric nodes, human and bovine type 
18 years. 
Bronchial node, human type. 
Mesenteric nodes, human and bovine type 
Generalized tuberculosis. 
30 years. 
9 months. 
Bronchial nodes, bovine type. 
Mesenteric nodes, human type. 
Generalized tuberculosis, 
including meninges 
(alimentary origin). 
4 years. 
Meninges, human type. 
Bronchial nodes, human type. 
Mesenteric nodes, bovine type. 
Generalized tuberculosis, 
including meninges. 
4 years. 
Bronchial nodes j 
Spleen, J 
injected together, 
bovine and human types. 
Meninges, human type. 
Lung, human type. 
Mesenteric nodes, human type. 
Bronchial nodes, human and bovine types. 
4^ years. 
Total Cases, 1033. 
1 Summary of cases reported up to July, 1912, exclusive of cases examined at the 
Research Laboratory (See Table II). In contrast to the next table, the above con- 
tains a large percentage of selected cases of alimentary types of tuberculosis, many of 
which showed only slight lesions. BOVINE INFECTION IN MAN 
401 
Table II.-The Relative Proportion of Human and Bovine Tubercle Bacilli Infections 
in a Large Series of Unselected Cases1 Examined at the Research Laboratory. 
Diagnosis of 
eases ex- 
amined. 
Adults sixteen 
years arid over. 
Children five to 
sixteen years. 
Children under 
five years. 
Notes. 
Human. 
Bovine. 
Human. 
Bovine. 
Human. 
Bovine. 
Pulmonary 
tuberculosis. 
281 
- 
8 
- 
7 
- 
Clinical diagnosis only 
known and therefore no 
positive details as to the 
extent of lesions else- 
where. 
Tuberculous 
adenitis in- 
guinal and 
axillary 
1 
4 
- 
- 
- 
See next. 
Tuberculous 
adenitis cer- 
vical . 
9 
19 
8 
6 
13 
In two cases cultures were 
from axillary nodes but 
the primary focus was 
cervical. Another case 
died shortly afterward 
with pulmonary tuber- 
culosis. 
Abdominal 
tuberculosis 
1 
- 
1 
1 
1 
3 
Milk supply of one child 
subsequently examined. 
Tubercle bacilli isolated. 
Generalized 
tuberculosis 
alimentary 
origin . 
1 
2 
Only three cases given 
under this heading. 
Many of the cases in the 
following subdivisions 
showed marked intes- 
tinal lesions and some 
possibly were of aliment- 
ary origin. 
Generalized 
tuberculosis 
2 
- 
1 
- 
18 
4 
One bovine case had tuber- 
culous osteomyelitis of 
the metatarsal bone. 
Generalized 
tuberculosis 
including 
meninges 
1 
- 
25 
1 
Tubercular 
meningitis. 
1 
- 
2 
- 
26 
2 
No autopsy. Extent of 
lesions elsewhere un- 
known. 
Tuberculosis 
of bones 
and joints. 
1 
- 
10 
7 
- 
Genito-urinary 
tuberculosis 
6 
1 
1 
- 
- 
- 
The adult bovine case was 
tuberculosis of kidney. 
Removal of kidney. 
Complete recover}'. 
Tuberculosis 
of sk'in 
1 
- 
- 
- 
- 
- 
Tuberculous 
abscess 
1 
- 
- 
- 
- 
- 
Possibly primary in bone. 
Totals 
305 
1 
46 
9 
91 
25 
Double infection. Both types isolated. Generalized tuberculosis including meninges, 13 months. 
Mesenteric nodes gave human type. Meningeal fluid gave bovine type. 
Total cases, 478. 
1 Unselected cases from the hospitals of New York City. For full resume and discus- 
sion of results see Park and Krumwiede, Journal of Medical Research, vols. xxiii, xxv, 
and xxvii. 402 
PATHOGENIC MICROORGANISMS 
commonly affected by the bovine type than by the human type. Infec- 
tion of adults is very uncommon; and, though cases of pulmonary 
tuberculosis due to the bovine type of bacillus have been reported, such 
cases are rare. 
A careful study of all the factors leads us to estimate that about 
10 per cent, of all deaths caused by tuberculosis in children under five 
is due to bovine infection. 
The tables on pages 400 and 401 give a summary of the results 
obtained in the larger investigations so far carried out. 
Hypothesis of Transmissibility of Tubercle Bacilli to the Fetus.--The 
transmission of tubercle bacilli from the mother to the fetus in animals 
occurs occasionally. With regard to tuberculosis in the human fetus 
the evidence is not so clear, though some 20 cases have been recorded 
of tuberculosis in newly born infants, and about a dozen cases of pla- 
cental tuberculosis. As to the infection of the fetus from the paternal 
side, where the father has tuberculosis of the scrotum or seminal vessels, 
we have no reason to suppose that such can occur. There are, how- 
ever, grounds for belief that infection in this way may take place from 
husband to wife. 
Attenuation.-Tubercle bacilli when subjected to deleterious in- 
fluences slowly decrease in virulence. Some strains lose their virulence 
when artificially cultivated for some time; some quickly, some slowly. 
Others retain their virulence indefinitely. 
Mixed Infection.-In regions where tuberculous processes are on the 
surface, such as lung and skin infections, and also when the infection 
itself is multiple, as in disease of the glands of the neck from tonsillar 
absorption, the tubercle bacilli are frequently associated with one or 
more other varieties of organisms. Those of most importance are the 
streptococcus, pneumococcus, and influenza bacillus. Besides these 
many other varieties are met with occasionally in individual cases. 
What the influence of this secondary or mixed infection is, under all 
circumstances, is not exactly known; but generally the effect is an 
unfavorable one. For the technique employed in examining sputa 
for mixed infection see page 418. 
Individual Susceptibility.-It was believed by many that in demonstrat- 
ing that tuberculosis was due to a specific bacillus that its occurrence 
was sufficiently explained; but they left out another important factor 
in the production of disease-individual susceptibility. That this 
susceptibility, or "predisposition," as it is improperly called, may be 
either inherited or acquired is now an accepted fact in medicine. It 
has even been thought that the physical signs and characters-the 
phthisical habit-which, indicate this susceptibility can be externally 
recognized. At first the inherited susceptibility was considered more 
important than the acquired, but now much that was attributed to the 
former is known to be explained by the fact of living in an infected area. 
The acquired susceptibility may arise from faulty physical develop- 
ment or from -depression, sickness, overwork, excessive use of alcohol, 
etc. Unquestionably, vast differences exist in different individuals in IMMUNIZATION 
403 
the intensity of the tuberculous process in the lung. That this does 
not depend chiefly upon a difference in virulence of the infection is evi- 
dent from the fact that individuals contracting tuberculosis from the 
same source are attacked with different severity, and that there is, as 
a rule, no great difference in degrees of virulence for animals in the 
tubercle bacilli obtained from different sources. As is seen from the 
results of postmortem examinations in which, according to the com- 
pleteness of the examinations, the remains of old tuberculous processes 
have been found in the lungs of about one-third to one-half of all the 
bodies examined, many cases of pulmonary infection must occur without 
showing any visible evidence of disease, and heal of their own accord. 
The possibility of favorably influencing in many an existing tubercu- 
losis by treatment also proves that, under natural conditions, there is 
a varying susceptibility to the disease. Clinical experience teaches, 
likewise, that good hygienic conditions, pure air, good food, freedom 
from care, etc., increase immunity to phthisis. Animal experiments 
have shown that not only are there differences of susceptibility in vari- 
ous animal species, but also an individual susceptibility in the same 
species. The doctrine of individual susceptibility, therefore, is seen 
to be founded on fact, although the reasons for it are only partially 
understood. Certain infectious diseases reduce the resistance to tuber- 
culous infection, the most noteworthy example being measles. 
Immunization.-As in other infectious diseases, many attempts 
have been made to produce an artificial immunity against tuberculosis, 
but so far the results have been disappointing. The great majority 
of mankind has in a varying degree some natural immunity against 
tuberculosis. In many individuals this immunity is only relative, and 
is maintained only as long as the health is kept at a high standard or 
the exposure to infection is not too intense or prolonged. 
Acquired immunity against many bacterial diseases occurs within 
a very few days or weeks after the development of infection. This 
immunity may be complete or slight and vary greatly in its duration. 
There is little at first glance in the clinical history of tuberculosis to 
show that acquired immunity occurs in this disease, since relapse is the 
rule. For this reason the production of an artificial immunity against 
tuberculosis has always been looked upon by many as a result possibly 
never to be achieved. The careful study of tuberculosis seems, however, 
to indicate an attempt on the part of nature at the production of acquired 
immunity in this disease. It is known that by the eighteenth year of 
life many, as shown by postmortem examination and tuberculin reac- 
tions are infected, although the general condition gives no evidence 
of infection. In certain classes this may constitute as high as 95 per 
cent, of the children examined. The small proportion of these which 
progress to serious lesions immediately after infection, or later through 
relapse or reinfection indicates a degree of acquired immunity. 
An attempt to imitate nature's methods, by the inoculation of a 
living culture or of toxins and dead bacilli has been made. The injec- 
tion by Koch of the heat-resistant toxins, as in his original tuberculin 404 
PATHOGENIC MICROORGANISMS 
treatment, produced in animals a certain degree of acquired resistance 
to larger doses of toxins, but did not protect to any appreciable degree 
from subsequent living tubercle bacilli, or produce in animals a pro- 
tective serum. In 1892 Trudeau succeeded in producing in rabbits 
an appreciable immunity by inoculations of living avian cultures. 
The rabbits so treated supported, as a rule, inoculation of virulent 
tubercle bacilli in the anterior chamber of the eye, while in controls 
the eyes were invariably lost. Later, attenuated human cultures were 
used with the same results. Behring, Pearson and Gilliland and others 
have treated cows by giving each of them intravenous injections of 
human tubercle bacilli. The animals so treated are immune to an 
otherwise fatal dose of bovine tubercle bacilli. The human bacilli 
cause a relatively slight amount of tuberculosis and remain alive in 
the encapsulated lesions. Behring has had prepared an emulsion of 
attenuated human bacilli to use in cattle, and advised its use for 
general immunization. Unfortunately calves so vaccinated may later 
in life excrete virulent human tubercle bacilli in their milk. 
The observations of Koch, later elaborated by Romer and others, 
on reinfection in guinea-pigs fit in with the above results. Thus they 
found that if guinea-pigs with a non- or slowly progressive tuberculosis 
were reinjected with virulent tubercle bacilli they developed only a 
local lesion, but this reinjection did not cause generalized lesions and 
death as in the non-tuberculous controls. Death only resulted when 
the reinfecting dose was very large and then resulted rather from 
toxemia than from progressive tuberculous lesions. 
From the above observations the generalization has been drawn 
that immunity only exists when a tuberculous lesion is present, and 
that such immunity disappears after the lesion heals. The injection, 
therefore, of non-virulent tubercle bacilli or dead bacilli or their 
products should give immunity only in so far as they are capable of 
producing tubercle lesions. This has been found to be the case in 
experimental animals. The use of related acid-fast bacilli treatment 
as in the case of the Friedmann vaccine is based on the fact that they 
produce tuberculous-like lesions, but their use has not been followed by 
any appreciable results. Such results as were obtained in the treatment 
of tuberculosis by the injection of bacillary products (see tuberculins) 
are probably due not.to the production of an immunity against the 
bacillus, but to other factors. If the generalization "No immunity 
without a lesion" is accepted then the presence of a tuberculin reaction 
in man is an indication of immunity. 
On the basis of the experiments in animals the course and type of 
infection in man find a ready explanation. Man when first infected, 
if he succumbs, usually dies of a more or less generalized tuberculosis. 
This is seen in the infections during the early years of life and likewise 
among the adults of races where tuberculosis has recently been imported. 
The less susceptible children develop latent lesions, and with the wide- 
spread opportunity for infection we find that the majority of children 
of eighteen years of age are so infected, but this remains latent as long TUBERCULIN, THEORETICAL CONSIDERATIONS 
405 
as general good health is maintained. All so infected are immune not 
only from reinfection from without but are also partially protected from 
reinfection from within due to dissemination of bacilli from these existing 
lesions. The bacilli even from latent lesions find their way into the blood- 
stream at times. Should, however, the immunity be reduced dissemina- 
tion will result in fresh lesions which may progress. The lungs are the 
most likely point of arrest of the bacilli and are the more susceptible to 
infection, although the other organs may be protected by the immunity 
present, although lowered in degree. If the immunity were completely 
lost, general miliary tuberculosis would probably result. 
An analogy to this is seen in the rabbit. This animal has little resist- 
ance to infection by the bovine type, and however infected a generalized 
type of disease usually results. There is, however, a marked resistance 
to infection with the human type, and however the infectious agent 
may be introduced, the lungs are the first point, as a rule, where exten- 
sive lesions develop. 
The original lesion may have been in the lung, but the dissemination 
may still be through the blood-stream. 
On this basis, the great majority of us become infected during the 
early years of life and most of such infections remain latent. As long 
as these latent lesions exist and the general resistance is good, a relative 
immunity to reinfection persists. Should, however, this immunity be 
lowered by some factor as ill health, fatigue, etc., dissemination occurs, 
which may then progress, or in turn with recovery of the general bodily 
resistance, regress and the immunity again rises and protection is 
reestablished. 
We can conclude, therefore, that childhood is the most frequent 
time of infection, that the disease in later life is usually an autoinfection 
from the lesions so acquired and that most adults have little reason 
to fear contact infection. The precautions now taken should not be 
relaxed for there are some, who, because of the complete healing of a 
previously existent lesion or having escaped infection, are susceptible. 
There is reason to believe, however, that those who have lost their 
immunity through the complete healing of their lesions are on the average 
more protected than those who have escaped infection. Should they 
become reinfected they would probably reestablish this immunity more 
promptly and thus limit or destroy the bacilli which found entry. 
Tuberculin,1 Theoretical Considerations.-Tuberculin is the term 
used for fluids containing the products of growth, the watery soluble 
substances of or a suspension of the ground up bodies of the bacilli 
or a combination of two or more of these. Such products when injected 
into normal animals, even in large amounts are not toxic. When injected 
into tuberculous animals even in minute amounts they produce a reaction 
which is shown by hyperemia of the tuberculous lesions and a general 
reaction as evidenced by a rise of temperature. Given in larger doses 
1 For a fuller discussion of Tuberculin, see Hamman, Forschheimer's Therapeutics, 
vol. v, or Baldwin and Brown, Osler's Practice of Medicine, vol. ii. 406 
PATHOGENIC MICROORGANISMS 
marked symptoms of toxemia develop, and in still larger doses death 
results. This sensitiveness of the tuberculous animal is also manifested 
when tuberculin is applied to the skin, as by scarification or to the mucous 
membranes, as by instillation in the eye; a local inflammatory reaction 
resulting. This reaction is specific and is employed for diagnosis. 
Induced hypersensitiveness to foreign protein, anaphylaxis, can be 
produced in animals by the injection of the proteins of the tubercle 
bacillus or of tuberculin, the animal showing symptoms of intoxication 
when a second dose is given. However animals have been sensitized, 
there has never developed a skin sensitiveness unless tubercles were 
produced. In discussing the production of artificial immunity a similar 
condition was described. If the two are compared, we are compelled 
to deduce from the parallelism that tuberculin sensitiveness is closely 
related to the mechanism of immunity, although dead bacilli may pro- 
duce tuberculin hypersensitiveness but no appreciable immunity. What 
relation this phenomenon, tuberculin sensitiveness or allergy, bears 
to the induced anaphylaxis following the injection of tubercle bacillus 
protein is not clear, further than that they are similar phenomena, but 
one is associated with immunity the other not. The opposite of allergy 
is anergy and this term is used to denote the disappearance of tuberculin 
sensitiveness during measles or in miliary tuberculosis; associated in 
the first, with a temporary depression of the immunity often followed by 
dissemination or progress of the tuberculous lesions already present, 
and in the latter is an index of the total loss of immunity which must 
occur before such generalized lesions can develop. 
The theories that have been advanced to explain the tuberculin 
reaction are not altogether satisfactory. The tuberculin may be toxic 
in itself for the tuberculous animal either directly or because of an 
acquired ability to split the tuberculin into toxic products. The general 
symptoms, however, are probably due to the reaction of the highly 
sensitive tissues at the site of infection resulting in the local production 
of toxic substances. 
Why the careful administration of tuberculin as a therapeutic agent 
should result in improvement is still more difficult to understand. 
By the injection of tuberculin we do not, as far as can be determined, 
cause the production of antibodies active against the tubercle bacillus. 
What we do produce is a tolerance to tuberculin, either raising the thresh- 
hold of sensitiveness or causing it to disappear. If we return to the 
discussion of allergy and its parallelism to immunity, this should be 
the last thing we should care to cause, for fear of the possibility of super- 
infection. The general symptoms in tuberculosis, however, are due to 
the action of hypersensitized tissues at the site of infection on the bacilli 
or their products. We have, therefore, the paradoxical condition, that 
the mechanism associated with protection is also the mechanism of 
intoxication when overdeveloped or overactive. On this basis, if we 
are able by the administration of tuberculin to lower this hypersensi- 
tiveness, clinical improvement should result, which is usually the case. 
In this case allergy is reduced or latent. If tuberculin is given recklessly TUBERCULIN 
407 
in large doses regardless of reactions, however, the mechanism of allergy 
is destroyed and harm results. 
The localization of a tuberculous lesion is the response of the surround- 
ing tissues to the irritation of the poisons formed. When tuberculin 
is given the reactions in the lesions stimulate further the production 
of connective tissue, but such stimulation to be of value must not be 
accompanied by general reaction. 
The fear has been maintained that tuberculin causes the dissemina- 
tion of bacilli from the existing lesion. Recently Rabinowitsch has again 
raised this question. When we remember how much more frequently 
tubercle bacilli find their way into the blood, than was formerly believed, 
and consider the mechanism of protection against such dissemination, 
it is doubtful if such results are to be feared when proper dosage is 
employed. 
The tuberculins made from the bovine and human types of bacilli 
show no quantitatively different reaction when used in man or animals. 
The tuberculin from avian bacilli, however, is much less active. Although 
the tuberculin reaction is in general specific there is evidently a group 
reaction in infections with the acid-fast group. Further examples of 
this are the reaction of cattle with chronic enteritis to avian tuberculin, 
and of lepers to ordinary tuberculin. 
Various types of tuberculins have been employed, the endeavor being 
to change the constituents of the bacillus or its products as little as 
possible either by manipulation or heat. The original tuberculin was 
heated, the reactive substances being heat resistant. The newer tuber- 
culins for treatment are heated moderately or not at all. 
Tuberculin.-Preparation.-Various tuberculins have been used, the 
following, however, are the only ones that are used generally: 
Tuberculin, Koch'1 s"Old"or "Crude" (0. T.)--Culturesof tubercle bacilli 
after six weeks' growth on 5 per cent, glycerin broth are heated in the 
Arnold sterilizer to kill the bacilli and filtered to remove the bacilli. 
This filtrate is evaporated to one-tenth its bulk and after filtering 
through paper to remove the sediment that forms is ready for use. 
The tuberculin is therefore a heated 50 per cent, glycerin solution of 
the products of the bacilli in the culture fluid and the portions of 
the bacilli which go into solution. It is used chiefly for diagnostic 
purposes. 
Tuberculin B. F. (Bouillon Filtrate) is made from a broth culture as 
above. The culture is not heated, but filtered first through paper and 
then through a Berkefeld filter to insure sterility. It differs from the 
original tuberculin in that it is neither heated nor concentrated. 
Tuberculin B. E. (Bacillus Emulsion) is produced by grinding up 
dried tubercle bacilli until no intact bacilli can be found on microscopic 
examination. This powder is then suspended in glycerin water and 
heated to 60° C. for an hour or more to kill any viable tubercle bacilli. 
The proportion of bacillus to water is such that 1 c.c. of fluid contains 
5 mgs. of bacillus substance. 
The undiluted products keep for a long time if kept cool and pro- 408 
PATHOGENIC MICROORGANISMS 
tected from the light. The low dilutions keep for at least one month, 
but the high dilutions should not be used after two weeks. 
Method of Diluting Tuberculin.-The following describes a .method of 
dilution in terms of volume of finished tuberculin, and gives the weight equiva- 
lent. Although in the case of bouillon filtrate many use dilutions taking into 
consideration the fact that the bouillon filtrate is not concentrated or in the 
case of bacillus emulsion dilute according to the weight of solid substance con- 
tained, a uniform method for each seems advisable for reasons of simplicity. 
If we consider the finished product in terms of cubic centimeters or grams 
regardless of the contents, the following is the method of dilution. Dilutions 
should be made with sterile saline to which 0.25 per cent, carbolic has been added. 
Dilu- 
tions. 
Amount of tuber- 
culin. 
Amount 
of 
diluent. 
- 
Content of tuberculin terms 
of finished product. 
Using bacillus emulsion c< 
of solid substance in 
dilution will be. 
mtent 
each 
A 
1 c.c. 
9 c.c. 
1 c.c. = 0.1 c.c. or gms., or 
100 c.mm. or mgs. 
1 c.c. = 0.5 mgm., or 
0.5 mgm. 
B 
1 c.c. of dilution A 
9 c.c. 
1 c.c. = 0.01 c.c. or gms., or 
10 c.mm. or mgs. 
1 c.c. = 0.05 mgm., or 
0.05 mgm. 
C 
1 c.c. of dilution B 
9 c.c. 
1 c.c. = 0.001 c.c. or gms., 
or 1 c.mm. or mg. 
1 c.c. = 0.005 mgm., 
0.005 mgm. 
or 
D 
1 c.c. of dilution C 
9 c.c. 
1 c.c. = 0.0001 c.c. or gms., 
or 0.1 c.mm. or mgm. 
1 c.c. = 0.0005 mgm., 
0.0005 mgm. 
or 
E 
1 c.c. of dilution D 
9 c.c. 
1 c.c. = 0.00001 c.c. or gms., 
or 0.01 c.mm. or mgm. 
1 c.c. = 0.00005 mgm., 
0.00005 mgm. 
or 
F 
1 c.c. of dilution E 
9 c.c. 
1 c.c. = 0.000001 c.c. or gm., 
or 0.001 c.mm. or mgm. 
1 c.c. = 0.000005 mgm. 
0.000005 mgm. 
or 
Diagnostic Use of Tuberculin.-Tuberculin, Old or Crude, is used 
for this purpose and it is advised that it be used either as a cutaneous 
test (von Pirquet) or as the intracutaneous test (Mantoux) or as a 
subcutaneous test. 
The Cutaneous Test (von Pirquet).-This is performed as follows: 
The inner side of the forearm is cleansed with alcohol and ether and two 
scarifications or similar scratches are made about three inches apart. 
Oozing of blood is to be avoided. On one spot or scratch a drop of 
tuberculin is placed and allowed to soak in. The tuberculin may be 
diluted to 25 per cent, if desired. The other spot is kept as a control. 
Both spots should be examined at the end of twelve, twenty-four, and 
thirty-six hours. A positive reaction appears after three to twenty- 
four hours and is usually at its height at thirty-six to forty-eight hours, 
and consists then of a slightly raised reddening of the skin somewhat 
circular in outline and usually about 10 mm. in diameter. Reactions 
under 5 mm. in diameter should be regarded as doubtful. 
Intracutaneous Reaction (Mantoux).-The hypodermic needle is inserted 
only beneath the superficial layers of the skin, and enough old tuber- 
culin (1: 5000 dilution) is inoculated to form a bleb about the size of 
a pin-head. A salt solution control is inoculated in the same way a few 
inches beyond. In a few hours an infiltration appears, and in twenty- 
four hours, a red papule with a marked hyperemic zone is seen. The 
height of the reaction is forty-eight hours. 
Ophthalmic Test (Calmette).-This test, due to the occurrence of serious 
accidents, is little used. A drop of a 2 per cent, solution of tuberculin THE TUBERCULIN TEST FOR. DIAGNOSIS 
409 
is applied to the lower conjunctival sac. The reaction is indicated by 
secretion and reddening of the inner canthus caruncle or lower lid 
which may include the entire conjunctiva with edema of the lids. 
Moro Test.-An ointment is made of equal parts of lanolin and tuber- 
culin. A little of this is rubbed into the skin. A positive reaction is 
shown by the development of reddening and papules. 
The Subcutaneous Test.-This is performed as follows: The tempera- 
ture should be taken four times at three-hour intervals, on each of two 
days before the injection and for two days after the injection. The 
temperature before injection should not exceed 99° F. The injection 
should be given with a sterile syringe in the back below the level of the 
shoulder blades. The time for injection which is most convenient is 
10 to 11 p.m. The temperature taking being started early the following 
morning covers the period at which a rise is to be expected. 
The initial dose for adults should not exceed 0.5 mgm. or c.mm., 
viz., 0.5 c.c. of dilution C. This dose is large enough to cause a reaction 
in many cases but is small enough to avoid violent or unpleasant symp- 
toms. If no reaction follows a second injection is given at the end of 
three to four days. This should be 1 mgm. or c.mm., viz., 1 c.c. of 
dilution C. If this is not followed by a reaction a third dose of 2 gms. 
or cm., viz., 2 c.c. of dilution C or 0.2 c.c. of dilution B. If this dose 
gives no reaction there is probably no tuberculosis present sufficient 
to be of interest clinically. If this dose gives a doubtful reaction a 
fourth injection of 5 mgms. or c.mm. may be given. The doses for 
children are one-half to one-fifth of the above, depending on the age. 
Reactions are of three types: (a) generalized and febrile; (6) a focal 
at the site of the lesion; (c) local at the site of injection. 
(a) A reaction may be considered positive where there is a rise of 1° 
F. over the highest point reached before the injection. The rise usually 
begins six to eighteen hours after the injection, very occasionally the 
rise is delayed. Other general symptoms may be present, viz., headache, 
malaise, loss of appetite or nausea, chilliness or chills, giddiness, pains 
in the back and limbs, and insomnia. 
(6) A focal reaction may be absent even though the general reaction 
be marked. If present, is evidenced by an increase in the signs at the 
site of the lesion. 
(c) The local reaction is also variable in its appearance. If present, 
it consists in redness, swelling, induration or soreness at the site of 
injection. 
Contra-indications to the subcutaneous test are, certainty of diagnosis, 
fever, recent hemoptysis, advanced heart or renal disease, epilepsy, 
diabetes, and convalescence from a severe infection. 
Deductions and Limitations of the Tuberculin Test for Diagnosis.- 
Advanced cases commonly fail to react, but in these cases other methods 
make diagnosis certain without resort to tuberculin. The reaction when 
positive gives us only one item of information, viz., a tuberculous fecus 
exists in the body. It tells us nothing as to its location, except one 
should elicit an appreciable focal reaction, and nothing as to the extent 410 
PATHOGENIC MICROORGANISMS 
or activity of the lesion. The site of the lesion, the degree of involve- 
ment and activity of the disease must be elicited by other means, by 
careful consideration of the history and physical examination. 
Tuberculin Treatment. Tuberculin B. E. and B. F. Used for this 
Purpose.-The patients of choice for treatment are those who are 
afebrile and generally well-nourished, especially if the pulmonary con- 
dition is incipient or if elsewhere localized and not extensive. They 
are especially suited if they have reached a stationary stage under general 
treatment. Patients with slight fever unaffected by rest if carefully 
injected may obtain a. normal temperature. The febrile and more 
advanced cases may be benefited, but the use of tuberculin in these 
cases requires much skill and experience. 
Dosage.-The fundamental rule is to give the patient increasing 
doses as large as can be borne without eliciting any reaction. The begin- 
ning dose should therefore be very small. In the scheme of dilutions 
given 0.0001 mgm. or c.mm. is the beginning dose. One-tenth of this 
amount is sufficient if any fever is present. This is increased by 0.0001 
mgm. until 0.001 mgm. or c.mm. is given. This dose is then increased 
by 0.001 mgm. until 0.01 mgm. or c.mm. is given and so on until 0.1 
c.c. or even 1 c.c. of the undiluted tuberculin is given. 
The progression in terms of the dilution is as follows: 0.1 c.c. of dilu- 
tion F. or 0.0001 mgm. or c.mm. increased by 0.1 c.c. until 1 c.c. is 
given, then 0.2 c.c. of dilution E or 0.002 mgm. or c.mm. increased by 
0.1 c.c. of this dilution until 1 c.c. is given. Then start with 0.2 c.c. 
of dilution D, increased by 0.1 c.c. at each succeeding dose of this dilu- 
tion to 1 c.£. Then start with 0.2 c.c. dilution C, and increase by 0.1 
as above until 1 c.c. is given. Then give dilutions B and A in the same 
manner and finally the undiluted tuberculin. 
It should be noted that in the dilutions the rate of increase is ten 
times as large as one progresses from one dilution to the next. 
The interval between injections is usually three to four days, or twice 
a week. The interval is wisely increased as the doses are increased and 
the rate of increase becomes correspondingly higher. 
The duration of the treatment is long. There is no use starting such 
treatment unless it can be persisted in at least for several months. 
The more advanced cases will require a more prolonged course of 
treatment. 
Reactions.-As has been said, every effort should be made to avoid 
a reaction. The temperature should be taken three times a day, and also 
at any time the patient feels indisposed. If at any time there is a rise 
in temperature no tuberculin should be given until the temperature has 
returned to the previous range and stayed there for two or three days. 
If the rise has not exceeded 1° F., and has been unaccompanied by other 
symptoms, the next dose may be the same as the preceding dose. If 
accompanied by general symptoms, the dose should be reduced one- 
half to one-quarter of the reacting dose and the increase made as before. 
Increase in pulse rate calls for decrease of the dose and increase of the 
interval. Dyspnea is an indication for the same reduction. DIRECTIONS FOR INSPECTING HERDS FOR TUBERCULOSIS 
411 
Increase in the local signs, continuous loss of weight, hemoptosis 
or acute pleuritic pains mean that one should either not increase the 
dose or even lower it and increase the intervals. If the symptoms are 
marked, cessation of dosage for a time is called for. 
There are also certain indefinite symptoms which point to the toxic 
action of the tuberculin and are evidences of intolerance. They are 
fatigue, loss of appetite, indefinite pains, headache, and malaise. If 
they occur, delay increasing the dose, or if they persist, diminish the 
dose and increase the interval. 
A local reaction is usually disregarded in the absence of any other 
symptoms of reaction. 
Contra-indications.-Very advanced tuberculosis, tuberculous menin- 
gitis, and general miliary tuberculosis. Rapid loss of weight or a per- 
sistent pulse rate above 100. 
Tuberculin should never be sprayed about from a syringe. It may 
cause serious reactions in those exposed. 
Results of Tuberculin Treatment.-Tuberculin is not a cure for tuber- 
culosis. It promotes healing and relapses are less frequent after its 
use. It should be used as an addition not as a substitute for the recog- 
nized methods of treatment. It is a two-edged weapon and should be 
employed only by those who have a thorough understanding of its 
possibilities for good and, unfortunately, for harm. 
United States Government Directions for Inspecting Herds for Tuberculosis.- 
"Inspection should be carried on while the herd is stabled. If it is necessary 
to stable animals under unusual conditions or among surroundings that make 
them uneasy and excited, the tuberculin test should be postponed until the 
cattle have become accustomed to the conditions they are subjected to, and 
then begin with a careful physical examination of each animal. This is essen- 
tial, because in some severe cases of tuberculosis, on account of saturation with 
toxins, no reaction follows the injection of tuberculin, but experience has 
shown that these cases can be discovered by physical examination. This 
should include a careful examination of the udder and of the superficial 
lymphatic glands, and auscultation of the lungs. 
"Each animal should be numbered or described in such a way that it can be 
recognized without difficulty. It is well to number the stalls with chalk and 
transfer these numbers to the temperature-sheet, so that the temperature of 
each animal can be recorded in its appropriate place without danger of con- 
fusion. The following procedure has been used extensively and has given 
excellent results: 
" (a) Take the temperature of each animal to be tested at least twice, at 
intervals of three hours, before tuberculin is injected. 
"(b) Inject in the evening, preferably between the hours of six and nine, 
0.4 c.c. of Koch's tuberculin previously diluted to 5 c.c. with sterile water. The 
injection should be made with a carefully sterilized hypodermic syringe. The 
most convenient point for injection is back of the left scapula. Prior to the 
injection the skin should be washed carefully with 5 per cent, solution of car- 
bolic acid or other antiseptic. 
"(c) The temperature should be taken nine hours after the injection, and 
temperature measurements repeated at regular intervals of two or three hours 
until the sixteenth (eighteenth)1 hour after the injection. 
1 The directions allow temperatures to be stopped the sixteenth hour, but even when 
there is no reaction at all it is much safer to always take temperatures for eighteen 
hours. We have found now and then a tuberculous cow that reacted on the eighteenth 
hour for the first time. 412 
PATHOGENIC MICROORGANISMS 
" (d) When there is no elevation of temperature at this time the examination 
may be discontinued; but if the temperature shows an upward tendency, meas- 
urements must be continued until a distinct rection is recognized or until the 
temperature begins to fall. 
" (e) If a cow is in a febrile condition tuberculin should not be used, because 
it would be impossible to determine whether, if a rise of temperature occurred, 
it was due to the tuberculin or to some transitory illness. 
" (/) Cows should not be tested within a few days before or after calving, for 
experience has shown that the result at these times may be misleading. 
" (ff) In old, emaciated animals and in retests, use twice the usual dose of 
tuberculin, for these animals are less sensitive. 
" (Ji) Condemned cattle must be removed from the herd and kept away from 
those that are healthy. 
" (J) In making postmortems the carcasses should be thoroughly inspected, 
and all the organs should be examined. " 
Antituberculous Serum.-Every conceivable way of obtaining the true 
products of the tubercle bacilli has been tried, so as to cause the in- 
jected animals to produce antibodies both antitoxic and bactericidal. 
In spite of much conflicting testimony, it is probably safe to assert 
that no sera now obtainable have any great value. This view, how- 
ever, in no way lessens the necessity of continued endeavor until every 
method conceivable has been tried. 
Prophylaxis.-Meanwhile all energies should be directed to the 
prevention of tuberculosis, not only by the enforcement of proper 
sanitary regulations as regards the care of sputum, milk, meat, dis- 
infection, etc., but also by continued experimental work and by the 
establishment of free consumptive hospitals, and by efforts to improve 
the character of the food, dwellings, and conditions of the people in 
general, we should endeavor to build up the individual resistance to the 
disease. It may be years before the public are sufficiently educated 
to cooperate with the sanitary authorities in adopting the necessary 
hygienic measures to stamp out tuberculosis entirely; but, judging 
from the results which have already been obtained in reducing the 
mortality from this dread disease, we have reason to believe that in 
time it can be completely controlled. 
Among the numerous medical agents that have been tried without 
avail to protect animals against the action of the tubercle bacillus 
may be mentioned tannin, menthol, sulphuretted hydrogen, mercuric 
chloride, creosote, creolin, phenol, arsenic, eucalyptol, etc. 
Agglutination Reaction for Diagnosis.-The results obtained by 
various observers have been very conflicting. Two methods are 
employed in making the test. In one a vigorous growth of bacilli is 
dried, ground up, and an emulsion made. In the other Arloing and 
Courmont grow the culture for a time on potato and then in bouillon. 
In this way a homogeneous culture of separate bacilli is obtained which 
can be used for agglutination. The examination is usually made macro- 
scopically, and requires twelve to twenty-four hours. At present the 
test cannot be advised as useful in diagnosis as the sera of cases suffer- 
ing from tuberculosis frequently fail to give a reaction, while the sera 
from those having no detectable tuberculosis frequently cause a good RABBIT VIRULENCE 
413 
reaction. A positive agglutination test is thought by some to be a 
favorable sign as indicating resistance to infection by the body. A 
reaction in dilutions of 1: 10 or 1: 15 is considered a positive test. 
The Tubercle Bacillus of Domestic Animals, and its Relation to 
Human Tuberculosis.-Among the domestic animals tuberculosis is 
most common in cattle. On account of the milk which they provide 
for our use, and which is liable to contain bacilli, the relation of these 
to human tuberculosis is a matter of extreme importance. 
The chief seat of the lesions is apt to be the lungs, and with them 
the pleura; less often the abdominal organs and the udder are affected. 
In pigs the abdominal organs are most often involved, then the lungs 
and lymphatic glands. In sheep, horses and goats tuberculosis is 
rare. 
Differences between Tubercle Bacilli of Human and Bovine Type. 
-As has been already noted in the tables given of the incidence of 
bovine and human infection, it is possible to tell in any case the type 
of infection. The essential differences are in cultural characteristics 
and in virulence for rabbits and calves. 
Cultural Differences.-The bovine bacillus grows very poorly when 
isolated, the human bacillus very freely. This is noted on plain egg, 
but to a less extent than on glycerin egg. The glycerin restrains or 
adds little to the growth of bovine bacilli, but increases markedly the 
amount of growth of the human bacillus. In fact, primary cultures 
on glycerin egg of bovine material commonly fail. This difference is 
very noticeable in the first few generations and is sufficient in the great 
majority of instances for differentiation to one who has had some experi- 
ence with such cultures. Further, the majority of human strains can 
be transplanted to glycerin potato or glycerin broth and give vigorous 
growth in the first few generations, whereas the bovine bacillus fails 
or growth is very slight. After further cultivation the bovine bacillus 
gradually increases its amount of growth until it is indistinguishable 
from the human type. This increase in luxuriance may be rapid or 
very slow. 
Rabbit Virulence.-The bovine bacillus is exceedingly virulent for 
rabbits by any method of inoculation; the human bacillus only slightly 
so. The best method of differentiation is by intravenous inoculation. 
A small amount of culture is weighed after the moisture has been 
extracted with filter paper and a suspension made in normal saline and 
diluted so that 1 c.c. = 0.01 mg. of culture; this amount is then injected 
into the ear vein of a rabbit. If the rabbit survives for from forty to 
fifty days, and on autopsy shows only lesions in the lungs or kidneys 
or both, the strain is of the human type. With the bovine type of bacillus 
the rabbit will die in the majority of instances before or about this 
time, if not it may be killed. On autopsy a progressive generalized 
tuberculosis will be found. The lesions in the lungs will be very marked, 
the tubercles having become confluent with caseous centres. The liver 
or spleen or both will be peppered with tubercles. Tubercles will be 
present in the great majority of cases in the superficial lymph nodes 414 
PATHOGENIC MICROORGANISMS 
and also in those of abdomen and thorax. There may be tubercles on 
the heart, in the rib marrow, or over the peritoneum. 
These two differences alone are sufficient to differentiate in every 
case the type of bacillus. It must be insisted upon again that the cul- 
tural characteristics be observed in the early generation and, further, 
that the virulence be tested in early generations. In case the bovine 
culture does not afford sufficient material for weighing, a suspension 
can be made and compared with a weighed suspension. 
Virulence for Calves.-In proving the non-identity of the two bacilli, 
calf experiments were resorted to. This was necessary as the supposed 
bovine cultures from children would have to be virulent for calves to 
the same extent as cultures from bovine material. The commonly 
used method was the subcutaneous inoculation in the side of the neck 
with 50 mg. of culture. The human type of bacillus caused only a 
local lesion or at most a spreading to the nearest lymph node. The 
bovine bacillus, on the other hand, caused a generalized tuberculosis 
which was or was not fatal. Sufficient data has been accumulated 
to make this test practically unnecessary for the determination of type. 
Differences in Morphology.--The bovine bacillus tends to be shorter, 
thicker, and solidly stained; the human type tends to be longer, slimmer, 
usually bent, and shows beading and irregularities in staining. We have 
found this difference most marked on glycerin egg, slight or impercep- 
tible on other media. 
Besides the above differences Theobald Smith made the interesting 
discovery that the production of acid differed with the two types when 
grown on glycerin broth. The bovine type renders the bouillon less 
and less acid; this may even progress until the medium becomes slightly 
alkaline to phenolphthalein. The human type causes a preliminary 
fall in the acidity; as growth progresses the acidity is then gradually 
increased, and may exceed the original acidity of the broth used. This 
difference is evident in tuberculin made from the two types of 
bacilli. The bovine tuberculin is alkaline or very slightly acid while 
human tuberculin is markedly acid. The change is only noticed when 
glycerin is used in the media. Whether this difference is specific is 
doubtful. The work of more recent investigators would seem to show 
that this difference, like all differences between the types, is purely 
quantitative, and that different strains vary in their reactions and give 
intermediate reactions between these two extremes. 
Bird (Avian) Tuberculosis.-Tuberculosis is very common and infec- 
tious among fowl. The bacillus grows easily and freely on glycerin 
media. It tends to form a moist or even slimy growth, and commonly 
produces an orange pigment. It is able to grow at a higher tem- 
perature than mammalian tubercle bacilli, the latter failing to grow 
above 41° C.; the former growing at even higher temperatures. Guinea- 
pigs are less susceptible to inoculation with avian tubercle bacilli, and 
the virulence for these animals is usually quickly lost. Rabbits are much 
more susceptible. Rats and mice are spontaneously infected with avian 
tubercle bacilli and are supposed to be an important factor in spreading METHODS OF EXAMINATION FOR TUBERCLE BACILLI 
415 
the disease. Birds are refractory, with few exceptions, to infection 
with the mammalian tubercle bacillus. Parrots, however, are msceptible 
to infection with all three types and commonly have spontaneous 
tuberculosis caused by the human type of bacillus. 
Stability of the Different Types of Bacilli.-The fact that the agglu- 
tination reactions and the tuberculin reactions of the different types is 
similar shows their close relationship. This has led to the endeavor 
to change one type into the other. This is usually done by passage 
through animals. The results have been peculiar. Some cultures 
have been passed through a series of calves without any change ex- 
cept for a moderate increase in virulence. Other cultures seem to 
have completely changed their type. We believe that this is not a 
change of type, but an additional bovine infection. Strong negative 
evidence is the fact that the bovine bacillus when infecting man loses 
none of its characteristics, though present in the human body for years. 
Tuberculosis of Cold-blooded Animals.-The bacilli of this group 
are of interest mainly because of the recent claims of Friedmann that 
immunity against the tubercle bacillus is produced by their injection, 
and the publicity given to his claims that vaccines of these organisms 
have a curative influence in tuberculosis. The results of the use of 
the so-called Friedmann vaccine have, however, been unsatisfactory. 
A similar attempt to employ the cold-blooded types for immunization 
is the use in cattle as advised by Klimmer. 
These types have been isolated from spontaneous, tuberculous-like 
lesions of frogs, lizards, turtles, fishes, etc. They have little resemblance 
to the mammalian types of tubercle bacilli other than their acid-fastness. 
They grow rapidly and luxuriantly on ordinary media and their optimum 
of growth is at 20° to 30° C., higher temperatures inhibiting their growth. 
They are not pathogenic for warm-blooded animals, although toxic 
symptoms or limited lesions may be produced by the injection of large 
doses as with most other non-pathogenic acid-fasts. Tuberculins 
prepared from them are not toxic for tuberculous mammalia except 
in large doses, so that a specific reaction can be excluded. It is possible 
but very improbable that they produce some antibodies which would 
protect against mammalian bacilli to a limited extent. 
Methods of Examination for Tubercle Bacilli and Other Associated 
Bacteria.-One of the most important results of the discovery of the 
tubercle bacillus relates to the practical diagnosis of tuberculosis. 
The staining peculiarities of this bacillus renders it possible by the 
bacteriological examination of microscopic preparations to make an 
almost absolutely positive diagnosis in the majority of cases. A still 
more certain test in doubtful cases is the subcutaneous or intraperi- 
toneal injection of guinea-pigs, which permits of the determination 
of the presence of numbers of bacilli, so small as to escape detection 
by microscopic examination. For the animal test, however, time is 
required-at least three weeks, and, if the bacilli present are very few 
in number, at least six weeks-before any positive conclusion can be 
reached, for when only a few bacilli are present tuberculosis develops 416 
PATHOGENIC MICROORGANISMS 
slowly in animals. In disinfection experiments where many dead bacilli 
are injected, care must be taken to exclude the local effect of dead 
bacilli. In doubtful cases a second guinea-pig should be injected from 
the first. 
Microscopic Examination of Sputum for the Presence of Tubercle 
Bacilli.-1. Collection of Material.-The sputum should be collected 
in a clean bottle (two-ounce) with a wide mouth and a water-tight 
stopper, and the bottle labelled with the name of the patient or with 
some other distinguishing mark. The expectoration discharged in 
the morning is to be preferred, especially in recent cases, and the 
material should be coughed up from the lungs. Care should be taken 
that the contents of the stomach, nasopharyngeal mucus, etc., are 
not discharged during the act of expectoration and collected instead 
of pulmonary sputum. If the expectoration be scanty the entire 
amount discharged in twenty-four hours should be collected. In 
pulmonary tuberculosis the purulent, cheesy, and mucopurulent spu- 
tum usually contains bacilli; while pure mucus, blood, and saliva, as 
a rule, do not. When hemorrhage has occurred, if possible, some 
purulent, cheesy, or mucopurulent sputum should be collected for 
examination. The sputum should not be kept any longer than neces- 
sary before examination, for, though a slight delay or even until putre- 
faction begins, does not vitiate the results so far as the examination 
for tubercle bacilli is concerned, it almost destroys any proper inves- 
tigation of the mixed infection present; it is best, therefore, to ex- 
amine it in as fresh a condition as possible, and it should be kept on ice 
until examined if cultures are to be made. 
2. Methods of Examination.-Examination for Tubercle Bacilli.- 
Pour the specimen into a clean, shallow vessel, having a blackened 
bottom-a Petri dish placed upon a sheet of dull black paper answers 
the purpose-and select from the sputum some of the true expectora- 
tion, containing, if possible, one of the small white or yellowish-white 
cheesy-looking masses or "balls." From this make rather thick cover- 
glass or slide "smears" in the usual way. In doubtful cases a number 
of these coarse or fine particles should be placed on the slide. The 
material being thick should be evenly spread and very thoroughly 
dried in the air before heating. (For methods of staining see p. 389.) 
Occasionally one is able to demonstrate the presence of tubercle 
bacilli with the Hermann stain where Ziehl's carbol fuchsin gives nega- 
tive results. At least two smears should be made and examined, if 
possible, before a negative report is given. Many of the incipient cases 
will require several examinations before bacilli are found. Some will 
remain consistently negative. It must be remembered that lesions may 
exist and that without ulceration the bacilli do not find their way into 
the sputum. 
Methods for Concentrating the Bacilli.-Uhlenhuth advises the use 
of antiformin. This is a patented preparation consisting of a mixture 
of sodium hydroxide and sodium hypochlorite solution. If this is 
mixed with sputum so that the total strength is about 15 per cent. METHODS FOR CONCENTRATING THE BACILLI 
417 
of antiformin, the sputum quickly becomes fluid. This should be 
thinned with water or alcohol to help reduce the specific gravity of 
the mixture and centrifuged. The sediment is then mixed with 
water and recentrifuged, and the washed sediment used for smears. 
Besides the dissolving action, antiformin kills most of the bacteria in 
the sputum, but not the tubercle bacilli, though they are slowly 
affected, so that sediment may be used for cultural purposes or 
injection into guinea-pigs. 
A comparison of the above methods made by us gave the follow- 
ing results: Of twenty-eight sputa negative with carbol-fuchsin, two 
showed bacilli after a few minutes search with the crystal violet stain. 
On restaining with carbol-fuchsin and giving only a light counterstain 
with methylene blue the negative slides were also positive. Of the 
remaining twenty-six, four (15 per cent.) were quickly positive in the 
antiformin sediment when stained with crystal violet, whereas only 
three were positive with carbol-fuchsin and only after restaining as 
above. It is advisable, therefore, in using carbol-fuchsin to have only 
a light counterstain to make the method most efficient, and control 
the results with crystal violet if negative. 
In place of sedimenting the bacilli, the dissolved sputum may be 
shaken up with a hydrocarbon. When the hydrocarbon separates out 
from the sputum the waxy tubercle bacilli adhere to it and are collected 
in a layer between the dissolved sputum and the hydrocarbon. If 
the hydrocarbon is heavy, chloroform, they are carried down, if light, 
ligroin, they are carried up. 
Kinyoun has modified the original ligroin method as follows: We 
have had very satisfactory results with its use. As a routine method 
it saves time and gives a high percentage of positive results. 
Bottles of about 15 c.c. capacity containing about 2 c.c. of a 1 per 
cent, solution of cresol are used for collection. The cresol is added to 
limit decomposition of the sputum if its transit to the laboratory is 
delayed. When received 1 c.c. of ligroin (specific gravity, not less than 
0.715 or more than 0.72) is added and the bottle filled with an alkaline 
solution of hypochloride of lime. If the bottle is full, about one-third 
must be poured out to allow for the addition of the solution. The 
hypochlorite solution is prepared as follows: Three packages of chlori- 
nated lime are weighed, and for each 90 grams, 65 grams of sodium car- 
bonate are taken. The lime is mixed with 500 to 600 c.c. of water and 
the carbonate is dissolved in 1500 c.c. of water by boiling. The carbonate 
is then added to the lime and thoroughly mixed. After standing twelve 
to twenty-four hours the solution is filtered off. The amount of avail- 
able chlorine is estimated and the solution diluted so that the chlorine 
is 5.6 per cent. Then 7.5 grams of caustic soda are added to each 100 
c.c. of the filtrate. The solution should be kept cool and in the dark. 
Fresh lots should be prepared about every three months. 
After the addition of the ligroin and the solution of chlorine the 
bottles of sputum are placed in a shaking machine and thoroughly shaken 
for five to ten minutes. The bottles can then be allowed to stand until 
27 418 
PATHOGENIC MICROORGANISMS 
the ligroin rises, which takes several hours, or this can be hastened, 
placing the bottles in a centrifuge, with special cups to accommodate 
the bottles, and run for about ten minutes at moderate speed. 
When the ligroin rises to the top a soapy layer develops at the 
point of contact with the fluidified sputum and the tubercle bacilli are 
collected in this layer. The soapy layer is taken up with a platinum 
loop and smears made on glass slides fixed by heat and stained. Indi- 
vidual slides must be used and the slides must be stained separately 
or error will result, as the bacilli are not firmly fixed to the slides. 
For this reason, staining in the cold is more convenient, and the fol- 
lowing formulae is especially suited for this purpose: 
Carbolic acid (crystals) 8 grams. 
Alcohol, 95 per cent . 20 c.c. 
Water 100 c.c. 
The staining requires three minutes. The slides are then decolorized 
with acid alcohol and counterstained in the usual way. A counterstain 
is desirable to aid in focussing on the small amount of detritus present. 
Care must be exercised so that the bacilli are not carried from one slide 
to another. 
All the antiformin methods must be used with caution, as it is easy 
to see how error can creep in from contamination with other acid-fast 
bacilli. 
Detection of Tubercle Bacilli in Urine and Feces, Etc.-The cath- 
eterized urine is centrifuged. If little sediment appears, the upper 
portion of the fluid is removed and more urine added and again 
centrifuged. If the urine is rich in salts of uric acid, the same may 
be diminished by carefully warming the urine before treating it. If 
too alkaline add a little acetic acid. A possible source of error is the 
presence of smegma bacilli. 
The feces are examined for any purulent or mucous particles. If 
none are found, larger masses of feces are removed and then the rest 
diluted and centrifugalized. The antiformin methods are a great aid 
in the examination of feces. The examiner must remember that 
bacilli swallowed with the sputum may appear in the feces. In examin- 
ing cerebrospinal fluid for tubercle bacilli it must be remembered 
that the majority of the bacilli are entangled in the delicate clot that 
forms. Wherever possible the fluid after withdrawal should be allowed 
to stand until this filmy clot develops, which is then fished out and 
examined. If this is impossible the fluid should be centrifuged and the 
sediment stained. This is also the case in other serous fluids, but in 
ascitic or pleuritic fluid they are usually very few in number. 
Examination for Other Bacteria (Mixed Infection).-With regard 
to the bacteriological diagnosis of pulmonary phthisis, many consider 
that it is not enough to show only the presence of tubercle bacilli; 
it is held to be of importance, both for purposes of prognosis and 
treatment, that the presence of other microorganisms which may be 
associated with the tubercle bacillus should also be determined. It CULTIVATION 
419 
is now usual to distinguish pure tuberculosis of the lungs from a 
mixed infection. Phthisis due to the tubercle bacillus alone, which 
constitutes but a small percentage of all cases, may occur almost 
without febrile reaction; or when fever occurs the prognosis is un- 
favorable, thus indicating that the disease is already advanced. It 
is in the uncomplicated forms of phthisis, moreover, where one must 
expect if anywhere the best results from treatment. The majority 
of cases, however, of pulmonary tuberculosis show a mixed infection, 
especially with varieties of the streptococcus, pneumococcus, or in- 
fluenza bacilli. These cases may be active, with fever, or passive, 
without fever, according, perhaps, as the parenchyma of the lung is 
invaded by the bacteria; or they are only superficially located in cavities, 
bronchi, etc. Mixed infection with the staphylococcus, other micrococci, 
and with the influenza bacilli have also been frequently met with by us. 
The tetragenus has not been often detected by us in thorougly washed 
fresh sputum. At present the facts seem to prove that the tubercle 
bacilli have in the great majority of cases, at least shortly before death, 
a much more important role than the associated bacteria. 
Sputum Washing.-Some of the associated bacteria found in the expectora- 
tion come from the diseased areas of the lungs,, while others are merely added 
to the sputa as it passes through the mouth or are developed after gathering. 
To endeavor to separate the one from the other we wash the sputa. The first 
essential is that the material is to be washed within a few minutes, and certainly 
within an hour after being expectorated. If a longer time is allowed to inter- 
vene, the bacteria from the mouth will penetrate into the interior of the mucus, 
and thus appear as if they came from the lungs. Sputum treated twenty-four 
hours after its expectoration is useless for examining for anything except the 
tubercle bacillus. A rough method is to pour some of the specimen of sputum 
to be examined into a convenient receptacle containing sterile water, and with- 
draw, by means of a sterilized platinum wire, one of the cheesy masses or thick 
"balls" of mucus. Pass this loop five times through sterile water in a dish; 
repeat the operation in fresh water in a second and third dish. Spread what 
remains of the mass on cover-glasses and make smear preparation; stain and 
examine. With another mass inoculate ascitic bouillon in tubes and agar in 
plates. 
When we wish thoroughly to exclude mouth bacteria, a lump of the sputum 
raised by a natural cough is seized by the forceps and transferred to a bottle of 
sterile water and thoroughly shaken; it is then removed to a second bottle of 
bouillon and again thorougly shaken. From this it is passed in the same way 
through four other bottles of bouillon. A portion of the mass is now smeared 
over cover-glasses, and the rest inoculated in suitable media, such as agar in 
Petri dishes, and ascitic fluid bouillon in tubes. If desired, the bacteria washed 
off in the different washings are allowed to develop. 
Inoculation of Animals.-The inoculation of suspected material into 
guinea-pigs produces tuberculosis; even if the number of bacilli is very 
small. When no bacilli can be detected by microscopic examination 
this can be done for diagnostic purposes. The material should be 
injected subcutaneously as already described. 
Cultivation.-This requires so much time that it is not used except 
in important investigations upon the nature of the tubercle bacilli. 
The special methods have already been given. CHAPTER XXIX. 
OTHER ACID-FAST BACILLI: BACILLUS OF LEPROSY, 
BACILLUS OF RAT LEPROSY, BACILLUS OF JOHNE'S 
DISEASE IN CATTLE, AND THE GROUP OF NON- 
PATHOGENIC ACID-FAST BACILLI. 
LEPROSY BACILLUS-B. LEPRAE. 
The bacillus of leprosy was discovered by Hansen and Neisser 
(1879) in the leprous tubercles of persons afflicted with the disease. 
This discovery was confirmed by many subsequent observers. 
Morphology (in Tissues).-Small, slender rods resembling the tubercle 
bacilli in form, but somewhat shorter and not so frequently curved. 
The rods have pointed ends, and in stained preparations unstained 
spaces, similar to those observed in the tubercle bacillus, are seen. 
They stain readily with the aniline colors and also by Gram's method. 
Although differing slightly from the tubercle bacillus in the ease with 
which they take up the ordinary aniline dyes, they behave like tubercle 
bacilli in retaining their color when subsequently treated with strong 
solutions of the mineral acids and alcohol. The slight difference in 
staining characteristics is too little to be relied upon for diagnostic 
purposes (see Plate VI). 
Bacilli Isolated from Leprous Lesions.-Clegg reported in 1908 that 
he had been able to cultivate an acid-fast bacillus by growing it in 
symbiosis with ameba and cholera. Since then Duval, Kedrowski, 
Twort, and many others have reported their results. Various organisms 
have been isolated which may be grouped as follows:1 
Bacilli of the Diphtheroid Type.-In serum media the colonies are 
yellowish white and develop best at 37° C., although a slight growth 
occurs at room temperature. When growth occurs on broth, the medium 
remains clear, and a pellicle is produced. The morphology is variable. 
They are either solidly staining or irregularly stained like other types 
of diphtheroids. They are gram-positive and may show some resist- 
ance to decolorization after staining with carbol-fuchsin, especially the 
metachromatic granules. 
Pathogenicity none or questionable. 
Acid-fast Chromogenic Bacilli.-This type of bacillus is difficult 
to isolate but after isolation grows freely at both 37° and 20° C. on 
most of the ordinary media. The growth is luxuriant, moist, and a 
1 The results of all the investigations cannot be given. For a fuller discussion and 
bibliography see the excellent resume of Wolbach and Honeij, Journ. Med. Research, 
1914, xxix, 367. LEPROSY BACILLUS 
421 
yellow to deep orange color develops. They vary in morphology from 
coccoid to filamentous bacilli, some showing metachromatic granules, 
others showing clear areas. They are acid-fast but less so than the 
tubercle bacillus. 
Pathogenicity- Lesions similar to leprosy produced in Japanese 
dancing mice and in monkeys. 
Anaerobic Bacilli.-In this place it is sufficient to state that such 
organisms have been isolated. 
Acid-fast Non-chromogenic Bacilli.-These types are characterized 
by their feeble, slow growth on artificial media, and growth only takes 
place at 37° C., and then only on special media. 
Morphologically they vary from plump to long, slender bacilli, often 
beaded or bipolar in appearance. 
Pathogenicity, none. 
Fig. 147 
Leprosy bacilli in nodule. (Kolle and Wassermann.) 
What conclusion is to be drawn from such variable results is difficult 
to say. Has the diphtheriod bacillus any relationship to the acid-fast 
types? This is a possibility when one considers that under certain cir- 
cumstances it shows some resistance to decolorization. Then, too, the 
leprosy bacilli in tissue may decolorize easily although they are abundant, 
as shown by staining with polychrome methylene blue. On the other 
hand, the repeated isolation of diphtheroids from the lymph nodes in 
Hodgkin's disease and even from the lymph nodes in other conditions 
raises a strong element of doubt as to the etiologic significance of 
this organism. A careful comparison of the diphtheroids from various 
conditions, especially their resistance to decolorization, might give us 
some help. 
As to the chromogenic acid-fasts, the character of their growth, viz., 
the luxuriant growth upon ordinary media at low temperature is not 
that of a highly specialized parasite. As to the apparently successful 
animal inoculations it must be remembered that lesions have been 
produced by acid-fast bacilli, known not to be pathogenic. Why such 422 
PATHOGENIC MICROORGANISMS 
bacilli should be frequently insolated from leprous lesions is still to be 
explained. 
The non-pigmented types are more consistent with our idea of what 
the bacillus should be like, but whether it is actually the etiologic 
organism remains to be seen. 
The serum reactions, such as agglutination and complement-fixation, 
have added no evidence as to the etiologic significance of any one 
of the bacilli isolated. Each of the bacilli mentioned have been agglu- 
tinated by sera of lepers. The complement-fixation reaction awaits 
a successful specific test for the individual acid-fast organisms. 
Pathogenesis-Numerous inoculation experiments have been made 
on animals with portions of leprous tubercles, but there is no con- 
clusive evidence that leprosy can be transmitted to the lower animals 
by inoculation. The inference that this bacillus bears an etiologic 
relation to the disease with which it is associated is based chiefly upon 
the demonstration of its constant presence in leprous tissues (Fig. 147). 
The bacilli are found in all the diseased parts, and usually in large 
numbers, especially in tubercles on the skin, in the conjunctiva and 
cornea, the mucous membranes of the mouth, gums, and larynx, 
and in the interstitial processes of the nerves, testicles, spleen, liver, 
and kidneys. The rods lie almost exclusively within the peculiar 
round or oval cells of the granulation tissue which composes the lep- 
rous tubercles, either irregularly scattered or arranged parallel to 
one another. In old centres of infection the leprosy cells containing 
the bacilli are larger and often polynuclear. Giant cells, such as are 
found in tuberculosis, are claimed to have been observed by a few 
investigators (Boinet and Borrel). In the interior of the skin tuber- 
cles, the hair follicles, sebaceous and sweat-glands are often attacked, 
and bacilli have sometimes been found in these (Unna, etc.). Quite 
young eruptions often contain a few bacilli. A true caseation of the 
tubercles does not occur, but ulceration results. During acute exacer- 
bations with development of new lesions bacilli have been observed in 
the blood. 
In the anesthetic forms of leprosy the bacilli are found most com- 
monly in the nerves and less frequently in the skin. They have been 
demonstrated in the sympathetic nervous system, in the spinal cord, 
and in the brain. The Bacillus leprce occurs also in the blood, partly 
free and partly within the leukocytes, especially during the febrile 
stage which precedes the breaking out of fresh tubercles (Walters 
and Doutrelepont). The bacilli have also been found in the intestines, 
in the lungs, and in the sputum, but not in the urine. 
With regard to the question of the direct inheritance of the disease 
from the mother to the unborn -child there is considerable difference 
of opinion. Some cases have been reported, however, in which a 
direct transmission of the bacillus during intra-uterine life seems to 
be the only or most plausible explanation of the infection. At the 
same time, we have no positive experimental evidence to prove that 
such an infection does take place. Although many attempts have LEPROSY BACILLUS 
423 
been made to infect healthy individuals with material containing the 
bacilli of leprosy, the results are not conclusive. Even the experi- 
ments made by Arning, who successfully infected a condemned crim- 
inal in the Sandwich Islands with fresh leprous tubercles, and which 
have been regarded as positive evidence of the transmissibility of the 
disease in this way, are by no means conclusive; for, according, to 
Swift, the man had other opportunities for becoming infected. These 
negative results, together with the fact that infection does not more 
frequently occur in persons exposed to the disease, may possibly be 
explained by the assumption that the bacilli contained in the 
tuberculous tissue are mostly dead, or much more probably that 
an individual susceptibility to the disease is requisite for its pro- 
ductions. 
The widespread idea, before the discovery of the leprosy bacillus, 
that the disease was associated with the constant eating of dried fish or 
a certain kind of food, has now been entirely abandoned. 
The relation of leprosy to tuberculosis is sufficiently evident from 
their great similarity in many respects. This is rendered still more 
remarkable by the fact that leprosy reacts, both locally and generally, 
to an injection of tuberculin in the same manner as tuberculosis, but to 
a somewhat less extent. 
Rat Leprosy.-The interest in this disease lies in the fact that diph- 
theroid and a chromogenic acid-fast bacilli similar to those described 
above, have been isolated from leprous rats. 
Bacillus of Johne's Disease, Chronic Enteritis or Paratubercular 
Dysentery of Cattle.-This disease is comparatively common in this 
country and is characterized by chronic diarrhea and emaciation, 
commonly leading to death. The intestinal mucosa is thickened, and 
the lesions are not limited. Tubercle formation and necrosis are absent. 
The bacilli are present in the lesions in enormous numbers. Twort 
succeeded in cultivating the organism and his work was verified by 
Holth and Meyer. A tuberculin made from this organism will probably 
be of diagnostic value. Animals suffering with disease do not react 
to the ordinary tuberculin test but do react to large doses of tuberculin 
made from the avian type of tubercle bacillus. The bacillus is not 
pathogenic for guinea-pigs and rabbits, although local abscesses may 
be produced. 
Non-pathogenic Acid-fast Bacilli. - These have no importance 
further than historical interest and the fact that they may be present 
in materials suspected of containing tubercle bacilli and thus lead to 
error. They vary widely in their acid-fastness, especially when artifi- 
cially cultivated. Differential staining methods have been divised to 
separate them from the tubercle bacillus and although in a general way 
the decolorization by prolonged action of acid and alcohol is presumptive 
evidence against suspected bacilli being tubercle bacilli, it is an unsafe 
procedure. Tubercle bacilli vary in their acid-fastness but the non- 
pathogenic types vary even more widely, some being extremely resist- 
ant to decolorization. Many of the non-pathogenic types grow rapidly 424 
PATHOGENIC MICROORGANISMS 
at low temperatures and in cultures can thus be quickly differentiated 
from tubercle bacilli. 
They can be separated from tubercle bacilli by inoculating ani- 
mals in which no progressive lesions will develop, although limited 
lesions may be produced if they are injected in large numbers. The 
guinea-pig may be injected with 2 c.c. of tuberculin and if infected with 
tuberculosis will die, but if by other acid-fast bacilli, will show little or 
no reaction. If a second group of guinea-pigs are inoculated with a 
small amount of the infected tissue from the inoculated pigs there will 
develop progressive tuberculosis, if the doubtful bacilli were tubercle 
bacilli, and practically no lesions if they were grass bacilli. Cultures 
from the lesions may also be an aid in differentiation. 
Bacillus of Lustgarten.-This bacillus was found by Lustgarten in 
1884 in syphilitic lesions or ulcers. It is undoubtedly a saprophyte. 
It is very similar morphologically to the smegma bacillus and may be 
identical with it. It is of historical interest only. 
Smegma Bacillus.-This bacillus is present in smegma from the prepuce 
or vulva. Its only interest is the danger of mistaking it for tubercle 
bacilli in the examination of urine, especially if the latter be carelessly 
collected. 
Timothy and Other Grass Bacillus.-On various grass, in cow manure, 
in butter and in milk there have been found bacilli with varying degrees 
of acid-fastness. Similar bacilli have also been demonstrated in water. 
They make the direct microscopic examination of such material for 
tubercle bacilli of little value and the nature of any acid-fast organisms 
so found must be determined by animal inoculations. CHAPTER XXX. 
GLANDERS BACILLUS (BACILLUS MALLEI). 
Tins bacillus was discovered and proved to be the cause of glanders, 
by isolation in pure culture and inoculation into animals, by several 
bacteriologists almost at the same time (1882). The bacilli were first 
obtained in impure cultures by Bouchard, Capitan, and Charrin, and 
first accurately studied in pure culture by Loftier and Schutz. They 
are present in the recent nodules in animals affected with glanders, 
and in the discharge from the nostrils, pus from the specific ulcers, 
etc., and occasionally in the blood. 
Morphology.-Small bacilli with rounded or pointed ends, from 
nutrient agar cultures, 0.25m to 0.5m broad and from 1.5m to 5m long; 
usually single, but sometimes 
united in pairs, or growing out 
to long filaments, especially in 
potato cultures. The bacilli 
frequently break up into short 
almost coccus-like elements (Fig. 
148). 
Staining.-The bacillus mallei 
stains with difficulty with the ani- 
line colors, best when the aqueous 
solutions of these dyes are made 
feebly alkaline; it is decolorized 
by Gram's method. This bacillus 
presents the peculiarity of losing 
very quickly in decolorizing solu- 
tions the color imparted to it by 
the aniline staining solution. For 
this reason it is difficult to stain 
in sections. Loffler recommends his alkaline methylene-blue solution 
for staining sections, and for decolorizing, a mixture containing 10 c.c. 
of distilled water, 2 drops of strong sulphuric acid, and 1 drop of a 5 
per cent, solution of oxalic acid; thin sections to be left in this acid 
solution for five seconds. 
Biology.-An aerobic, non-motile bacillus, whose molecular move- 
ments are so active that they have often been taken for motility. It 
grows on various culture media at 37° C. Development takes place 
slowly at 22° C. and ceases at 43° C. The bacillus does not form 
spores. Exposure for ten minutes to a temperature of 55° C., or for 
five minutes to a 3 to 5 per cent, solution of carbolic acid, or for two 
Fig. 148 
Glanders bacilli. Agar culture. 
X 1100 diameters. 426 
PATHOGENIC MICROORGANISMS 
minutes to a 1 :5000 solution of mercuric chloride, destroys its vitality. 
As a rule, the bacilli do not grow after having been preserved in a desic- 
cated condition for a week or two; in distilled water they may live 25 
days. It is doubtful whether the glanders bacillus finds conditions in 
nature favorable to a saprophytic existence. 
A solution of chlorinated lime, containing 1 part of free chlorine per 
1000, is useful as a disinfectant of stables and utensils; it kills the bacillus 
in from one to two minutes. 
Cultivation.-(For obtaining pure cultures see page 428.)-It grows 
well in the incubating oven on glycerin agar. Upon this medium, 
at the end of twenty-four to forty-eight hours, whitish, transparent 
colonies are developed, which in six or seven days may attain a diameter 
of 7 or 8 mm. On blood-serum a moist, opaque, slimy layer develops, 
which is of a yellowish-brown tinge. The growth on cooked potato 
is especially characteristic. At the end of twenty-four to thirty-six 
hours at 37° C. a moist, yellow, transparent layer develops; this later 
becomes deeper in color, and finally takes on a reddish-brown color, 
while the potato about it acquires a greenish-yellow tint. In bouillon 
the bacillus causes diffuse clouding, ultimately with the formation of 
a more or less ropy, tenacious sediment. It grows on media about 1.5 
per cent, acid to phenolphthalein, and both with and without oxygen. 
Milk is coagulated with the production of acid. 
Pathogenicity.-The bacillus of glanders is pathogenic for a num- 
ber of animals. Among those which are most susceptible are horses, 
asses, guinea-pigs, cats, dogs, ferrets, moles, and field mice; sheep, 
goats, swine, rabbits, white mice, and house mice are much less sus- 
ceptible; cattle are immune. Man is susceptible, and infection not 
infrequently terminates fatally. 
When pure cultures of Bacillus mallei are injected into horses or 
other susceptible animals true glanders is produced, The disease is 
characterized in the horse by the formation of ulcers upon the nasal 
mucous membrane, which have irregular, thickened margins, and 
secrete a thin, virulent mucus; the submaxillary lymphatic glands 
become enlarged and form a tumor which is often lobulated; other 
lymphatic glands become inflamed, and some of them suppurate and 
open externally, leaving deep, open ulcers; the lungs are also involved, 
and the breathing becomes rapid and irregular. In farcy, which is a 
more chronic form of the disease, circumscribed swellings, varying in 
size from a pea to a hazel-nut, appear on different parts of the body, 
especially where the skin is thinnest; these suppurate and leave angry- 
looking ulcers with ragged edges, from which there is an abundant 
purulent discharge. The bacillus of glanders can easily be obtained 
in pure cultures from the interior of suppurating nodules and glands 
which have not yet opened to the surface, and the same material will 
give successful results when inoculated into susceptible animals. The 
discharge from the nostrils or from an open ulcer may . contain com- 
paratively few bacilli, and these being associated with other bacteria 
which grow more readily on the culture media than the Bacillus mallei, GLANDERS BACILLUS 
427 
make it difficult to obtain pure cultures from such material by the 
plate method. In that case, however, guinea-pig inoculations are 
useful. 
Of test animals guinea-pigs and field mice are the most susceptible. 
In guinea-pigs subcutaneous injections are followed in four or five 
days by swelling at the point of inoculation, and a tumor with case- 
ous contents soon develops; then ulceration of the skin takes place, 
and a chronic purulent ulcer is formed. The essential lesion is the 
granulomatous tumor, characterized by the presence of numerous 
lymphoid and epithelioid cells, among and in which are seen the 
glanders bacilli. The lymphatic glands become inflamed and gen- 
eral symptoms of infection are developed in from two to four weeks; 
the glands suppurate, and in males the testicles are involved; finally 
purulent inflammation of the joints occur, and death ensues from 
exhaustion. The formation of the specific ulcers upon the nasal 
mucous membrane, which characterizes the disease in the horse, is 
rarely seen when guinea-pigs are inoculated. In these-the process 
is often prolonged or remains localized on the skin. They succumb 
more rapidly to intraperitoneal injection, usually in from eight to ten 
days, and in males the testicles are invariably affected (see p. 428). 
Mode of Spread.-Glanders occurs as a natural infection only in 
horses and asses; the disease is occasionally communicated to man by 
contact with affected animals, usually by inoculation on an abraded sur- 
face of the skin. The contagion may also be received on the mucous 
membrane. Infection has sometimes been produced in bacteriologic 
laboratories. In man, as in horses, an acute and chronic form of 
glanders may usually be recognized. The disease in human beings is 
fatal in about 60 per cent, of the cases. It is transmissible also from 
man to man. Washerwomen have been infected from the clothes of 
a patient. The infective material exists in the secretions of the nose, 
in the pus of glanders nodules, and frequently in the blood; it may 
occasionally be found in the secretions of glands not yet affected, as in 
the urine, milk, and saliva, and also in the fetus of diseased animals 
(Bonome). From recent observations it appears that glanders is by 
no means an uncommon disease among horses, sometimes taking a mild 
course and remaining latent for a considerable time. Horses apparently 
healthy, therefore, may possibly spread the disease, through the public 
drinking troughs and blacksmith shops. 
Attenuation of virulence occurs in cultures which have been kept 
for some time, and inoculations with such cultures may give a nega- 
tive result, or, when considerable quantities are injected, may pro- 
duce a fatal result at a later date than is usual when small amounts 
of a recent culture are injected. 
Immunity.-Attempts have been made to produce artificial immu- 
nity against glanders, but so far with unsatisfactory results. Ac- 
cording to Strauss, by intravenous inoculations of small quantities 
of living bacilli, dogs may be protected against an injection of quanti- 
ties which usually kill them. Fenger has found that animals inoculated 428 
PATHOGENIC MICROORGANISMS 
with glanders bacilli react less powerfully to fresh injections; and that 
rabbits which have recovered from an injection of glanders are sub- 
sequently immune, the immunity lasting for from three to six weeks. 
Ladowski has obtained positive results also in rabbits and cats by in- 
travenous injections of sterilized cultures. Other observers have 
reported not only the production of immunity, but also cures by the 
use of mallein. 
Vaccine.-The therapeutic and preventive value of the vaccine 
has yet to be demonstrated, and, since its indiscriminate use causes 
confusion in the blood-tests, it is not now being distributed by the 
Health Department of New York. 
Use of Guinea-pigs for Cultures and Diagnosis.-It is often diffi- 
cult to demonstrate microscopically the presence of the bacillus of 
glanders in the nodules which have undergone purulent degenera- 
tion, in the secretions from the nostrils, or in the pus from the specific 
ulcers and suppurating glands. It is then necessary to make im- 
mediate cultures and also animal tests of these discharges by inocu- 
lating susceptible animals, as guinea-pigs and mice, and then from 
those to obtain a pure culture; but this requires time, and in clinical 
work it is of great importance for the diagnosis to be established as 
quickly as possible. With this view Strauss has prepared a method 
which is prompt and which has given satisfactory results. This 
consists in introducing into the peritoneal cavity of a male guinea- 
pig some material or a culture from the suspected products. If it be 
a case of glanders, the diagnosis may usually be made within two to 
five days from the tumefaction of the testicles, which become swollen, 
and show, evidences of pus formation. An objection to this method, 
however, is that occasionally from the injection of impure material, 
as in the nasal secretion, the animal may die of septicemia; but if pure 
matter can be obtained, as from the lymphatic glands of the horse, 
this method is satisfactory according to most authors. Nevertheless, 
while a positive result is conclusive evidence of glanders, failure of the 
pig to develop lesions is not proof of its absence, for other workers find 
such inoculations fail in about one-half the animals injected, even after 
a prolonged period. 
Preparation of Mallein.-This preparation is like that of tuberculin. 
It consists of the glycerinated bouillon in which the glanders bacilli 
have grown and contains the products of their growth and activity.1 
Mallein for subcutaneous injection is produced by growing the glanders 
bacillus for six weeks in a 5 per cent, glycerin nutrient veal bouillon, 
about 1.5 per cent, acid to phenolphthalein. The culture is then steamed 
in the Arnold sterilizer for one hour. It is then filtered through paper- 
pulp and carbolic acid added to give 0.5 per cent. Sterilized in Arnold 
sterilizer two successive days. The dose for diagnostic purposes in the 
horse is 2.5 c.c. 
For eye mallein an acid broth made of equal parts of potato juice and 
veal broth, and containing 4.5 per cent, glycerin, is used. After growing 
1 The methods given are those in use in the New York City Health Department. GLANDERS BACILLUS 
429 
the glanders bacilli in this for six weeks, the culture is killed (as above), 
and filtered through paper-pulp. The liquid is evaporated to one-tenth 
its bulk and the supernatant fluid poured off, centrifuged if not clear, 
and used (see below). 
Diagnostic Use of Mallein.-The diagnosis of glanders in horses 
in which the usual symptoms of the disease have not yet manifested 
themselves, or in which it is suspected, may often be made by the 
use of mallein. Following a subcutaneous injection of mallein in a 
glanderous horse (best made about midnight) there will be a local 
reaction, and a general reaction with a rise of temperature. The tem- 
perature usually begins to rise three or four hours after the injection, 
and reaches its maximum between the tenth and twelfth hour. Some- 
times, however, the highest point is not reached until fifteen or eighteen 
hours after the injection. This elevation of temperature is from 1.5° 
to 2° C. (2° to 3.5° F.), above the normal mean temperature, which 
should be taken twice, at intervals three hours apart, before the injec- 
tion of the mallein. If there is fever, mallein should not be given. In 
a healthy animal the rise of temperature, as a rule, amounts to only a 
few tenths of a degree, but it may reach 1° C. The rise of tempera- 
ture, however, should be considered always in connection with the 
general and local reactions. In a glanderous animal, after an injection 
of mallein, the general condition is more or less profoundly modified. 
In healthy animals, on the contrary, the general symptoms do not 
occur. The local reaction around the point of injection in a glanderous 
animal is usually very marked. A few hours after the injection there 
appears a large, warm, tense, and very painful swelling, and running from 
this will be found hot, sensitive lines of sinuous lymphatics, directed 
toward the neighboring lymphatic nodes. This edema increases for 
twenty-four to thirty-six hours and persists for several days, not dis- 
appearing entirely for eight or ten days. In healthy animals, at the 
point of injection, mallein produces only a small edematous tumor, 
and the edema, instead of increasing, diminishes rapidly and disappears 
within twenty-four hours. The value of this test has been demon- 
strated by numerous experiments. There are some exceptions to the 
rule as described above, but they are infrequent, and mallein is useful 
as a diagnostic aid in detecting the existence or absence of glanders 
in doubtful or obscure cases. Since injected mallein interferes with the 
serodiagnostic tests, these should be made before its injection. 
Ophthalmo-reaction.-When 2 to 3 drops of concentrated mallein 
are instilled into the conjunctival sac, no reaction save a slight lacrima- 
tion and congestion results in healthy horses. In glanderous animals 
this goes on, at the end of from five to seven hours, to profuse lacrima- 
tion, redness, edema, and the formation of pus. There may be only 
a drop at the inner canthus of the eye, or all degrees to profuse purulent 
discharge. Unless pus is present, the reaction is not considered positive. 
There is a slight rise in temperature in those cases showing a good 
reaction, but as the local reaction is very distinct, the tedious task of 
taking temperatures is superfluous. Another advantage of this method 430 
PATHOGENIC MICROORGANISMS 
over the subcutaneous inoculation, aside from its simplicity, is the 
fact that it may be repeated after twenty-four hours in doubtful cases, 
and does not interfere with subsequent serodiagnostic tests. With very 
few exceptions, a second test in a glanderous horse gives a prompt 
reaction. 
Collection of the Blood for the Serodiagnostic Tests.-In obtaining blood from 
horses a large-sized hypodermic needle, which has been sterilized, is inserted into 
the jugular vein which has been brought into view by pressing the thumb upon it 
from below; the blood is allowed to flow through the needle into a sterile tube or 
flask, 8 to 10 c.c. being sufficient. In the case of human beings it is obtained by 
drawing 5 c.c. of blood with a sterile hypodermic syringe from the median 
basilic vein at the bend of the elbow. 
The macroscopic agglutination test may be carried out either with the 
incubator or the centrifuge method. 
Incubator Method.-The procedure of Schutz and Meissner, with slight modi- 
fications, is as follows: A forty-eight-hour acid-glycerin-agar culture of B. mallei 
is washed off with normal saline solution containing 0.5 per cent, pure carbolic 
acid. This suspension is heated at 60° C. for two hours. It is then filtered 
through cotton, and enough of the carbolic salt solution added to reduce it to a 
faintly cloudy suspension. It should be standardized by comparing it with a 
known test fluid, if possible, and testing it with known negative and positive 
sera. This test fluid will keep in the ice-box for several weeks. 
The serum to be tested is then made up with the normal carbolic salt solution 
to a 1 to 40 dilution. From this the final dilutions of 1 to 500, 1 to 800, 1 to 1000, 
etc., are made by adding 0.24 c.c., 0.15 c.c., and 0.12 c.c. respectively to 3 c.c. 
of the standardized test fluid in each test-tube. The tubes are well shaken and 
incubated with positive and negative control sera in the same dilutions for forty- 
eight to seventy-two hours. If a reaction occurs the upper part of the fluid is 
clear, while a veil-like sediment is found at the bottom. A strong positive reac- 
tion (1 to 1000) may occur in twenty-four hours. A negative reaction shows the 
sediment in a definite "button" at the bottom of the tube and the fluid above is 
cloudy. 
Schiitz and Meissner claim that a culture recently passed through 
a guinea-pig is essential for a good test fluid. In our hands, cultures 
kept in artificial media do well, but should be passed through a guinea- 
pig about once in two months. A univalent test fluid is most logical 
for agglutination work, for in a polyvalent fluid a strain not agglutinated 
by a particular serum would cloud the supernatant fluid at the same 
time that a reaction occurred in the bottom of the tube. 
With the centrifuge method1 the time factor is greatly reduced. The tests are 
incubated for a half-hour at 37° C., then cehtrifugalized at 1600 revolutions for 
ten minutes, and kept at room temperature for two hours before reading. This 
is done by looking down on the tubes from above toward a dark background. 
Indefinite reactions may be read the next day after standing at room temperature. 
Special tubes with perfectly rounded bottoms are essential for this method, and 
the dilutions are made up in only 2 c.c. of the test fluid. 
1 Mohler and Eichhorn, Bureau of Animal Industry, Circular No. 191. GLANDERS BACILLUS 
431 
The limit of agglutination of the normal horse is 1 to 500; since, 
however, some cases of chronic glanders do not react above this, it 
should be regarded with suspicion and checked by the use of eye mallein 
and the complement-fixation test. So, also, with horses reacting in 
1 to 800. A 1 to 1000 reaction may be considered positive; yet no one 
laboratory test should be relied upon wholly1 where clinical symptoms 
are absent. In human cases a reaction of 1 to 100 and above is con- 
sidered positive, the normal blood not reacting above 1 to 50. In very 
acute cases that run their course in a few days the agglutination reaction 
may be entirely absent. 
The complement-fixation test has given satisfactory results both here 
and abroad, and has replaced the agglutination test as required by the 
Government. Here a polyvalent antigen is preferable. (For preparation 
and test see p. 190.) Like the agglutination and mallein tests, however, 
the complement-fixation fails in a certain number of cases, especially 
early in the disease. It is advisable to check its results with the oph- 
th almo-reaction. 
Appearance of the Reaction of the Various Tests after Infection.2- 
Agglutinins appear from four to five days and continue to increase in the 
early stages of the disease and diminish as the disease becomes chronic. 
Specific amboceptors for the complement-fixation test may be demon- 
strated in from seven to ten days and remain during the entire course 
of the disease. Subcutaneous mallein test may, as a rule, be relied 
upon fifteen days after injection. Conjunctival mallein test may be 
relied upon three weeks after infection. 
Effect of One Test on the Others.3-All blood-serum tests are 
influenced in three to six days after a subcutaneous injection of mallein 
or any glanders antigen, including glanders vaccines, for a period varying 
from six to eight weeks following injection of mallein and up to three 
months and even longer following injection of glanders antigen or 
vaccines. All blood-samples, therefore, should be taken prior to or 
at the time of such injections. The subcutaneous mallein test or injec- 
tion of glanders antigen and vaccines may influence the conjunctival 
mallein test. The conjunctival mallein test should, therefore, be with- 
held for thirty days after application of the subcutaneous mallein. 
1 Anthony and Grund, Collected Studies, Bureau of Laboratories, Department of 
Health, New York, vol. vii. 
2 Report on the Detection of Glanders, Fiftieth Meeting American Veterinary Medical 
Association, 1913. 
' Ibid. CHAPTER XXXI. 
THE GROUP OF HEMOGLOB INOPHILIC BACILLI. 
BORDET-GENGOU BACILLUS. 
THE INFLUENZA BACILLUS. 
A disease called influenza can be traced back to the fifteenth 
century and probably existed at a much earlier date. 
At times but few endemic cases occur, and then a great epidemic 
spreads over the civilized world. The last great epidemic reached 
Russia from the East in the fall of 1889 and gradually spread over 
Europe and to America, reaching the latter country in December of 
that year. Since then we have had more or less of it, especially dur- 
ing the winter months. Many acute inflammations of the respiratory 
mucous membranes, due to pneumococci and streptococci, give symp- 
toms similar to those due to the influenza bacillus. 
The rapidity of the spread 
of epidemics of influenza sug- 
gested that persons were the 
carriers of the infection, while 
the location of the disease 
pointed to the respiratory tract 
as the location of, and to the ex- 
pectoration as the chief source of 
infection by, the microorganisms. 
After numerous unsuccessful 
attempts, during the epidemic of 
1889 and succeeding years, to 
discover the specific cause of 
influenza, Pfeiffer (1892) suc- 
ceeded in isolating and growing 
upon blood-agar a bacillus which 
abounded in the purulent bron- 
chial secretion of patients suffer- 
ing from epidemic influenza, which 
he showed was the probable cause of the disease. Canon, working at 
the same time, found a similar bacillus in the blood of several cases 
of the disease. Though 77. influenzae has been shown to have definite 
pathogenic powers, its specificity in epidemic influenza has not been 
fully proved. 
Morphology. Very small, moderately thick bacilli (0.2/z to 0.3/z) 
in thickness to 0.5^ to 2/x in length), usually occurring singly or united 
Fig. 149 
Influenza bacilli. X 1100 diameters. THE INFLUENZA BACILLUS 
433 
in pairs, but show threads are occasionally found in spreads from the 
sputum and young cultures. In later cultures threads may be produced 
in great abundance. No capsule has been demonstrated. 
Staining.-The bacillus stains rather faintly with the ordinary aniline 
colors-best with dilute Ziehl's solution (water 9 parts to Ziehl's solu- 
tion 1 part), or Loffler's methylene-blue solution, with heat. Giemsa's 
method stains them* well and brings out the polar granules which some- 
times develop in these bacilli. They are not stained by Gram's method. 
Biology.-An aerobic, facultative anaerobic (contrary to the accepted 
opinion), non-motile bacillus; does not form spores; no growth occurs 
with most cultures below 22° C., or above 41° C. 
Cultivation.-This bacillus is best cultivated at 37° C., and on ordinary 
nutrient culture media containing hemoglobin (p. 101). At the end 
of eighteen hours in the incubator very small circular colonies are 
developed, which, under a low magnification (100 diameters), appear 
as shining, transparent, homogeneous masses, and even under a No. 7 
lens scarcely show at all the individual organisms. Older colonies are 
sometimes colored yellowish brown and in the centre are characteristic 
heapings. Another characteristic feature of the influenza bacillus is 
that the colonies tend to remain separate from each other, although 
when they are thickly sown in a film of moist blood upon nutrient 
agar they may become confluent. Transplantation of the original 
culture to ordinary agar or serum cannot be successfully performed, 
owing to the want, of sufficient hemoglobin; but if sterile rabbit, pigeon, 
or human blood be added to these media, even in very small amounts 
(1 : 500), especially if the agar is very hot (90° C.) at the time 
the blood is added, transplantation may be indefinitely performed, 
provided it is done every three or four days. Cultures if kept moist, 
may remain alive for a variable time. By a series of beautifully 
carried out experiments, Pfeiffer showed that not only were the red- 
blood cells the necessary part of the blood needed for the growth of the 
influenza bacillus, but that it was the hemoglobin in the cells that was 
the essential element. 
Though in pure cultures this organism will not grow without hemo- 
globin, in the presence of certain other organisms it will grow abun- 
dantly for successive culture generations without blood. Meissner and 
others found that it would grow well with members of the diphtheria 
group, and with the pyogenic cocci. We have found, contrary to 
others, that staphylococci tend to inhibit growth. 
Resistance and Length of Life.-The influenza bacillus is very sensi- 
tive to desiccation; a pure culture diluted with water and dried is 
destroyed with certainty in twenty-four hours; in dried sputum the 
vitality, according to the completeness of drying, is retained from 
twelve to forty-eight hours. It does not grow, and soon dies in water. 
In blood-bouillon cultures at 20° C. they retain their vitality for several 
weeks. In moist sputum it is difficult to determine the duration 
of their life, since the other bacteria overgrow and make it impossible 
to find them. It is probable that they can remain alive for at least 434 
PATHOGENIC MICROORGANISMS 
two weeks. The bacilli are very readily killed by chemicals, disinfec- 
tants, and succumb to boiling within one minute and to 60° C. within 
five minutes. 
Effect on Animals.-The bacillus of influenza is only slightly virulent 
for experimental animals. It is, however, definitely pathogenic for 
some animals, especially the rabbit, and several observers have found 
that such pathogenic power (from one blood-agar slant to y-g of a slant) 
may be decidedly increased by successive passage through the sus- 
ceptible animal. Guinea-pigs are also quite susceptible to some 
strains. Pure, abundant cultures are always obtained from the heart 
after intraperitoneal inoculation. Wollstein has been able to pro- 
duce cerebrospinal meningitis in the monkey by subdural inoculation. 
Toxic products cause toxic symptoms in rabbits only when inoculated 
in large quantities. 
Immunity.-Short immunity may be established after an attack. 
That rabbits may be hyperimmunized is shown in the process of obtain- 
ing antibodies. At least in three experiments made by Pfeiffer on 
monkeys, these animals, after recovering from an inoculation with 
bacilli, seemed to be much less susceptible to a second injection. 
Complement Fixation.-Specific antibodies may be obtained in most 
rabbits, which definitely fix complement. The greatest difficulty in 
demonstrating the phenomenon of complement deviation with this group 
of organisms, is the preparation of suitable antigens. We have found 
that with the use of only small quantities of heated blood in the medium 
and by employing the method of shaking the cultures and then incu- 
bating overnight to help the autolysis, very satisfactory antigens have 
been obtained (p. 190). Definite groups among these bacilli have been 
demonstrated by this reaction. 
Pathogenicity for Man.-The invasion of the body by the influ- 
enza bacillus is more widespread than was earlier supposed. Very 
frequently the influenzal process invades portions of the lung tissue. In 
severe cases a form of pneumonia is the result, which is lobular and 
purulent in character and accompanied by symptoms which may be 
somewhat characteristic for influenza, or, again, almost identical 
with bronchopneumonia due to the pneumococcus. The walls of the 
bronchioles and alveolar septa become densely infiltrated with leuko- 
cytes, and the spaces of the bronchial tubes and alveoli become filled. 
The influenza bacilli are found crowded in between the epithelial 
and pus cells and also penetrate the latter. There may be partial 
softening of the tissues or even abscess formation. Bacilli are found 
in fatal cases to have penetrated from the bronchial tubes not only 
into the peribronchitic tissue, but even to the surface of the pleura, 
and rarely they have been obtained in pure cultures in the pleuritic 
exudation. The pleurisy which follows influenza, however, is usually 
a secondary infection, due to the streptococcus or pneumococcus. 
Presence in Other Parts of the Body.-Influenza bacilli are found 
at times in the blood during the early days of an acute infection, while 
there is marked fever (Ghedini found them in 50 per cent, of his cases); THE INFLUENZA BACILLUS 
435 
and sometimes in bad cases in young children a septicemia develops 
before death. It is found at times in otitis media accompanying 
influenza, and has been found in cases of meningitis (Wollstein) in 
cases of acute and subacute conjunctivitis (Williams) and in several 
cases of peritonitis, appendicitis, and cystitis. The general, cerebral, 
gastric, and other symptoms produced are usually due to the absorp- 
tion of the toxic products of the specific organism, these poisons being 
particularly active in their effects on the central nervous system. 
Presence of Influenza Bacilli in Chronic Influenza and in Tuber- 
culosis.-Ordinarily influenza runs an acute or subacute course, and 
not infrequently it is accompanied by mixed infections with the 
pneumococcus and streptococcus. Pfeiffer was the first to draw 
attention to certain chronic conditions depending upon the influenza 
bacillus. Bacilli may be retained in the lung tissue for months at 
a time, remaining latent a while, and then becoming active again, 
with a resulting exacerbation of the disease. Consumptives are 
liable to carry influenza bacilli for years and are particularly sus- 
ceptible to attacks of influenza. Williams, in the examination of 
sputa in cases of pulmonary tuberculosis, has found abundant in- 
fluenza bacilli to be present in a large proportion of the samples of 
sputum from consumptives, and this not only in winter but also in the 
summer, when no influenza was known to be present in New York. 
Taken together with results elsewhere, this indicates that at all times 
of the year many consumptives carry about with them influenza ba- 
cilli, and that very likely many healthy persons as well as persons 
suffering from bronchitis also harbor a few. Given favorable con- 
ditions, we have at all times the seed to start an epidemic. 
Epidemiology.-The discovery of this bacillus enables us to explain 
many things, previously unaccountable, in the cause of epidemic in- 
fluenza. We now know, from the inability of the influenza bacillus 
to exist for long periods in dust, that the disease is not transmissible to 
great distances through the air. We also know that the infective ma- 
terial is contained only in the secretions. Sporadic cases or the sudden 
eruption of epidemics in any localities from which the disease has been 
absent for a' long time, or where there has been no new importation 
of infection, may possibly be explained by assuming that the bacilli, 
as already mentioned, often remain latent in the lungs or bronchial 
secretions or secretions of the conjunctiva for many months, and per- 
haps years, and then become active again, when under favorable 
circumstances they may be communicated to others. 
Bacteriologic Diagnosis.-This is of importance for the identifica- 
tion of clinically doubtful cases, which, from their symptoms, may be 
mistaken for bronchitis, pneumonia, or tuberculosis. 
In acute uncomplicated cases the probable diagnosis can be fre- 
quently made by microscopic examinations of stained preparations of 
the sputum. In chronic cases or those of mixed infection few or many 
bacilli may be found and the culture method may be necessary to give 
even a probable diagnosis. The bacillus of influenza is not readily 436 
PATHOGENIC MICROORGANISMS 
separated by its morphologic, staining, and cultural peculiarities from 
other bacteria belonging to the influenza group, and at present by the 
spread method it is almost impossible to identify it certainly. 
Examination of Sputum for Influenza Bacilli.-1. Sputum coughed from the 
deeper air passages and not from throat scraping should be used. 
2. The sputum should be received in a sterile bottle, which should then be 
placed immediately in cracked ice. 
3. Blood-agar plates should be made by dropping a drop of fresh rabbit's 
blood, obtained aseptically, on the centre of a hardened agar plate. 
4. One of the more solid masses of the sputum should be taken from the 
bottle with sterile forceps and placed on a plain agar plate. A small portion of 
this mass should be separated with a sterile platinum needle and drawn through 
the blood on the blood-agar plate from the centre out in different directions. 
The larger part of what is left of this small portion is then placed in a similar 
manner over a second blood agar, and from this to a third, sterilizing the needle 
between the transfers. The plates should be placed in the thermostat for 
twenty-four hours. 
5. After the plates are planted two smears should be made, one stained by 
Gram and the other by weak carbol-fuchsin. 
6. After twenty-four hours the plates are examined under low power. The 
influenza colonies use up the hemoglobin, and in parts of the blood-agar plate 
where the blood is of right thickness such colonies show as almost clear areas 
surrounded by the red blood. With a higher power (No. 6 or 7 objective), if 
such areas seem to be made up of fine indefinite granulations, they are prac- 
tically sure to be influenza colonies. Most influenza colonies are more highly 
refractive than other light colonies, and they show this characteristic best when 
they grow on the edge of a blood-mass. Many influenza colonies also show heap- 
ings in the centre. Influenza colonies growing away from the blood cells are 
less characteristic in appearance and less easily differentiated from other similar 
bacteria. 
7. Fishings from the influenza-like colonies should be planted on blood- 
agar tubes, and if, after twenty-four hours in the thermostat, the resulting 
growth should consist of influenza-like organisms, plantings should be made 
on plain agar. The first generation on plain agar may show slight growth 
because of the blood carried over from the original tube, but the second 
generation should show no growth if the organism is the influenza bacillus. 
8. The agglutination characteristics of the cultures should be tested in the 
serum from a rabbit injected with a single typical culture, and in the serum 
from one injected with a number of cultures. The agglutination tests should 
be carried out in order to gain information. The cultures tested in the Research 
Laboratory have shown considerable variation. 
Other Bacilli Resembling the Influenza Bacillus.-There are a 
number of bacilli which differ slightly in morphology and growth in 
culture from the characteristics of the typical influenza bacillus. These 
were grouped under the name "pseudo-influenza bacilli." But so far 
there have been shown no characteristics distinct enough to separate 
these hemoglobinophilic organisms into other than "strains" or 
"varieties" of the one specie B. influenza. For example, the influenza- 
like bacilli found first in whooping cough by Jochmann and others, 
Muller's "trachoma bacillus," Koch-Weeks' bacilli, the bacilli found 
by Cohen in meningitis, and those reported occasionally in other parts 
of the body-all of them seem to be so closely related that they should 
be considered one species or, at the most, varieties of one species until 
more specific characteristics can be demonstrated. INFLUENZA-LIKE BACILLI IN CONJUNCTIVITIS 
437 
Relation of the Clinical Symptoms to the Bacterial Excitant.- 
There is no doubt that other infections are also included under the 
clinical forms of influenza, and during an epidemic of bronchopneu- 
monias, irregular types of lobar pneumonias, and cases of bronchitis 
frequently have symptoms so closely alike that the nature of the bac- 
teria active in the case is very frequently different from that supposed 
by the clinician. Thus in four consecutive autopsies examined by the 
writer the influenza bacillus was found almost in pure culture in one 
case believed, from the symptoms, to be due to the pneumococcus, and 
entirely absent in two of the three believed to be due to it. Except for 
these examinations the clinician would be of the opinion that he had 
clearly diagnosed bacteriologically the cases, while in fact he had been 
wrong in three of the four. 
The striking symptoms in acute respiratory diseases are frequently 
more due to the location of the lesions than to the special variety of 
organisms producing them. In epidemics of influenza there are, 
of course, many cases which, on account of their characteristic symp- 
toms, can be fairly certainly attributed to the influenza bacilli. Even 
under these circumstances error may be made, as, for instance, two 
cases of apparently typical influenza were reported in a household 
and both showed a total absence of influenza bacilli. The pneumo- 
coccus was present in almost pure culture. 
For Testing the Agglutination of Influenza Bacilli in the Hanging 
Drop.-Grow the cultures on slanted blood-agar tubes (defibrinated 
blood) added to melted agar at 90° C., then slanted and cooled. When 
twenty to twenty-four hours old, make a suspension of the bacilli in 
normal salt solution, controlling the number of bacilli by examining 
a hanging-drop preparation. The influenza bacilli seem to aggluti- 
nate rather slowly, so it usually takes four to five hours to get a good 
reaction.. 
Serum Therapeutics.--No protective serum has been produced which 
has been used successfully in human cases, but Wollstein obtained one 
which was of value in experimentally produced infection in monkeys. 
Vaccine Treatment.-So far this has not been proven to be of marked 
value. Certain cases may be slightly helped, but too little is still 
known about the best conditions for use to make it of universal value. 
Sensitized vaccines have been said to give good results. 
INFLUENZA LIKE BACILLI IN CONJUNCTIVITIS (INCLUDING 
TRACHOMA). 
The Koch-Weeks' Bacillus.-This bacillus was first observed by R. 
Koch in 1883 while making certain investigations into inflammation 
of the eye occurring during an epidemic of cholera in Alexandria. It 
was later, in 1887, more specifically described by Weeks1 in New York. 
Weeks obtained it in cultures in growing with the xerosis bacillus from 
1 New York Med. Rec., 1887, xxvi, 571. 438 
PATHOGENIC MICROORGANISMS 
cases of "pink eye," or acute contagious conjunctivitis. Morax stated 
that he was able to obtain pure cultures only until the third culture 
generation. Others state that on human serum or hydrocele fluid they 
have obtained growths for many generations. Kamen concludes that 
it is a strict hemoglobinophile. Our studies led us to agree with this 
last conclusion. The successive cultures obtained from some sera are 
probably due to the presence of hemoglobin in amounts too small to be 
easily detected, but large enough to allow growths of hemoglobinophilic 
bacilli. The few differential points claimed between this bacillus and 
influenza bacilli do not hold.1 
Fig. 150 
Fig.151 
Koch-Weeks' bacillus ("pink-eye")- 
third generation. X 1000 diameters. 
(Weeks.) 
Secretion of mucopus from conjunc- 
tiva in "pink-eye." X 1000 diameters. 
(Weeks.) 
Other Microorganisms in Conjunctivitis.-Many organisms are found 
in diseases of the eye, but few of these present evidence of specific 
etiology. 
Morax-Axenfeld Bacillus.-In certain subacute inflammations of the 
conjunctiva, especially noticeable about the angle of the eyes (angular 
conjunctivitis), Morax (1896) and later Axenfeld found a bacillus which 
they consider the cause of the disease. 
Morphology.-Short (about 2/z long), thick, non-motile bacilli, gen- 
erally in twos, but sometimes single or in short chains. They take the 
ordinary stains easily, but are decolorized by Gram's stain. 
Cultures.-At 37° C. the bacillus produces a delicate growth on media 
containing blood or serum. Later cultures grow slightly on nutrient 
veal agar. It grows slightly, if at all, at room temperature. 
Upon serum agar, it forms delicate grayish colonies. 
Upon Lofller's blood-serum after twenty-four to thirty-six hours the 
growth appears as an indentation of the medium due to liquefaction. 
This liquefaction continues slowly for a variable time. 
Tn ascitic broth cloudiness is produced within twenty-four hours. 
1 Williams and Co-workers, Jour. Inf. Dis., 1914, xiv, 2G1. INFLUENZA-LIKE BACILLI IN CONJUNCTIVITIS 
439 
Pathogenicity.-Lower animals so far have shown themselves refrac- 
tory. In human beings inoculations of pure cultures have produced 
subacute conjunctivitis. 
Bacillus of Zur Nedden.-This bacillus has been found by Zur Nedden 
in certain ulcers of the cornea and is supposed to produce the disease. 
Morphology.-Small (usually less than ly long) single, sometimes 
slightly curved, non-motile bacilli. They may occur as diplobacilli, but 
they do not form chains. 
They stain easily, sometimes faintly at ends. They are decolorized 
by Gram. 
Cultures.-They are easily grown on all laboratory media. Upon agar, 
within twenty-four hours they produce rounded, raised, translucent, 
slightly fluorescent colonies, which are more or less confluent and, under 
the low power, are rather coarsely granular. Upon potato they form a 
thick, yellowish growth. Milk is coagulated; gelatin is not liquefied; 
dextrose is fermented without gas. No indol is produced. 
Pathogenicity.-Pure cultures inoculated into the cornea of guinea- 
pigs produced ulcers. 
Trachoma.-Many studies have been made on the etiology of 
trachoma (progressive follicular inflammation of the conjunctiva fol- 
lowed by cicatrization) and allied conjunctival affections. Halber- 
stadter and Prowazek (1907) state that the cause of trachoma is a 
small germ which grows in a characteristic way in the conjunctival 
epithelial cells. The organism itself, they say, is so small that at first 
it cannot be seen, only the mantle which it produces is demonstrable. 
This stains blue with Giemsa, and as the organisms grow in bunches, 
one sees at first in the neighborhood of the nucleus only a bunch of small, 
blue, coccus-like bodies. The organism finally appears as a minute 
red granule within the blue body. As it continues to increase in numbers 
and size the blue mantles finally disappear, leaving a mass of small 
rounded or slightly elongated red bodies. The bodies are only found in 
the early acute cases. Prowazek named them Chlamydozoa on account 
of their mantle, and thinks they should occupy a place between bacteria 
and protozoa. 
We have called attention to the similarity of these inclusions to nests 
of growing hemoglobinophilic bacilli. 
The Koch-Weeks' bacillus has been frequently reported as occurring 
in trachomatous eyes, from the time of Koch (Collins, Morax, Muller, 
etc.). Markus states that the Koch-Weeks' bacillus is the cause of 
"Schwellungskatarrh" (our papillary conjunctivitis). Miitermilch 
goes further in declaring that "often repeated infection with the same 
microorganism, e. g., bacillus of Koch-Weeks, produces a series of exac- 
erbations on an already inflamed conjunctiva and finally produces 
the picture of trachoma." 
Muller, who isolated hemoglobinophilic bacilli from the largest series 
of trachoma cases reported, had positive results chiefly in his "acute 
trachoma" cases. Muller thought at first that his bacillus was the cause 
of trachoma, but now, most authors have decided that it is an influenza 440 
PATHOGENIC MICROORGANISMS 
bacillus and has nothing specifically to do with trachoma (Zur Nedden, 
Morax, and others). 
We have demonstrated the continued presence of hemoglobinophilic 
bacilli in cases showing successively acute, subacute, and chronic 
inflammation of the conjunctiva and their increase in numbers during 
acute exacerbations of chronic cases. We have further shown the 
bacilli found in cases of "pink eye" are indistinguishable, so far, from 
hemoglobinophilic bacilli found in the chronic cases. 
Relation of Hemoglobinophilic Bacilli to Trachoma Inclusions.- 
In studying closely the morphology in cultures of these bacilli, 
we were struck by the fact that they frequently grew in more or less 
dense clumps of extremely minute and irregular coccoid forms. This 
led us to the conclusion that possibly they formed the Prowazek inclu- 
sions found in trachoma, and when we found that these bacilli and the 
inclusions were found coincidently and repeatedly in so many cases 
diagnosed as acute papillary trachoma, the possibility became a 
probability and we proceeded to study the morphology of the cultures 
more minutely. 
The morphology varies somewhat with the age of the inoculated 
culture, the date from isolation, the kind of medium, and the strain. 
In 48 hours the forms become somewhat more irregular. Then in 3 
days most of the bacilli have become extremely minute, many showing 
only as reddish granules (the "elementary bodies of Prowazek"), while 
scattered through the culture are swollen spheroidal bodies taking 
a fainter clear blue stain (the larger "initial bodies of Prowazek"), 
in some of which are minute reddish granules (more of the "elemen- 
tary bodies of Prowazek"). A number of irregular light blue bodies 
are also scattered through the culture. Where the bacteria are densely 
grouped more red granules may appear in the centre of the group 
than at the periphery and more blue bodies at the periphery than in 
the centre. In short, all of the changes described by Prowazek and 
others as characteristic of trachoma inclusions are seen in the growing 
cultures of these hemoglobinophilic bacilli. 
Similar day-to-day studies were undertaken with the other types 
of bacteria found most frequently in the eyes diagnosed as trachoma, 
e. g., streptococci, staphylococci, gonococci, a minute Gram-negative, 
non-hemoglobinophilic bacillus not before described, found in a few 
cases of papillary conjunctivitis, xerosis bacillus,, and Micrococcus 
tetragenus, but in none of these varieties except the gonococcus were 
similar changes found in the same marked degree. 
The fact that the gonococcus cultures showed such definite appear- 
ances similar to the trachoma inclusions led us to make a special study 
of a series of ophthalmia neonatorum cases. 
In all of the inclusion cases where gonococci are found, apparent 
transition forms between go'nococcus and inclusion are very evident, 
and we find from a further microscopic study of these slides that the 
inclusions on the whole present certain characteristics different from 
those found in our series produced, according to our hypothesis, by 
nests of growing hemoglobinophilic bacilli. THE BORDET-GENGOU BACILLUS 
441 
From this comparative study of "inclusions" and cultures we have 
reached the following conclusion: 
In many cases of "papillary conjunctivitis" and a certain number 
of cases of ophthalmia neonatorum, as well as in a certain number of 
cases of inflammation of the mucous membranes of other parts of the 
body (e. g., vagina, urethra), the trachoma inclusions found are due to 
one or more varieties of hemoglobinophilic bacilli; in a certain number 
of cases of gonorrheal ophthalmia as well as in gonorrheal inflammation 
of the mucous membranes of other parts of the body, the trachoma 
inclusions are due to the gonococcus. According to one of the later 
reports of Leber and Prowazek and the recent report of Noguchi and 
Cohen, certain inclusion conjunctivitis cases may be caused by micro- 
organisms other than the two mentioned above. 
THE BORDET-GENGOU BACILLUS (B. PERTUSSIS). 
In 1906 Bordet and Gengou announced that they had discovered the 
etiologic factor of whooping cough to be a small bacillus found in pre- 
dominating numbers in whooping-cough sputum. To this organism 
they gave the name Bacillus pertussis. Their claim that this bacillus 
is the real cause of whooping cough they based upon their results with 
the complement-fixation test which they had been first to describe. 
Morphology.-The pertussis bacillus is a short, oval rod, varying in 
size from about 0.2^ to 0.3/x in diameter and from 0.5^ to 2^ in length. 
It occurs singly, sometimes in twos joined at the ends, and very 
exceptionally in short chains. 
Motility.-It is non-motile. 
Staining.-The pertussis bacillus is decolorized by Gram's method. 
It is stained faintly by the ordinary aniline dyes. Bi-polar staining 
is demonstrated very well by Gram's method and by Toluidine's 
methylene blue. 
Cultivation.-The pertussis bacillus grows best at 35° C. to 37° C. 
It grows slowly at room temperature. 
It is aerobic, facultative anaerobic. When first isolated in pure 
culture it grows only upon the glycerin-potato-blood-agar, recom- 
mended by Bordet and Gengou. In later generations it grows upon 
the ordinary culture media. 
Isolation.-The pertussis bacillus is isolated from sputum with 
difficulty, owing to the fact that in cultures it is frequently overgrown 
by other organisms found in the respiratory tract. The sputum should 
be collected from the patient during the early stage of the disease, 
not over one week. The thick grayish portion of the sputum should be 
selected for the culture. This material is streaked over the surface 
of a plate of Bordet-Gengou medium. The plates are incubated at 
35° C. to 37° C. In forty-eight hours to three days very minute, dis- 
crete, elevated colonies appear. When these colonies occur in abun- 
dance, an area of hemolyzed blood may be seen at their periphery. 
In pure cultures the hemolysis is marked. 442 
PATHOGENIC MICROORGANISMS 
Identification.-(a) Differential Diagnosis by Culture.-Occurring in 
whooping-cough sputum are other bacilli so closely resembling the 
pertussis bacillus in morphologic and staining characteristics that 
they cannot be distinguished in smears. These bacilli, however, can 
be differentiated by their growth upon various culture media. The 
most important of these organisms is the influenza bacillus, which will 
never grow without the presence of hemoglobin in the culture medium. 
There is frequently found a Gram-negative bacillus which makes a pro- 
fuse growth upon all media from the first generation in pure culture. 
The following table gives the chief points of differentiation: 
Growth on 
Bordet-Gengou 
plates. 
Bordet- 
Gengou 
slants. 
Coagulated 
horse-blood 
slants. 
Glycerin- 
ascites-agar 
slants. 
Plain- 
agar slants. 
B. pertussis . 
Hemolyzes or 
First generation 
After several 
After several 
After several 
lightens the 
medium. 
in pure culture 
abundant 
moist growth 
in twenty-four 
hours. 
generations 
abundant 
tenacious 
growth in 
forty-eight 
hours. 
generations 
abundant 
tenacious 
growth in 
forty-eight 
hours. 
generations 
tenacious 
growth 
occurs 
slowly. 
B. influenza; . 
Darkens the 
medium. 
First generation: 
delicate growth. 
First gene- 
ration: 
abundant 
moist spread 
in twenty- 
four hours. 
Never grows. 
Never 
grows. 
Intermediate group of 
Hemolyzes the 
First generation: 
First genera- 
First genera- 
First gene- 
Gram-negative bacilli. 
medium. 
abundant 
moist spread 
in twenty-four 
hours. 
t'.on: 
profuse 
moist spread 
in twenty- 
four hours. 
tion: 
profuse 
moist 
spread in 
twenty- 
four hours. 
ration: 
profuse 
moist 
spread in 
twenty- 
four hours 
(&) Complement Fixation.-Bordet and Gengou regarded the com- 
plement fixation as the main support for their claim of the specificity of 
their organism. They report positive complement fixation in the 
majority of human cases tested by them. Other investigators, how- 
ever, have reported negative tests of complement fixation. 
The irregular reports given of the complement-fixation test in human 
beings may be accounted for by different methods used without 
making comparative studies and sufficient controls, and, more impor- 
tant still, by the occurrence of distinct varieties in the species Bacillus 
pertussis. The question of the best method of making the complement- 
fixation test is still under experiment. Different observers have used 
different methods of preparing both antigen and serum and have em- 
ployed different hemolytic systems. In experimental animals, specific 
antibodies are produced which give a positive complement-fixation test. 
Pathogenicity.-The pathogenicity of the pertussis bacillus for man 
still lacks positive proof. Several investigators have reported its patho- 
genicity for monkeys, dogs, rabbits, and guinea-pigs. 
Mallory claims that a certain number of bacilli between the cilia 
of the epithelial cells in the trachea and bronchi constitute the specific 
lesion. He claims that he has fulfilled Koch's laws by the findings of 
these bacilli in experimental animals in the same situation and by the THE BACILLUS OF SOFT CHANCRE 
443 
recovery of the culture from these animals. lie acknowledges, however, 
that his results in animals are complicated by the fact that these animals 
are frequently infected by the Bacillus bronchisepticus (accepted as the 
cause of distemper in dogs) which is morphologically similar to the 
pertussis bacillus and that it apparently has the same power (as Theo- 
bold Smith and others have pointed out) to cling to the cilia of the 
epithelial cells in the respiratory tract. In human beings Mallory 
states, judging from the examination of hundreds of controls, only 
pertussis cases showed this lesion. 
THE BACILLUS OF SOFT CHANCRE. 
This bacillus was first specifically described and obtained in pure 
culture by Ducrey in 1889. An experimental inoculation is followed 
in one to two days by a small pustule. This soon ruptures and a small 
round depressed ulcer is left. About this other pustules and ulcers 
develop which tend to become confluent. The base of the ulcer is 
covered with a gray exudate and its edges are undermined. There 
is no induration such as in the syphilitic chancre. The secretion is 
seropurulent and very infectious. The process usually extends to the 
neighboring lymphatics, which become swollen and may result in 
abscesses. These are known as "buboes." 
Morphology.-About 1.5^ long and O.Au thick, growing often in 
chains and in cultures, sometimes twisted together in dense masses. 
It stains best with carbol-fuchsin, and shows polar staining. It is 
Gram-negative. 
Cultural Characteristics.-The following method of cultivation has 
given the best results: Two parts of liquefied agar at 50° C. are 
mixed with one part human, dog, or rabbit blood. The blood from 
the cut carotid of a rabbit may be allowed to run directly into the 
agar tube, to which the pus from the ulcerated bubo is then added 
in proper proportion, and the whole placed in the incubator at 35° 
C. The pus may be obtained by puncture and aspiration from the 
unbroken ulcer, or if the ulcer is already open it is first painted with 
tincture of iodine and covered with collodion or sterile gauze. After 
twenty-four to forty-eight hours, some pus having collected under the 
bandage, inoculations are made from it. The bacillus grows well 
also in uncoagulated rabbit-blood serum or in condensation water of 
blood agar. In twenty-four to forty-eight hours, on the surface of the 
media, well-developed, shining, grayish colonies, about 1 mm. in diameter 
may be observed. The colonies remain separate, but only become 
numerous after further transplantation. The best results are obtained 
when the pus is taken close to the walls of the abscess. Glass smears 
show isolated bacilli or short parallel chains with distinct polar 
staining. 
After the eleventh generation of the culture, and from all old cul- 
tures, on inoculation the characteristic soft chancre is produced in 444 
PATHOGENIC MICROORGANISMS 
man. Animals in general cannot be infected, but positive results have 
been obtained with monkeys and cats. 
The organisms are especially characteristic in the water of con- 
densation from blood-agar, the bacilli being thinner and shorter, with 
rounded ends; sometimes long, wavy chains are found. In rabbit- 
blood-serum at 37° C. a slight clouding of the medium is produced and 
small flakes are formed, consisting of short bacilli or moderately long, 
curved chains, showing polar staining. 
The bacillus lives several weeks on blood-agar at 37° C., but it soon 
dies in cultures on coagulated serum. All other ordinary culture media 
so far tried have given negative results, and even with the media 
described development is difficult and often fails entirely. 
The chancre bacillus possesses but little resistance to deleterious 
outside influences. Hence, the various antiseptic bandages, etc., used 
in treatment of the affection soon bring about recovery by preventing 
the spread of inoculation chancre. CHAPTER XXXII. 
MICROORGANISMS BELONGING TO THE HEMORRHAGIC 
SEPTICEMIA GROUP. 
A number of bacilli of similar characteristics have been described 
as causing certain infectious diseases of lower animals, marked by the 
appearance of hemorrhagic areas throughout the body (hemorrhagic 
septicemia of Hueppe). The bacilli are short, bipolar-staining, non- 
motile, non-sporebearing organisms. They are Gram-negative and 
do not liquefy gelatin. They are found in rabbit septicemia, fowl 
cholera, swine plague,1 and a similar disease in cattle. The bacillus 
of bubonic plague seems to be closely related to the bacteria of this 
group. 
BACILLUS OF CHICKEN CHOLERA. 
(Bacillus Avisepticus.) 
Iii 1880 Pasteur carried on some fundamental studies on the bacillus 
of chicken cholera.. The bacillus was isolated from a widely disseminated 
acute disease of fowls and smaller birds. 
Characteristics.-It is a short (0.5-1 n long), non-motile bacillus with 
marked polar staining. In general, its characteristics are similar to 
those of other numbers of this group. 
Pathogenicity.-Pure cultures are very pathogenic for chickens and 
rabbits, they are less so for sheep, pigs, and horses. Chickens are 
infected even by feeding minute amounts. A septicemia is produced 
which is rapidly fatal. It is accompanied by diarrhea, depression, and 
finally coma. The lesions are hemorrhagic inflammation of the mucous 
membrane of the intestines and sometimes of lungs, with enlargement 
of the spleen and liver. 
Immunity.-Pasteur immunized chickens with attenuated cultures. 
The method, however, has not been found practicable. Kitt and Mayer 
state the serum of horses immunized with the bacillus of chicken cholera 
shows some protection against the bacillus of swine plague. 
BACILLUS OF SWINE PLAGUE (SCHWEINESEUCHE) 
(Bacillus Seuisepticus.) 
This organism is morphologically and culturally similar to the B. 
avisepticus. It differs in pathogenesis in that it is naturally a disease 
of swine, characterized by a more or less chronic bronchopneumonia 
1 Gei-nian, Schweinseuche. 446 
PATHOGENIC MICROORGANISMS 
followed by septicemia. The gastro-intestinal tract is not markedly 
affected. The disease is generally fatal in young pigs. 
A polyvalent serum has been produced which has given some practical 
results. 
The "bacillus of hog cholera'' (schweinepest) may often be found as a 
mixed infection with the B. seuisepticus. 
BACILLUS OF BUBONIC PLAGUE (BACILLUS PESTIS). 
Historically we can trace the bubonic plague back to the third 
century. In Justinian's reign a great epidemic spread over the Roman 
Fig. 152 
Fig. 153 
Bacillus pestis from agar culture. 
X 1100 diam. 
Bacillus pestis from bouillon culture. 
X 1100 diam. 
empire and before it terminated destroyed in many portions of the 
country nearly 50 per cent, of the people. The fourteenth century 
saw the whole of Europe stricken with this "black death." Europe 
and America have of late been practically free, but in India the disease 
still breaks out in all its horrors so that at the present time over 500,000 
persons die annually from it. Among the most fatal forms of infection 
is that of the lungs. Pneumonic cases are not alone very serious, but 
they readily spread the infection. The bacillus exciting the disease 
was discovered simultaneously by Kitasato and Yersin (1894) during 
an epidemic of the bubonic plague in China. It is found in large numbers 
in the seropurulent fluid from the recent buboes characteristic of this 
disease and in the lymphatic glands; more rarely in the internal organs 
except in pneumonic cases when the lungs and sputum contain im- 
mense numbers, it occurs in the blood in acute hemorrhagic cases and 
shortly before death. It also occurs in malignant cases in the feces 
of men and animals. The bacillus, as we have stated, is closely allied 
to the hemorrhagic septicemia group. BACILLUS OF BUBONIC PLAGUE 
447 
Morphology.-The bacilli in smears from acute abscesses or infected 
tissues are, as a rule, short, thick rods with rounded ends. The central 
portion of the bacillus is slightly convex. When lightly stained the two 
ends are more colored than the middle portion. The bacilli are mostly 
single or in pairs. Bacilli in 
short chains occur at times. 
The length of the bacilli 
varies, but on the average is 
about 1.6/z (1.5m to 1.7m), 
breadth 0.5m to 0.7m. Besides 
the usual oval form, the plague 
bacillus has many exceptional 
variations which are character- 
istic of it. In smears, especi- 
ally from old buboes, one looks 
for long bacilli with clubbed 
ends (similar to involution 
forms (Fig. 154), yeast-like 
forms, and bladder shapes. 
Some of these stain with dif- 
ficulty. When obtained from 
cultures the bacilli present not 
only the forms already men- 
tioned, but also long chains. 
Staining.-They stain readily with the ordinary aniline dyes, and 
especially well with methylene blue, the ends being usually more 
deeply colored than the central portion; they do not stain by Gram's 
method. 
Biology.-An aerobic, non-motile bacillus. Grows best at 30° to 
35° C. Does not form spores. Grows on the usual culture media, 
which should have a slightly alkaline reaction. Does not liquefy 
gelatin. Grows well on blood-serum media. It grows rapidly on 
glycerin agar, forming a grayish-white surface growth. The bacilli 
appear, as a rule, as short, plump, oval bacilli, but a few present 
elongated thread forms which are very characteristic. In bouillon 
which is kept still a very characteristic appearance is produced, the 
culture medium remaining clear while a pellicle forms on the sur- 
face from which projections sprout downward (stalactite formation) 
toward a granular or grumous deposit which forms on the walls and 
on the bottom of the tube. In bouillon and most fluid media the 
growth is in the form of short or medium chains of very short, oval 
bacilli, which look almost like streptococci. It does not coagulate 
milk, but produces a slight acidity. It produces no indol in peptone 
media. 
Pathogenicity.-This bacillus is pathogenic for rats, mice, squirrels, 
guinea-pigs, monkeys, and rabbits. 
Fleas, flies, and other insects may also become infected. The chief 
lower animal infected is the rat, and the chief carrier of like disease 
Fig. 154 
Involution forms on salt agar. (Kolle and 
Wassermann.) 448 
PATHOGENIC MICROORGANISMS 
is the flea {X enopay cla cheopis), which by its bites usually die within 
two or three days after inoculation. Then at the point of inocula- 
tion is found a somewhat hemorrhagic infiltration and edema, with 
enlargements of the neighboring lymph glands, hemorrhages into the 
peritoneal cavity, and parenchymatous congestion of the organs. The 
spleen sometimes shows minute nodules resembling miliary tubercles. 
Microscopically the bacilli are found in all the organs and in the blood. 
The disease is rapidly communicated. During epidemics, rats, mice, 
and flies, in large numbers, become infected and die, and the disease is 
frequently transmitted through them to man. Very small wounds are 
sufficient to allow entrance of the germs. Mere rubbing of a culture 
on the skin of rats and guinea-pigs may produce the disease. The 
organism is found at times in the feces of sick animals, in the dust of 
infected houses, and in infected soil. 
Fig. 155 
Bacilli in smear from acutely inflamed gland. 
Ground squirrels in California have been shown to be susceptible 
to infection and they are supposed to help spread the disease. 
The virulence of the bacilli in cultures and in nature seems to vary 
considerably. It may rapidly diminish when grown on artificial media. 
The growth in cultures becomes more abundant after frequent trans- 
plantation. The virulence of the organism is increased by successive 
inoculation in certain animal species, and then its pathogenic prop- 
erties for other species are less marked. 
In man the pneumonic form of the disease is most dangerous as a 
means of direct infection to man. 
Immunity.-Like typhoid infection, a single attack of the plague 
bacillus protects, with rare exception, from a second infection. Yersin, 
Calmette, and Borrel have succeeded in immunizing animals against 
the bacillus of bubonic plague by the intravenous or intraperitoneal 
injection of dead cultures, or by repeated subcutaneous inoculation. 
They also succeeded in immunizing rabbits and horses, so that the BACILLUS OF BUBONIC PLAGUE 
449 
serum afforded protection to small animals, after subcutaneous injection, 
of virulent cultures, and even cured those which had been inoculated, 
if administered .within twelve hours after injection. The serum has 
considerable antitoxic as well as bactericidal properties. It also contains 
specific agglutinins which may be made use of in diagnosis. More 
recently this serum has been applied to the treatment of bubonic plague 
in man, with promising results. Experience has shown that the treat- 
ment is more efficacious the earlier the stage of the disease. When 
treatment is begun in the first day of the attack, fever and all alarming 
symptoms sometimes disappear with astonishing rapidity. In cases 
treated at a later stage larger doses of the serum are required, and even 
in the favorable cases suppuration of the buboes is not always prevented. 
In some of the early cases and in many of the rather late ones the serum 
fails. When the disease is far advanced the serum is powerless. For 
immunizing purposes the serum should be valuable, and a single injection 
would probably give protection for several weeks. 
Vaccines.-Haffkine, in India, has applied his method of preventive 
inoculation to the bubonic plague, as he previously did with cholera, 
and apparently with equally good results. This method consists in 
an inoculation of dead cultures, and is essentially a protective rather 
than a curative treatment. It gives after six to ten days a consider- 
able immunity, lasting a month or more. 
Duration of Life Outside of the Body.-In cultures protected from 
the air and light the plague bacilli may live ten years (Wilson) or more. 
In the bodies of dead rats they may live for two months. In sputum 
from pneumonic cases the bacilli lived ten days. Upon sugar sacks, 
food, etc., they may live six to fifteen days. 
Resistance to Deleterious Influences.-The bacilli resemble the 
colon bacilli in their reaction to heat and disinfectants. Boiling for 
one to two minutes kills them. Carbolic acid, 5 per cent, solution, 
kills cultures in one minute. Mercuric chloride 1 to 1000 kills in ten 
minutes. 
Bacteriologic Diagnosis. - When the lymph glands are acutely 
inflamed but not yet suppurated, cut down on one and make cultures 
on nutrient agar slanted in tubes. If pus has formed withdraw a little 
by means of the hypodermic needle. There should also be made 
smears from the suspected bubo, or in case of pneumonia from the 
sputum. If the patient is dead, cultures from the spleen and heart's 
blood are also taken when possible. Suspected animals, such as rats 
and mice, when freshly killed, and after being immersed in an antiseptic 
solution to kill fleas, etc., are examined as in man; when decomposed, 
rats and guinea-pigs should be inoculated. 
Plague-like Bacilli in Rodents.-McCoy and Chapin (1912) found 
an organism (Bacillus tularense) in a disease of Californian ground 
squirrels, which show lesions similar to those of plague. The bacilli 
have been cultivated by McCoy and Chapin on an egg-yolk medium. 
They are very minute (0.3g to 0.7g long) and may show a capsule. 
They are most abundant in the spleen of victims of the disease. 
29 CHAPTER XXXIII. 
THE ANTHRAX BACILLUS AND THE PATHOGENIC 
ANAEROBES. 
BACILLUS ANTHRACIS. 
Anthrax is an acute infectious disease which is very prevalent 
among animals, particularly sheep and cattle. Geographically and 
zoologically it is the most widespread of all infectious disorders. It 
is much more common in Europe and in Asia than in America. The 
ravages among herds of cattle in Russia and Siberia and among sheep 
in certain parts of France, Hungary, Germany, Persia, and India 
are not equalled by any other animal plague. Local epidemics have 
occasionally occurred in England, where it is known as splenic fever. 
In this country the disease is rare. In infected districts the greatest 
losses are incurred during the hot months of summer. 
The disease also occurs in man as the result of infection, either through 
the skin, the intestines, or in rare instances through the lungs. It is 
found in persons whose occupations bring them into contact with 
animals or animal products, as stablemen, shepherds, tanners, butchers, 
and those who work in wool and hair. Two forms of the disease have 
been described-the external anthrax, or malignant pustules, and the 
internal anthrax, of which there are intestinal and pulmonary forms, 
the latter being known as "wool-sorters' disease." 
Owing to the fact that anthrax was the first infectious disease which 
was shown to be caused by a specific microorganism, and to the close 
study which it received in consequence, this disease has probably 
contributed more to our general knowledge of bacteriology than any 
other infectious malady. 
Pollender in 1849 observed that the blood of animals suffering from 
splenic fever always contained minute rod-shaped bacteria. Davaine 
in 1863 announced to the French Academy of Sciences the results of 
his inoculation experiments, and asserted the etiologic relations of 
the microorganism to the disease, with which his investigation showed 
it to be constantly associated. For a long time this conclusion was 
energetically opposed until, in 1877, Koch, Pasteur, and others estab- 
lished its truth by obtaining the bacillus in pure cultures, and showing 
that the inoculation of these cultures produced anthrax in susceptible 
animals as certainly as did the blood of an animal recently dead from 
the disease. 
Morphology.-Slender, cylindric, non-motile rods, having a breadth 
of Im to 1 .25m, aRd ranging from 2m or 3m to 20m or 25m in length. Some- BACILLUS AN TH RAC IS 
451 
times short, isolated rods are seen, and, again, shorter or longer chains 
or threads made up of several rods joined end-to-end. In suitable 
culture media very long, flexible filaments may be observed, which 
are frequently united in twisted or plaited cord-like bundles. (See 
Figs. 156 and 157.) These filaments in hanging-drop cultures, before 
the development of spores, appear to be homogeneous or nearly so; 
but in stained preparations they are seen to be composed of a series 
of rectangular, deeply stained segments. When obtained directly from 
the blood of an infected animal the free ends of the rods are slightly 
rounded, but those coming in contact with one another are quite square. 
In cultures the ends are seen to be a trifle thicker than the body of the 
cell and somewhat concave, giving the appearance of joints of bamboo. 
At one time much stress was laid upon these peculiarities as distinguished 
Fig. 156 
Fig. 157 
Anthrax bacillus. X 900 diameters. 
Agar culture. 
Spores heavily stained (in specimen red). 
Bodies of disintegrating bacilli faintly 
stained (in specimen blue). X 1000 
diameters. 
marks of the anthrax bacillus; but it has been found that they are the 
effects of artificial cultivation and not necessarily characteristic of the 
organism under all conditions. The bacillus is inclosed in a transparent 
envelope or capsule, which in stained preparations (from albuminous 
material) may be distinguished by its taking on a lighter stain than the 
deeply stained rods which it surrounds. 
I nder favorable conditions in cultures spores are developed in the 
bacilli. These spores are elliptic in shape and about one and a half 
times longer than broad. They first appear as small, refractive granules 
distributed at regular intervals, one in each rod. As the spore develops 
the mother-cell becomes less and less distinct, until it disappears alto- 
gether, the complete oval spore being set free by its dissolution. (See 
Fig. 157 and Plate III, Fig. 22). Irregular sporulation sometimes takes 452 
PATHOGENIC MICROORGANISMS 
place, and occasionally there is no spore formation, as in varieties of 
non-spore-bearing anthrax. 
Staining.-The anthrax bacillus stains readily with all the aniline 
colors, and also by Gram's method, when not left too long in the de- 
colorizing solution. In sections good results may be obtained by the 
employment of Gram's solution in combination with carmine, but when 
only a few bacilli are present this method is not always reliable, as some 
of the bacilli are generally decolorized. 
McFadyean-Heine Methylene-blue Reaction. In imperfectly fixed film 
preparations (pass through flame three times in a second with film side 
up) the capsule disintegrates. When a solid film is stained for a 
few seconds in an old solution of methylene blue, washed in water 
and dried with filter paper, the bacteria are surrounded by a varying 
amount of a reddish-purple amorphous or fixed granular deposit. 
McFadyean says this does not occur with other morphologically 
similar bacteria. 
Biology.-The anthrax bacillus grows easily in a variety of nutrient 
media at a temperature from 18° to 43° C., 37° C. being the most 
favorable temperature. Under 12° C. no development takes place, as 
a rule, though by gradually accustoming the bacillus to a lower tempera- 
ture it may be induced to grow under these conditions. Under 14° C. 
and above 43° C. spore formation ceases. The lower limit of growth 
and of sporulation is of practical significance in determining the question 
whether development can occur in the bodies of animals dead from 
anthrax when buried at certain depths in the earth. Kitasato has 
shown that at a depth 1.5 meters the earth in July has a temperature 
of 15° C. at most, and that under these conditions a scanty sporulation 
of anthrax bacilli is possible, but that at a depth of 2 meters sporulation 
no longer occurs. The anthrax bacillus is aerobic-that is, its growth 
is considerably enhanced by the presence of oxygen-but it grows also 
under anaerobic conditions, as is shown by its growth at the bottom 
of the line of puncture in stick cultures in solid media; but under these 
conditions it no longer produces the peptonizing ferment which it 
does with free access of air. Furthermore, the presence of oxygen is 
absolutely necessary for the formation of spores, while carbonic acid 
gas retards sporulation. This explains, perhaps, why sporulation does 
not take place within the animal body either before or after death. 
It is also capable of leading a saprophytic existence. The bacillus 
is non-motile. 
Growth in Gelatin.-In gelatin-plate cultures, at the end of twenty- 
four to thirty-six hours at 24° C., small, white, opaque coloneis are 
developed, which, under a low-power lens, are seen to be dark gray 
in the centre and surrounded by a greenish, irregular border, made 
up of wavy filaments. As the colony develops on the surface of the 
gelatin these wavy filaments spread out, until finally the entire colony 
consists of a light gray, tangled mass, which has been likened to a 
Medusa head (Fig. 158). 
At the same time the gelatin begins to liquefy, and the colony is BACILLUS ANTHRACIS 
453 
soon surrounded by the liquefied medium, upon the surface of which 
it floats as an irregular, white pellicle. In gelatin-stick cultures at 
first development occurs along the line of puncture as a delicate white 
thread, from which irregular, hair-like projections soon extend per- 
pendicularly into the culture medium, the growth being most luxuriant 
near the surface, but continuing also below. At the end of two or three 
days liquefaction of the medium commences at the surface and gradually 
progresses downward. 
Growth on Agar.-The growth on agar-plate cultures in the incubator 
at 37° C. is similar to that on gelatin, and is still more characteristic 
and beautiful in appearance. A grayish-white layer is formed on the 
surface within twenty-four hours, which spreads rapidly and is seen 
to be made up of interlaced threads. 
Growth in Bouillon.-The growth is characterized by the formation 
of flaky masses, w'hich sink as a sediment to the bottom of the tube, 
leaving the supernatant liquid clear. 
Fig. 158 
Colonies of Bacillus anthracis upon gelatin plates: a, at the end of twenty-four hours; 
b, at the end of forty-eight hours. X 80. (F. Fliigge.) 
Spore formation, as already noted, only takes place in the presence 
of oxygen, and at a temperature of 15° to 43° C. There is no develop- 
ment of spores at a greater depth than 1.5 meters in the earth, or in 
the bodies of living or dead animals; but spores may be found in the 
fluids containing the bacilli when these come in contact with the air, 
as in bloody discharges from the nostrils or from the bowels of the 
dead animal. 
There are certain non-spore-bearing species of anthrax. Sporeless 
varieties have also been produced artificially by cultivating the typical 
anthrax bacillus under certain conditions, among which may be 
mentioned the addition of antiseptics, as carbolic acid, and of continued 454 
PATHOGENIC MICROORGANISMS 
high temperature (43° C.). Varieties differing in their pathogenic 
power may also be produced artificially. Pasteur produced an "attenu- 
ated virus" by keeping his cultures for a considerable time before 
replanting them upon fresh soil. 
Anthrax cultures containing spores retain their vitality for years; 
in the absence of spores the vitality is much more rapidly lost. When 
grown in liquids rich in albumin the bacilli attain a considerable degree 
of resistance; thus dried anthrax blood has been found to retain its 
virulence for sixty days, while dried bouillon cultures only did so for 
twenty-one days. Dried anthrax spores may be preserved for many 
years without losing their vitality or virulence. They also resist a 
comparatively high temperature. Exposed in dry air they require a 
temperature of 140° C. main- 
tained for three hours to de- 
stroy them; but suspended in 
a liquid they are destroyed in 
four minutes by a temperature 
of 100° C. 
Pathogenesis.-The anthrax 
bacillus is pathogenic for cattle, 
sheep (except the Algerian 
race), horses, swine, mice, 
guinea-pigs, and rabbits. Rats, 
cats, dogs, chickens, owls, pig- 
eons, and frogs are but little 
susceptible to infection. Small 
birds-the sparrow particularly 
-are somewhat susceptible. 
Man, though subject to local 
infection and occasionally to 
internal forms of the disease, 
is not as susceptible as some 
of the lower animals. 
In susceptible animals the anthrax bacillus produces a true septicemia. 
Among test animals mice are the most susceptible, succumbing to very 
minute injections of a slightly virulent virus; next guinea-pigs, and 
lastly rabbits, both of these animals dying after inoculation with virulent 
bacilli. Infection is most promptly produced by introduction of the 
bacilli into the circulation or the tissues, but inoculation by contact 
with wounds on the skin also causes infection. It is difficult to produce 
infection by the ingestion even of spores; but it may readily be caused 
by inhalation, particularly of spores. 
Subcutaneous injections of these susceptible animals results in 
death in from one to three days. Comparatively little local reaction 
occurs immediately at the point of inoculation, but beyond this there 
is an extensive edema of the tissues. Very few bacilli are found in 
the blood in the larger vessels, but in the internal organs, and especially 
in the capillaries of the liver, the kidneys, and the lungs, they are present 
Fig. 159 
Section of liver from mouse dead of anthrax 
septicemia. X 1000 diameters. (From 
Itzerott and Niemann). BACILLUS ANTHRACIS 
455 
in great numbers. Tn some places, as in the glomeruli of the kidneys, 
the capillaries will be seen to be stuffed full of bacilli, and hemorrhages, 
probably due to rupture of capillaries by the mechanical pressure of 
the bacilli which are developing within them, may occur. The patho- 
logic lesions in animals infected by anthrax are not marked except in 
the spleen, which, as in other forms of septicemia, is greatly enlarged. 
Occurrence in Cattle and Sheep.-Cattle and sheep are affected 
chiefly with the intestinal form of anthrax, infection in these animals 
commonly resulting from the ingestion of food containing spores. 
The bacillus itself, in the absence of spores, is quickly destroyed by 
the gastric juice. The disease usually takes a rapid course, and the 
mortality is high-70 to 80 per cent. The pathologic lesions consist 
of numerous ecchymoses, enlargement of the lymphatic glands, serous, 
fatty, and hemorrhagic infiltration of the mediastinum and mesentery, 
of the mucous membranes of the pharynx and larynx, and particularly 
of the duodenum, great enlargement of the spleen, and parenchymatous 
changes in the lymphatic organs. The blood is very dark and tar-like. 
Bacilli are present, especially in the lymph spaces, in enormous masses. 
Sheep are also subject to external anthrax, infection taking place 
by way of the skin; cattle are seldom infected in this way. At the 
point of inoculation there develops a hard, circumscribed boil-the 
so-called anthrax carbuncle; or there may be diffuse edema with 
great swelling of the parts. When death occurs the appearances are 
similar to those in intestinal anthrax, except that the duodenum is 
usually less affected; but in all cases metastasis occurs in various 
parts of the body, brought about, no doubt, by previous hemorrhages. 
Occurrence in Man.-The disease does not occur spontaneously in 
man, but always results from infection, either through the skin, the 
intestines, or occasionally by inhalatioil through the lungs. It is 
usually produced by cutaneous infection through inoculation of exposed 
surfaces-the hands, arms, or face. Infection of the face or neck would 
seem to be the most dangerous, the mortality in such cases being 26 
per cent., while infection of the extremities is rarely fatal. 
External anthrax in man is similar to this form of the disease in 
animals. There are two forms: malignant pustule or carbuncle, and, 
less commonly, malignant anthrax edema. 
In malignant pustule, at the site of inoculations, a small papule 
develops, which becomes vesicular. Inflammatory induration extends 
around this, and within thirty-six hours there is a dark, brownish eschar 
in the centre, at a little distance from which there may be a series of 
small vesicles. The brawny induration may be extreme. There may 
also be considerable edema of the parts. In most cases there is no fever; 
or the temperature at first rises rapidly and the febrile phenomena are 
marked. Death may take place in from three to five days. In cases 
which recover the symptoms are slighter. In the mildest form there 
may be only slight swelling. 
Malignant anthrax edema occurs in the eyelids, and also in the head 
and neck, sometimes the hand and arm. It is characterized by the 456 
PATHOGENIC MICROORGANISMS 
absence of the papule and vesicle forms, and by the most extensive 
edema. The edema may become so intense "that gangrene results; 
such cases usually prove fatal. 
The bacilli are found on microscopic examination of the fluid from 
the pustule shortly after infection; later the typical anthrax bacilli 
are often replaced by involution forms. In this case resort may be 
had to cultures, animal inoculation, or examination of sections of the 
extirpated tumor. The bacilli are not present in the blood until just 
before death. Along with the anthrax bacilli pus cocci are often found 
in the pustule penetrating into the dead tissue. 
Internal anthrax is much less common in man; it does, however, 
occur now and then. There are two forms of this: the intestinal form, 
or mycosis intestinalis, and the pulmonic form, or wool-sorters' disease. 
Intestinal anthrax is caused by infection through the stomach and 
intestines, and results probably from the eating of raw flesh or unboiled 
milk of diseased animals. That the eating of flesh from infected animals 
is comparatively harmless is shown by Gerlief, who states that of 400 
persons who are known to have eaten such meat not one was affected 
with anthrax. On the other hand, an epidemic of anthrax was produced 
among wild animals, according to Jansen, by feeding them on infected 
horse-flesh. It is evident, therefore, that there is a possibility of infection 
being caused in this way. The recorded cases of intestinal anthrax 
in man have occurred in persons who were in the habit of handling 
hides, hair, etc., which were contaminated with spores; in those who 
were conducting laboratory experiments, and rarely it has been produced 
by the ingestion of food, such as raw ham and milk. The symptoms 
produced in this disease are those of intense poisoning: chill, followed 
by vomiting, diarrhea, moderate fever, and pains in the legs and back. 
The pathological lesions are similar to those described in animals. 
W ool-s otters' disease, or pulmonic anthrax, is found in large establish- 
ments in which wool and hair are sorted and cleansed, and caused by 
the inhalation of dust contaminated with anthrax spores. The attack 
comes on with chills, prostration, then fever. The breathing is rapid, 
and the patient complains of pain in the chest. There may be a cough 
and signs of bronchitis. The bronchial symptoms in some instances 
are pronounced. Death may occur in from two to seven days. The 
pathological changes produced are swelling of the glands of the neck, 
the formation of foci of necrosis in the air passages, edema of the lungs, 
pleurisy, bronchitis, enlargement of the spleen, and parenchymatous 
degenerations. 
Prophylaxis against Anthrax Infection.-Numerous investigations 
have been undertaken with the object of preventing infection from 
anthrax. The efforts of Pasteur to effect immunity in animals by 
preventive inoculations of "attenuated virus" of the anthrax bacillus 
opened a new field of productive original research. Following in his 
wake many others have devised methods of immunization against 
anthrax infection; but the one adopted by Pasteur, Chamberland, and 
Roux has alone been practically employed on a large scale. According BACILLUS ANTHRACIS SYMPTOMATICI 
457 
to these authors, two anthrax cultures of different degrees of virulence 
attenuated by cultivation at 42° to 43° C., are used for inoculation. 
Vaccine No. 1 kills mice, but not guinea-pigs; vaccine No. 2 kills 
guinea-pigs, but not rabbits. The animals to be inoculated-viz., 
sheep and cattle-are first given a subcutaneous injection of one to 
several tenths of a cubic centimeter of a four-day-old bouillon culture 
of vaccine No. 1; after ten to twelve days they receive a similar dose 
of vaccine No. 2. Prophylactic inoculations given in this way have 
been widely employed with apparently good results. 
Serum Treatment.-The serum of immunized animals has been 
used in Italy with apparently some good results. We have had no 
personal experience. 
Bacterial Cultures for Diagnosis.-The detection of the anthrax 
bacillus is ordinarily not difficult, as this organism presents morpho- 
logic, biologic, and pathogenic characteristics which distinguish it from 
all other bacteria. In the later stages of the disease, however, the 
bacilli may be absent or difficult to find, and cultivation on artificial 
media and experimental inoculation in animals are not always followed 
by positive results. Even in sections taken from the extirpated pustule 
it is sometimes difficult to detect the bacilli. In such cases only a 
probable diagnosis of anthrax can be made. It should be remembered 
that the bacilli are not found in the blood until shortly before death, 
and then only in varying quantity; thus blood examinations often 
give negative results, though the bacilli may be present in large numbers 
in the spleen, kidneys, and other organs of the body. The suspected 
material should be streaked over nutrient agar in Petri plates and 
inoculated in mice. 
Differential Diagnosis.-Among other bacteria which may possibly 
be mistaken for anthrax bacilli are Bacillus subtilis and the bacillus 
of malignant edema. The former is distinguished by its motility, 
by various cultural peculiarities, and by being non-pathogenic. The 
latter differs from the anthrax bacillus in form and motility, in being 
decolorized by Gram's solution, in being a strict anaerobe, and in 
various pathogenic properties. 
The diagnosis of internal anthrax in man is by no means easy, unless 
the history points definitely to infection in the occupation of the 
individual. In cases of doubt cultures should be made and inoculations 
performed in animals. 
BACILLUS ANTHRACIS SYMPTOMATICI (BACILLUS OF SYMP- 
TOMATIC ANTHRAX). 
Like the bacilli of anthrax and of malignant edema, both of which it 
resembles in other respects also, the bacillus of symptomatic anthrax 
is an inhabitant of the soil. It is found as the chief cause of the 
disease in animals-principally cattle and sheep-known as "black 
leg," "quarter evil," or symptomatic anthrax (rauschbrand, German; 
charbon symptomatique, French), a disease which is characterized by 458 
PATHOGENIC MICROORGANISMS 
a peculiar emphysematous swelling of the subcutaneous tissues and 
muscles, especially over the quarters. Clinically it is sometimes con- 
fused with anthrax. 
Morphology.-Bacilli having rounded ends, from 0.5ju to ().6/z broad 
and from 3^ to 5/z long; mostly isolated; also occurring in pairs, 
joined end-to-end, but never growing out into long filaments, as the 
anthrax bacilli in culture and the bacilli of malignant edema in the 
bodies of animals are frequently seen to do. In the hanging drop 
the bacilli are observed to be actively motile, and in stained prepara- 
tions flagella may be demonstrated surrounding the periphery. The 
spores are elliptic in shape, usually thicker than the bacilli, lying near 
the middle of the rods, but rather toward one extremity. This gives 
to the bacilli containing spores a somewhat spindle shape. 
Stains with the ordinary aniline dyes, but not with Gram's method 
or only with difficulty and after long treatment or intense colors. 
Biology.-Like the bacillus of malignant edema, this is a strict 
anaerobe, and cannot be cultivated in an atmosphere in which oxygen 
is present. It grows best under hy- 
drogen, and does not grow under 
carbonic acid. This bacillus de- 
velops at the room temperature in 
the usual culture media, in the ab- 
sence of oxygen, but it grows best 
in those to which 1.5 to 2 per cent, 
of glucose or 5 per cent, of glycerin 
has been added. 
Growth on Agar.-The colonies on 
agar are somewhat more compact 
than those of malignant edema, but 
they also send out projections very 
often. In agar-stick cultures, in the 
incubator, growth occurs after a 
day or two also some distance be- 
low the surface, and is accompanied 
by the production of gas and a 
peculiar disagreeable acid odor. 
Pathogenicity.-The bacillus of symptomatic anthrax is pathogenic 
for cattle (which are immune against malignant edema), sheep, goats, 
guinea-pigs, and mice; horses, asses, and white rats, when inoculated 
with a culture of this bacillus, present only a limited reaction; and 
rabbits, swine, dogs, cats, chickens, ducks, and pigeons are, as a rule, 
naturally immune to the disease. The guinea-pig is the most susceptible 
of test animals. When susceptible animals are inoculated subcutan- 
eously with pure cultures of this organism, or with spores attached to 
a silk thread, or with bits of tissue from the affected parts of another 
animal dead of the disease, death ensues in from twenty-four to thirty- 
six hours. At the autopsy a bloody serum is found in the subcutaneous 
tissues, extending from the point of inoculation over the entire surface 
Fig. 160 
Bacilli of symptomatic anthrax, showing 
spores. (After Zettnow.) BACILLUS ANTHRACIS SYMPTOMATICI 
459 
of the abdomen, and the muscles present a dark red or black appearance, 
even more intense in color than in malignant edema, and there is a 
considerable development of gas. The lymphatic glands are markedly 
hyperemic. 
The disease occurs chiefly in cattle, more rarely in sheep and goats; 
horses are not attacked spontaneously-i. e., by accidental infection. 
In man infection has never been produced, though ample opportunity 
by infection through wounds in slaughter-houses and by ingestion of 
infected meat has been given. The usual mode of natural infection 
by symptomatic anthrax is through wounds which penetrate not only 
the skin, but the deep, intercellular tissues; some cases of infection 
by ingestion have been observed. The pathologic findings present the 
conditions above described as occurring in the experimental infection. 
Distribution Outside of the Body.-Symptomatic anthrax, like anthrax 
and malignant edema, is a disease of the soil, but it shows a more 
limited endemic distribution than the former, and is differently dis- 
tributed over the earth's surface than the second of these diseases, 
being confined especially to places over which infected herds of cattle 
have been pastured. It is doubtful whether the bacilli are capable 
of development outside of the body like anthrax. In the form of spores, 
however, reproduction may take place; by contamination with these, 
through deep wounds acquired by animals in infected pastures, the 
disease is spread. 
Toxins.-Under favorable conditions extracellular toxins are formed 
so that the filtrate of cultures is very poisonous. Injections of the 
toxin into animals excite the production of antitoxins. 
Differential Diagnosis.-The principal points of differentiating this 
bacillus from the bacillus of malignant edema, which it closely resembles, 
are: it is smaller; it does not develop into long threads in the tissues; 
it is more actively motile, and forms spores more readily in. the animal 
body than does the bacillus of malignant edema. It is pathogenic 
for cattle, while malignant edema is not; and swine, dogs, rabbits, 
chickens, and pigeons, which are readily infected with malignant 
edema, are not, as a rule, susceptible to symptomatic anthrax. 
Preventive Inoculations.-It is well known to veterinarians that 
recovery from one attack of symptomatic anthrax protects an animal 
against a second infection. Artificial immunity to infection can also 
be produced in various ways: by inoculations with cultures which 
have been kept for a few days at a temperature of 42° to 43° C. and 
have thus lost their original virulence, or by inoculations of filtered 
cultures, or of cultures sterilized by heat. For the production of im- 
munity in cattle it is advised to use a dried powder of the muscles of 
animals which have succumbed to the disease, and which have been 
subjected to a suitable temperature to insure attenuation of the virulence 
of the spores contained therein. Two vaccines are prepared, as in 
anthrax-a stronger vaccine by exposing a portion of the powder to a 
temperature of 85° to 90° C. for six hours, and a weaker vaccine by 
exposing it for the same time to a temperature of 100° to 104° C. In- 460 
PATHOGENIC MICROORGANISMS 
oculations are made with this attenuated virus into the end of the tail 
•-first the weaker and later the stronger. These give rise to a local 
reaction of moderate intensity, and the animal is subsequently immune 
from the effects of the most virulent material and from the disease. 
Fourteen days are allowed to elapse between the two inoculations. The 
results obtained from this method of preventive inoculation seem to 
have been very satisfactory. According to the statistics, including 
many thousand cattle treated, the mortality, which among 22,300 
non-inoculated cattle was 2.20 per cent., has been reduced to 0.16 
per cent, in 14,700 animals inoculated. When danger of immediate 
infection exists, it is advisable to inject some antitoxin with the vaccine. 
This lessens the reaction and gives immediate immunity. 
If ah antitoxic serum is at hand it should be given in cases seen early 
in the disease. 
THE GROUP OF MALIGNANT EDEMA BACILLI. 
This group is widely distributed, being found in the superficial 
layers of the soil, in putrefying substances, in foul water, and by in- 
vasion from the intestine, in the blood of animals which have been 
suffocated. One such organism was discovered (1877) by Pasteur in 
animals after infection with putrid flesh, and named by him " vibrion 
septique." He recognized its anaerobic nature, but did not obtain it 
in pure culture. Koch and Gaffky (1881) carefully studied this micro- 
organism, described it in detail, and save it the name " Bacillus edematis 
maligni" (Fig. 161). 
Fig. 161 
Bacilli of malignant edema. 1, bacilli; 2, with spores; 3 and 4, deep colonies in 
dextrose nutrient agar. (Kolle and Wassermann.) 
In earlier times infection of man was quite often, but now with the 
surgical precaution used it rarely happens. This bacillus belongs to a 
group which have lateral flagellie, produce oval spores, and grow only 
anaerobically. 
Morphology.-The edema bacillus is a rod of from 0.8/z to l/x in 
width, and of very varying length, from 2^ to 10,u or more, according 
to the conditions of its cultivation and growth. It is usually found in THE GROUP OF MALIGNANT EDEMA BACILLI 
461 
pairs, joined end-to-end, but may occur in chains or long filaments. It 
forms spores, and these are situated in or near the middle of the body 
of the rods. Exceptionally the spores are near the ends. The spores 
vary in length and are oval in form, being often of greater diameter 
than the bacilli, to which they give a more or less oval shape. 
The bacilli stain readily by the usual aniline colors employed, but 
are usually decolorized by Gram's method. Freutag found that very 
young cultures were stained, while older ones were decolorized. 
Biology.-A strictly anaerobic, liquefying, motile bacillus. Forms 
spores which are very resistant. It grows in all the usual culture 
media in the absence of oxygen. Development takes place at 20° C., 
but more rapidly and abundantly at 37° C. 
Growth in Gelatin.-This bacillus may be cultivated in ordinary 
nutrient gelatin, but the growth is more abundant in glucose gelatin 
containing 1 or 2 per cent, of glucose. After two or three days small, 
almost transparent, circular colonies appear | to 1 mm. in diameter. 
Later, as liquefaction increases, the colonies become grayish and then 
confluent. Gas bubbles are formed and the gelatin liquefies. 
Growth on Agar.-On agar plates the colonies appear as dull, whitish 
points, irregular in outline, and when examined under a low-power 
lens are seen to be composed of a dense network of interlacing threads, 
radiating irregularly from the centre toward the periphery. 
Blood-serum is rapidly liquefied, with the production of gas. Cul- 
tures of the malignant edema bacillus give off gas with a peculiar, 
disagreeable odor. 
Resistance.-The spores are very resistant and because of this the 
soil remains infected. 
Pathogenicity.-The bacillus of malignant edema is especially patho- 
genic for mice, guinea-pigs, and rabbits, although man, horses, dogs, 
goats, sheep, calves, pigs, chickens, and pigeons are also susceptible. 
A small quantity of a pure culture injected beneath the skin of a sus- 
ceptible animal gives rise to an extensive hemorrhagic edema of the 
subcutaneous connective tissue, which extends over the entire surface 
of the abdomen and thorax, causing hyperemia and redness of the 
superficial muscles. No odor is developed, and there is little, if any, 
production of gas. In infection with garden earth, owing to the presence 
of associated bacilli, the effused serum is frothy from the development 
of gas and possesses a putrefactive odor. The disease, in natural 
infection caused by the contamination of wounds with earth or feces, 
runs the course above described. Simple abrasion of the skin is not 
sufficient to produce infection; owing to the bacillus being capable 
only of an anaerobic existence, the poison must penetrate deep into the 
tissues. Malignant edema is confined mostly to the domestic animals, 
the horse, sheep, cattle, and swine, but cases have also been reported 
in man. 
Animals which recover from malignant edema are subsequently im- 
mune. Artificial immunity may be induced in guinea-pigs by injecting 
filtered cultures of the malignant edema bacillus in harmless quantities. 462 
PATHOGENIC MICROORGANISMS 
In man the chief symptom is the sudden appearance of subcutaneous 
edematous swelling accompanied by high fever. In light cases this 
remains circumscribed; in severe cases it spreads widely and the 
case ends fatally. Appreciable quantities of gas usually fail. Autopsy 
shows a serous or hemorrhagic infiltration of the subcutaneous tissue 
and intramuscular connective tissue. In the inflamed tissue the bacilli 
with and without spores are found. 
Prevention.-Most cases are produced by injecting subcutaneously 
albuminous fluids infected by the bacilli. Care should be taken that 
fluids to be injected do not become infected by dust or dirt. 
BACILLUS AEROGENES CAPSULATUS (BACILLUS WELCHII). 
This bacillus was found by Welch in the bloodvessels of a patient 
suffering with aortic aneurysm; on autopsy, made in cool weather, 
eight hours after death, the vessels were observed to be full of gas 
bubbles. Since then it has been found in a number of cases in which 
gas has developed from within sixty hours of death until some hours 
after death. External cutting operations on the urethra and operations 
upon the uterus have been followed in a number of cases by infection. 
It has been found in ovarian abscesses and in infections of the genito- 
urinary tract. These cases are, as a rule, marked by delirium, rapid 
pulse, high temperature, and the development of emphysema and dis- 
Fig. 162 
Bacillus aerogenes capsulatus. 1, bacilli; 2, spores; 3, culture in dextrose nutrient agar. 
coloration of the diseased area or of marked abdominal distention 
when the peritoneal cavity is involved. This bacillus is present, as 
a rule, in the intestinal canal of man and animals and is apt to be found 
in the dust of hospitals and elsewhere. Herter1 has shown that it is 
present in excessive numbers in certain diseases of the digestive tract. 
These cases are apt to develop anemia. 
Morphology.-Straight or slightly curved rods, with rounded or 
sometimes square-cut ends; somewhat thicker than the anthrax bacilli 
1 Journal of Biolog. Chem., 1906, ii, p. 1. BACILLUS AEROGENES CAPSULATUS 
463 
and varying in length; occasionally long threads and chains are seen. 
The bacilli in the animal body, and sometimes in cultures, are enclosed 
in a transparent capsule. Spores are usually absent in the tissues and 
often in cultures. Dunham showed that the culture isolated by Welch 
formed spores when grown on blood-serum. Some strains since isolated 
make spores readily. It is possible that these differences may be due 
to the fact of there being several strains. 
Biology.-An anaerobic, non-motile, non-liquefying bacillus. Dif- 
ferent strains of this bacillus vary in their tendency to make spores. 
It is stained by Gram, but is more easily decolorized than many bacteria. 
Growth is rapid at 37° C., in the usual culture media in the absence of 
oxygen, and is accompanied by the production of gas. Nutrient gelatin 
is not liquefied by the growth of this bacillus, but it is gradually 
peptonized. If agar colonies are developed which are from 1 to 2 mm. 
or more in diameter, grayish-white in color, and in the form of flattened 
spheres, ovals, or irregular masses, beset with hair-like projections. 
Bouillon is diffusely clouded, and a white sediment is formed. Milk 
becomes acidified and coagulated, then partially digested, giving a 
worm-eaten appearance to the clot. 
Pathogenicity.-Usually non-pathogenic in healthy animals, although 
Dunham found that the bacillus taken freshly from human infection 
is sometimes very virulent. When quantities up to 2.5 c.c. of fresh 
bouillon cultures are injected into the circulation of rabbits and the 
animals killed shortly after the injection, the bacilli develop rapidly, 
with an abundant formation of gas in the bloodvessels and organs, 
especially the liver. This procedure is one of the best methods of 
obtaining the bacilli: The material suspected to contain the bacillus 
alone or associated with other bacteria is injected intravenously into 
rabbits, which are killed five minutes later and kept at 37° C. for sixteen 
hours, and cultures made from the liver and heart's blood. 
It is suggested by Welch that in some of the cases in which death 
has been attributed to the entrance of air into the veins the gas found 
at the atuopsy may not have been atmospheric air, but may have 
been produced by this or some similar microorganism entering the 
circulation and developing shortly before and after death. The same 
may be true for gas in the uterine cavity. 
Bacillus Enteritidis Sporogenes.-This resembles B. Welchii. It 
produces spores readily. Klein considers that when taken in milk it 
may produce diarrhea. This is disputed by others. It is also considered 
to be an evidence of sewage pollution, but this is not at all certain since 
it occurs in cultivated soils (Jordan). CHAPTER XXXIV. 
THE CHOLERA SPIRILLUM (CHOLERA VIBRIO) AND 
SIMILAR VARIETIES. 
In 1883 Koch separated a characteristically curved organism from 
the dejecta and intestines of cholera patients-the so-called "comma 
bacillus." It was absent from the blood and viscera, and was found 
only in the intestines; and the greater the number, it was said, the more 
acute the attack. Koch also demonstrated an invasion of the mucosa 
and its glands. The organisms were found in the stools on staining 
the mucus flakes or the fluid with methylene blue or fushsin, and 
sometimes alone; by means of cultivation on gelatin they were readily 
separated from the stools. Numerous-control observations made upon 
other diarrhoeic dejecta and upon normal stools were negative; the 
comma bacillus was found in choleraic material only, or occasionally 
in small numbers in the stools of healthy persons who came in contact 
with cholera. Soon, however, other observers described comma-shaped 
organisms of non-choleraic origin. Finkler and Prior, for instance, 
found them in the diarrheal stools of cholera nostras, Deneke in cheese, 
Lewis and Miller in saliva. All of these organisms, however, differed 
in some respects from Koch's comma bacillus, and it has since been 
proved that none of them is affected by the specific serum of animals 
immunized to cholera. After a time, therefore, the exclusive association 
of Koch's vibrio with cholera or those in contact with it became almost 
generally ackowledged, until now it is regarded by bacteriologists 
everywhere to be the specific cause of Asiatic cholera. Certain sporadic 
cases of cholera-like disease, however, are undoubtedly due to other 
organisms. 
Morphology.-Curved rods with rounded ends which do not lie 
in the same plane, of an average of 11^ in length and about 0.4/z in 
breadtht The curvature of the rods may be very slight, like that of a 
comma, or distinctly marked, particularly in fresh unstained prepara- 
tions where the adhesion of two individuals presents the appearance 
of a half-circle. By the inverse junction of two vibrios S-shaped forms 
are produced. Longer forms are rarely seen in the intestinal discharges 
or from the cultures grown on solid media, but in fluids, especially 
when grown under unfavorable conditions, long, spiral filaments may 
develop. The spiral forms are best studied in the hanging drop, for 
in the dried and stained preparations the spiral character of the long 
filaments is often obliterated. In film preparations from the intestinal 
contents in typical cases it will be found that the organisms are present 
in enormous numbers, and often in almost pure culture. In old cultures BIOLOGY 
465 
irregularly clubbed and thickened involution forms are frequent, and 
the presence in the organisms of small, rounded, highly refractile bodies 
is often noted. 
Staining.-The cholera spirillum stains with the aniline colors 
usually employed, but not as readily as many other bacteria; a diluted 
aqueous solution of carbol fuchsin (1-10) is recommended as the most 
reliable staining agent. It is decolorized by Gram's method. The 
organisms exhibit one long, fine flagellum attached to one end 
(Cholera-like spirilla often have two or more end flagella.) In sections 
the vibrios are stained best by alkaline methylene-blue solution and 
washed in water slightly acidulated with acetic acid. 
Fig. 163 
Fig. 164 
Contact smear of colony of cholera 
spirilla from agar. X 700 diameters. 
(Dunham.) 
Cholera spirilla preparation from gela- 
tin-plate culture of cholera. -X 800 diam- 
eters. 
Biology.-The cholera spirillum is aerobic, liquifying, and extremely 
motile. It grows readily on ordinary culture media, best at 37° C., 
but also at room temperature, 22° C. 
In gelatin plate cultures, characteristic colonies are produced. After 
twenty-four hours' growth, there is a uniform granular appearance of 
the surface, which because of the high degree of refraction gives the 
appearance of being powdered with broken glass. Color is practically 
absent or there may be a slight yellowish tint. The absence of color 
aids one in separating the cholera colony from those developing from 
coli in the stool. If growth is continued, liquefaction appears about 
the colony and the appearance gradually changes. The characteristic 
colony in gelatin has been of the greatest practical importance. With 
the introduction of special selective media, however, the use of gelatin 
has been practically discontinued. In gelatin stab-culture, a small 
funnel of liquifaction appears after twenty-four hours. This deepens 
and broadens, until at the end of a week liquifaction may be complete. 
On agar a moist shiny grayish-yellow layer develops. On the surface 
of agar-plates the individual colonies are characteristic. They are 
30 466 
PATHOGENIC MICROORGANISMS 
round, transparent and have a rather distinctive opalescent sheen. 
This characteristic appearance is used practically for isolation. 
On Diendonne alkaline blood-agar the colonies are large and circular. 
On transmitted light there is a glassy transparency, on reflected light 
they are grayish. On alkaline egg-agar the colonies, when examined 
by transmitted light, appear to be deep in the agar and have a dis- 
tinctive hazy appearance due to the development of a halo about 
the colony. A zone of clearing may develop where growth is continued. 
As some fecal bacteria other than vibrios may develop on these media, 
the development of a typical colony is a great aid in isolation. 
Fig. 165 
Colera colonies in gelatin; twenty-four to thirty-six hours' growth. 
X about 20 diameters. 
Loffler's blood-serum is rapidly liquified at 37° C. Milk is not coagu- 
lated. Glucose and saccharose are fermented with great rapidity, 
acid only being formed. Growth in fluid media is abundant and usually 
characteristic, most so in peptone-water. Peptone-water is diffusely 
cloudy to a moderate degree, but at the surface the cloud is much 
denser. Due to the greater supply of oxygen, the vibrios seek the 
surface and multiply here more freely. Cholera strains which have 
been in cultivation for some time show changes in morphology and in 
the appearance of colonies. They grow less typically on selective media 
and fluid cultures may develop a wrinkled pellicle. Their digestive 
powers are also lessened, as gelatin-liquefaction or the liquefaction of 
Loffler's serum medium. Variations can also be induced by special 
conditions. 
Reaction of Media.-Cholera grows best on media that are sharply 
alkaline to litmus. They can grow in an excess of alkali sufficient to 
inhibit the growth of many of the bacteria found in feces. This is 
most evident where an alkali-albumin mixture is used to increase the 
alkalinity. (See Special Media.) 
Cholera-red Reaction.-All cholera strains give this reaction. This 
is important and many non-cholera spirillae do not give this reaction. 
Hemolytic Production.-Six strains were isolated by Gottshlich at 
El Tor from Pilgrims to Mecca, who died with diarrheal symptoms but 
had shown no clinical evidences of cholera. These strains are called RESISTANCE AND VITALITY 
467 
"El Tor" strains. They give the serum reactions of cholera vibrios 
but produce a strong hemolysin. Kraus and Ruffer believed them not 
to be true cholera, as hemolysin production was considered by the 
former as an attribute possessed only by non-cholera types. Further 
investigations have shown that cholera vibrios may develop, lose, or 
show wide variations in their ability to produce hemolysins. It is 
apparently a potential power of all vibrios and of no value in differen- 
tiation. For this reason the El Tor strains must be considered as 
cholera vibrios. 
Resistance and Vitality.-Development Outside of Body.-If a culture 
be spread on a cover-glass and exposed to the action of the air at room 
temperature the bacilli will be dead at the end of two or three hours, 
unless the layer of culture is very thick, in which case it may take 
twenty-four hours or more to kill all the bacilli. This indicates that 
infection is rarely if ever produced by means of dust or other dried 
objects contaminated with cholera bacilli. The transmission of these 
organisms through the air, therefore, can only take place for short 
distances, as by the spray from infectious liquids. 
The cholera bacillus is also injuriously affected by the abundant 
growth of saprophytic bacteria. It is true that when associated with 
other bacteria, if present in large numbers, and if the conditions for 
their development are particularly favorable, the cholera bacillus 
may at first gain the upper hand, as in the moist linen of cholera 
patients, or in soil impregnated with cholera dejecta; but later, after 
two or three days, even in such cases, the bacilli die off and other 
bacteria gradually take their place. Thus, Koch found that the fluid 
contents of privies twenty-four hours after the introduction of comma 
bacilli no longer contained the living organisms; in impure river- 
water they were not demonstrable for more than six to seven days, 
as a rule. In the dejecta of cholera patients they were found usually 
only for a few days (one to three days), though rarely they have been 
observed for twenty to thirty days, and on one occasion for one hundred 
and twenty days. In unsterilized water they may also retain their 
vitality for a relatively long time; thus, in stagnant well-water they 
have been found for eighteen days, and in an aquarium containing 
plants and fishes, the water of which was inoculated with cholera germs, 
they were isolated several months later from the mud at the bottom. 
Koch found them in the foul water of a tank in India, used by the 
natives for drinking purposes. In running river-water, however, they 
have not been observed for over six to eight days. In milk they are 
finally destroyed by acidity due to the growth of the milk bacteria, 
in sterilized milk they may survive eight to ten days. For the cholera 
organisms the conditions favorable to growth are a warm temperature, 
moisture, a good supply of oxygen, and a considerable proportion of 
organic material. These conditions are fully met with outside the 
body in but very few localities. 
The common bacillus has the average resistance of spore-free bacteria, 
and is killed by exposure to moist heat at 56° C. in half an hour, at 468 
PATHOGENIC MICROORGANISMS 
80° for five minutes, at 95° to 100° C. in one minute. The bacilli have 
been found alive kept for a few days in ice, but ice which has been 
preserved for several weeks does not contain living bacilli. 
Chemical disinfectants readily destroy the vitality of cholera vibrios. 
For disinfection on a small scale, as for washing the hands when con- 
taminated with cholera infection, a 0.1 per cent, solution of bichloride 
of mercury, or a 2 to 3 per cent, solution of carbolic acid or, better, 
lysol may be used. For disinfection on a large scale, as for the disin- 
fection of cholera stools, strongly alkaline milk of lime is an excellent 
agent. The wash of cholera patients, contaminated furniture, floors, 
etc., may be disinfected by a solution of 5 per cent, carbolic acid and 
soap water. For the disinfection of drinking water, chlorinated lime, 
1 to 2 parts per million of free chlorine may be used for fairly pure 
water. Five parts per million would probably be effective even in 
polluted water. 
Pathogenesis.-Not one of the lower animals is naturally subject to 
cholera. It has been shown that the comma bacillus is extremely 
sensitive to the action of acids, and is quickly destroyed by the acid 
secretions of the stomach of man or the lower animals, when these 
secretions are normally produced. Koch sought to produce infection 
in guinea-pigs per vias natu rales by first neutralizing the contents of 
the stomach with a solution of carbonate of soda and inhibiting peri- 
stalsis by the use of opium. Animals thus treated in six to eighteen 
hours after infection show an indisposition to eat and other signs of 
weakness, its posterior extremities become weak and apparently para- 
lyzed, and, as a rule, death occurs within forty-eight hours with the 
symptoms of collapse and fall of temperature. At the autopsy the small 
intestine is found to be congested and filled with a watery fluid, con- 
taining the spirillum in great numbers. These results, however, are 
somewhat weakened by the fact that experiments made with some 
other bacteria morphologically similar to the comma bacillus of Koch 
but specifically different, occasionally produced death when introduced 
in the same way into the small intestines of guinea-pigs. Later, several 
investigators have succeeded in producing in animals a disease similar 
to cholera. Thomas injected a dilute suspension of cholera spirilla 
into the ear vein of young rabbits, and thus caused symptoms and 
lesions of the intestinal mucous membrane resembling those of cholera 
in man. The spirillum was recovered from the feces. Metchnikoff 
was similarly successfid with suckling rabbits by rubbing a small 
amount of a culture on the teats of a mother rabbit. Intraperitoneal 
injections with cholera spirilla kill guinea-pigs acutely, but intestinal 
lesions are rarely found. 
There are several cases on record which furnish the most satisfactory 
evidence that the cholera spirillum is able to produce the disease in 
man. In 1884 a student in Koch's laboratory in Berlin, who was taking 
a course on cholera, became ill with a severe attack of cholera. At 
that time there was no cholera in Germany, and the infection could 
not have been produced in any other way than through the cholera COMM UNICABILIT Y 
469 
cultures which were being used for the instruction of students. In 
1892 Pettenkofer and Emmerich experimented on themselves by 
swallowing small quantities of fresh cholera cultures obtained from 
Hamburg. Pettenkofer was affected with a mild attack of cholerine 
or severe diarrhea, from which he recovered in a few days without any 
serious effects, but Emmerich became very ill. On the night following the 
infection he was attacked by frequent evacuations of the characteristic 
rice-water type, cramps, tympanites, and great prostration. His voice 
became hoarse, and the secretion of urine was somewhat diminished; 
this condition lasting for several days. In both cases the cholera 
spirillum was obtained in pure culture from the dejecta. Finally, there 
is the case of Dr. Oergel, of Hamburg, who accidentally, while experi- 
menting on a guinea-pig, allowed some of the infected peritoneal fluid 
to squirt into his mouth. He was taken ill and died a few days after- 
ward of typical cholera, though at the time of his death there was no 
cholera in the city. 
Lesions in Man.-Cholera in man is an infective process of the 
epithelium of the intestine, in which the spirilla clinging to and between 
the epithelial cells produce a partial or entire necrosis and final destruc- 
tion of the epithelial covering, which thus renders possible the absorp- 
tion of the cholera toxin formed by the growth of the spirilla. The 
larger the surface of the mucous membrane infected and the more 
luxuriant the development of bacilli and the production of toxin, the 
more pronounced will be the poisoning, ending fatally in a toxic paralysis 
of the circulatory and thermic centres. On the other hand, however, 
there may be cases where, in spite of the large number of cholera bacilli 
present in the dejecta, severe symptoms of intoxication may be absent. 
In such cases the destruction of epithelium is not produced or is so 
slight that the toxic substance absorbed is not in sufficient concentration 
to give rise to the algid stage of the disease, or for some reason the 
spirilla do not produce toxin to any extent. In no stage of the disease 
are living cholera spirilla found in the organs of the body or in the 
secretions. 
Distribution in the Body.-The cholera spirilla are found only in 
the intestines and are believed never to be present in the blood or 
internal organs. The lower half of the small intestine is most affected, 
a large part of its surface epithelium becoming shed. The flakes floating 
in the rice-water discharges consist mostly of masses of epithelial cells 
and mucus, among which are numerous spirilla. The spirilla also 
penetrate the follicles of Lieberkuhn, and may be seen lying between 
the basement membrane and the epithelial lining, which become loosened 
by their action. They are rarely found in the connective tissue beneath, 
and never penetrate deeply. In more chronic cases other microorganisms 
play a greater part and deeper lesions of the intestines may occur. 
Communicability. Origin of Epidemics.-The two fundamental 
epidemiological facts are, that the vibrio leaves the body only in the 
feces, and the mode of infection is by way of the mouth. The feces 
of the cholera patient during the acute stage of the disease is extremely 470 
PATHOGENIC MICROORGANISMS 
rich in vibrios, which are at times present in almost pure culture. As 
the case recovers they decrease in number, but persist after recovery 
for seven to fourteen days, in exceptional instances longer. In only 
one case have they persisted for three months, that is, chronic carriers 
do not exist. These persons constitute the "convalescent carriers." 
In this connection, the mild cases which are undiagnosed or overlooked 
are important. Another group of persons may act as sources of infec- 
tion, viz., excrete cholera in their stools. These are the " healthy carriers." 
Not all persons who ingest cholera develop the disease. In a number, 
the vibrio will multiply in the intestine to a limited extent and be 
excreted in the stools, although no clinical evidences of disease are 
present. These healthy carriers are important not only as insidious 
spreaders of infection, but they may be potential cases of cholera. 
Should their resistance be lowered they may develop the disease. 
The transfer of the infectious agent to the mouth may occur in 
several ways: by personal contact, by fomites, and by contamination 
of food and water. Where a moderate number of cases are developing 
in a district having fair sanitary conditions, contact, especially with 
mild cases and carriers, or indirectly, fomites or infection of food are 
the sources of infection. As other factors enter, such as the contamina- 
tion of the soil and privy-vaults with subsequent infection of well- and 
river-water and of green vegetables, the number of cases increase in 
number. Where a general water supply becomes contaminated an 
explosive, wide-spread epidemic follows. It is easy to understand 
how localized epidemics are kept active, by the insidious development 
of healthy carriers, who help maintain the contamination of their 
surroundings; and, given also the climatic conditions, such as heat 
and moisture, to favor the continued vitality or even multiplication 
outside of the body, how endemic foci are maintained. The transfer 
of infection by fomites, such as body and bed linen or dishes, etc., is 
only dangerous when direct. Drying quickly lessens the danger. 
Flies may be a factor by mechanical transfer of the virus. 
The susceptibility to infection of different individuals varies and 
conditions may lower or raise the resistance of the individual. The 
occurence of healthy carriers illustrates this. Such carriers may be 
very numerous. Abel and Clausen, for example, found that 14 of 17 
persons belonging to families of 7 cholera patients, had cholera vibrios 
in their stools. In a group of immigrants who were exposed, we found 
10 per cent, were healthy carriers. 
The resistance of the individual depends partly on antibodies and 
partly on his health. Gastric and intestinal disorders due to indiscreet 
eating or drinking, or other causes undoubtedly favor infection or in 
the case of healthy carriers may cause the development of cholera. 
At the New York Quarantine Station two such cases promptly developed: 
one after the administration of a dose of salts, the other after a drinking 
bout. 
Cholera Toxins.-Koch assumed that the severe symptoms of cholera 
were due to the absorption of a toxin produced by the growth of the PROPHYLACTIC INOCULATION 
471 
vibrio in the intestines/ The toxic effects are apparently due to sub- 
stances, which are an integral part of the organism and are only liberated 
by the breaking down of the vibrio. Suspensions of killed vibrios, when 
injected into animals, give the same symptoms as living cultures, 
although quantitatively less toxic. The endotoxin is labile, and is 
best shown in cultures which are killed by chloroform or by heating 
to 56° C. for one hour. More active chemicals or a higher degree of 
heat changes it from a more specific toxin to a more general protein 
poison. The bacteria-free filtrates of fresh fluid cultures is only slightly 
toxic; old cultures, however, due to breaking down of the vibrios may 
be very toxic. This toxicity is due mostly to substances similar in 
action to the general class of ptomaines. Kraus has, however, been 
able to demonstrate, what he considers an extra-cellular toxin, in young 
broth cultures. Metchnikoff and Roux have also attempted to prove 
the existence of an extra-cellular toxin by growing cholera vibrios 
in collodion sacs implanted in the peritoneum of guinea-pigs. The 
production of an antitoxin in the Ehrlich sense against such toxins has 
not been accomplished. 
Cholera Immunity.-Eight to ten days after recovery the serum 
of cholera patients contains protective substancs. If a guinea-pig is 
injected intraperitoneally with living cholera vibrios, and serum from 
the patient be given, the pig recovers. Similar protective substances 
are found in the serum of animals injected with sublethal doses of 
live vibrios or with killed organisms. The serum is not antitoxic, for 
although it will protect an animal from a lethal dose of living vibrios, 
by preventing their multiplication, it has little effect, when a fatal dose 
of killed organisms or toxic extracts is given. Similarly an animal 
may be highly immune to the injection of living cultures but on intestinal 
infection will show no resistance to the poisonous products absorbed. 
The antibodies present in the serum are precipitins, agglutinins, 
opsonins, bacterilytic and bactericidal substances. 
Prophylactic Inoculation.-Haffkine, Kolle, and others have demon- 
strated the value of the prophylactic inoculation against cholera. 
Haffkine used live cultures following the principles of Pasteur. He 
first injected a culture attenuated by prolonged cultivation at 39° C.; 
a second injection was given of a culture which was passed rapidly 
through animals. Kolle advised a more practicable method, viz., a 
vaccine killed by heating to 58° C. for one hour and standardized so 
that 1 c.c. contained 2 mgms. of culture. At first a dose of 1 c.c. was 
used later 2 c.c. were given. A marked fall in the number of cases 
among the vaccinated was noted when the dose was increased. An 
example of the results of vaccination is the following: Among 825,287 
persons, unvaccinated, 0.13 per cent, developed cholera; of 77,907 
vaccinated, 0.06 per cent, developed cholera. The mortality of those 
infected was 75 per cent, among the unvaccinated, but was reduced 
to 42 per cent, among the vaccinated. The duration of immunity 
is about a year. It is probable that if three doses of vaccine were given 
like in typhoid, better results and longer immunity would follow. 472 
PATHOGENIC MICROORGANISMS 
Serum Therapy.-An immune serum can be produced which will 
be very rich in immune bodies but have little antitoxic value. If 
guinea-pigs be inoculated intraperitoneally with a virulent culture, 
they can be saved by a small amount of such a serum. If, however, 
an hour elapse before the serum is given, a large dose of the serum 
may destroy the vibrios, but the endotoxin liberated from the organisms, 
which have multiplied in the interim, will kill the pig. Attempts have 
been made to obtain a more antiendotoxic sera by various methods of 
immunization. Such sera have been tried in cases of cholera. Intra- 
venous doses of 100 c.c. or more in 400 to 600 c.c. of saline solution 
given as early as possible seem to have some effect on the death rate. 
Specific Serum Reactions.-Agglutinins.-Because of the acuteness 
of the disease, the agglutionation reaction is valueless for diagnosis. 
It may be of help in recovered cases, where no bacteriological diagnosis 
has been made. Agglutination is used, however, for identification of 
the cholera vibrio. In this way cholera and non-cholera vibrios can 
be separated with certainty, because a cholera immune serum is very 
poor in group agglutinins for non-cholera types. Freshly isolated 
strains agglutinate freely; in fact, seem to give better reactions than 
stock cultures, although strains vary in their agglutinability; relatively 
inagglutinable strains such as are encountered among freshly isolated 
strains of typhoid are not found. 
Pfeiffer Phenomenon.-This phenomenon, which is a bactericidal 
test in vivo, can be employed to differentiate the cholera vibrio from 
other vibrios. The bactericidal serum should have a high titer, viz., 
1 c.c. of a 1 : 1000 dilution should be able-to dissolve a loop of cholera 
vibros when injected intraperitoneally together. Each factor in the 
test must be controlled. 
ISOLATION OF CHOLERA VIBRIO FROM STOOLS. OUTLINE 
OF PROCEDURE. 
Feces.- 
Peptone-water 
Peptone-water. 
Direct 
Microscopic 
Examination 
Selective Media 
Smear, if positive, hanging drop 
with and without serum. Plate 
। for pure cultures for verifica- 
I tion, etc. 
Agglutination of 
suspicious colony. 
Direct Microscopic Examination.-This is of great value in suspected 
cases but of no use in the examination of carriers. The presence of 
many typical, extremely motile vibrios warrants a tentative diagnosis 
of cholera. In exceptional cases, not cholera, a great abundance of 
vibrios may be found. This led us in one instance to unnecessarily 
isolate a nurse who, while caring for a cholera patient, developed nervous 
diarrhea. ISOLATION OF CHOLERA VIBRIO FRO AI STOOLS 
473 
Peptone-water.-Inoculate with feces and incubate six to twelve hours. 
Examine smears from surface growth. If the vibrios are numerous, pre- 
pare hanging drops with and without immune serum. If vibrios are not 
found in the smear, or if too few in number for hanging drop observa- 
tion, subinoculate into peptone-water, or selective media, or both. 
Subculture Peptone-water. - This second enrichment is probably 
never required except in the examination of suspected carriers. In 
four instances we have found cholera vibrios in subculture where they 
were not evident in smears from the first peptone culture. This second 
enrichment helps also to exclude some of the vibrios other than cholera, 
some will have died out, some will not have enriched beyond the amount 
present in the first peptone culture; these are not cholera. Smears 
from the surface growth are examined and if positive, examination 
of hanging drops carried on Where haste is not a factor as in 
carrier examinations the examination of the first peptone culture may 
be omitted. 
In making smears and hanging drops from peptone-water especial care 
should be taken not to shake the tubes and that the loop is just touched 
to the surface. In the examination of smears little time need be spent; 
if very few vibrios are present, further enrichment is necessary. If 
they are present in sufficient numbers for agglutination, they are found 
at once. 
The use of the surface growth for agglutination is open to certain 
criticisms, but in practical work it has given us accurate results. Where 
cholera or cholera-like vibrios, having the same ability to enrich as 
true cholera, are present, the surface growth is nearly a pure culture. 
The reliability of the peptone method, as outlined, was^best shown 
in a series of examinations where we found two carriers. In this series 
50 per cent, of the stools contained vibrios. The results were checked 
by the isolation of pure cultures. With some experience a great number 
of examinations can be carried through in this way, using peptone- 
water only, with a minimum of preparation and equipment. 
Selective Media.-Inoculation may be done directly from the feces 
or after enrichment in peptone-water. The advantages of such media 
is that they may be heavily inoculated. The colonies which develop 
are used for agglutination. Various modifications of Dieudonne's 
have been suggested. The alkaline-egg medium has the advantage 
that a distinctive vibrio colony develops allowing quick selection for 
agglutination should non-vibrios develop. A secondary plating on 
plain agar is necessary, if pure cultures are to be obtained from selective 
media. The other bacteria, which are only restrained, may be trans- 
ferred in fishing a colony and thus yield mixed cultures. 
Alkaline agar may be employed for plating either directly from the 
feces or from peptone enrichments. As stated, the colony is distinctive 
and no difficulty will be encountered if the vibrios are fairly numerous.1 
1 The direct plating of stools on agar or gelatin is sometimes of practical importance. 
In no disease but cholera do vibrio colonies approach or exceed in number those develop- 
ing from the ordinary fecal bacteria. Even without identification by agglutination, such 
a condition gives us a practically certain diagnosis of cholera. 474 
PATHOGENIC MICROORGANISMS 
Saccharose Peptone-water.-This medium has been suggested by 
Bendick to avoid the time-consuming microscopic examination of 
peptone tubes. The stool is first inoculated in peptone-water and the 
surface growth then inoculated into this medium. Because of the ability 
of the cholera vibrio to rapidly split saccharose, decolorization occurs 
in five to eight hours. The tubes which do not decolorize in this time 
can be discarded, the decolorized are examined for vibrios, which, if 
present, are isolated by plating. 
liecause of the presence of a fermentable sugar, the growth is diffused 
and the surface is not satisfactory for agglutination. To avoid this 
difficulty duplicate peptone tubes could be planted, however, and used 
for agglutination where the saccharose peptone tubes were decolorized. 
This method promises to be of the greatest value where many specimens 
must be examined in the search for carriers. 
Examination of Suspected Carriers.-The simplest procedures possible 
must be employed where the daily examinations may run into many 
hundreds. The peptone method outlined reduces the bacteriological 
work very much, especially if the first peptone tubes are not examined. 
The collection of stools under these circumstances is impracticable. 
Ordinary swabs moistened in peptone-water may be inserted into the 
rectum to obtain fecal material. Individual glass specula may be 
employed to aid in the introduction of the swabs. Where feasible much 
time is saved by giving an identification card a number and dropping 
the swab at the time of taking into a similarly numbered tube of peptone- 
water. The sterilization of the peptone tubes, supported in blocks of 
wood or racks with a cover instead of using cotton plugs and the use 
of individual wire loops (sterilized in bundles) for transfers or smears 
saves a great deal of time. The removal and replacing of cotton plugs 
and the burning of the platinum loop usually employed is thus avoided. 
In transferring the wire is dropped into the second tube. The exposure 
of the tubes when the cover is removed for inoculation or transfer 
does not lead to interference by contamination.1 
Isolation from Water.-As the cholera vibrios are few in number in 
water a large volume should be used. About a liter of water is taken 
and 100 c.c. amounts placed in Ehrlenmeyer flasks and to each is added 
10 c.c. of a tenfold strength peptone-water. These are shaken and 
incubated for eighteen hours. The surface growth is then subinoculated 
in peptone-water for further enrichment if necessary and plates made 
for isolation. Especial care must be taken in fishing as vibrios other 
than cholera may be present in the water, and there is some evidence 
that cholera vibrios lose their agglutinability to some extent after 
living in water. These difficulties may be surmounted by many fishings 
and the use of a high titer serum in low dilutions for selecting cultures 
for final identification. 
Spirilla (Vibrios) More or Less Allied to the Cholera Spirillum.-Various 
types of spirilla may be isolated from stools, water, and other sources. 
1 Creel, Amer. Journ. Public Health, December, 1911. ISOLATION OF CHOLERA VIBRIO FROM STOOLS 
475 
Some are practically identical, morphologically and culturally, with 
true cholera. It would be well to limit the term "cholera-like vibrios" 
to this group. Another group of vibrios, similar to cholera in some 
respects but differing in others might be termed " non-cholera vibrios. 
Much of the practical interest in these types was lost when the sero- 
logical methods for identification were introduced. In emergency 
work, where immune serum is not at hand, they are of extreme practical 
importance. A few of the types are of interest because of their patho- 
genicity for animals. 
Some of these vibrios enrich in mixed cultures in peptone-water like 
true cholera, others enrich to a limited extent or die out. The majority 
of the vibrios found in stools during routine examinations for cholera 
spirilla can be excluded culturally if serum be not available. Of 50 
vibrios thus isolated we found that 43 did not give the cholera-red 
reaction. Of the 7 that did, 2 produced no acid from glucose and 4 
produced acid and gas. One produced acid only, but could be excluded, 
as it formed a tenacious pellicle on peptone-water. 
The following is a short list of vibrios of interest historically or 
because of their pathogenicity for animals: 
Vibrio Metschnikovii; source, epizootic gastro-enteritis of fowls, present in in- 
testinal contents and in blood. Typical vibrio with one flagellum, liquifies 
gelatin and gives cholera-red reaction. A minute amount of culture inoculated 
into a cutaneous wound causes a fatal vibriosepticemia in pigeons and guinea- 
pigs; 
Vibrio Massa vah; source stools, considered cholera vibrio when first isolated, 
four flagellae, pathogenicity like vibrio Metschnikovii. 
Vibrio septicus; source, stools, case of cholerine, cholera-like culturally and 
morphologically, minute amounts cause a rapidly fatal septicemia in guinea-pigs. 
Vibrio Nasik, produces a very strong toxin, 0.5 to 1 c.c. of a four-day broth 
culture intravenously kills rabbits in 10 to 15 minutes, a strong hemolytic 
toxin is produced. 
Spirillum Finkler-Prior; sources feces, cholera nostras, does not give cholera- 
red reaction. 
Vibrio Ivanoff and vibrio Berlionensis; source, former, artificially inoculated 
stools for disinfection tests, latter, water artificially inoculated to determine 
viability of cholera vibrio in water. Both are probably variants produced by 
artificial conditions, they give the immune reactions of cholera spirilla. CHAPTER XXXV. 
PATHOGENIC MICROORGANISMS BELONGING TO THE 
HIGHER BACTERIA (TRICHOMYCETES). 
Observers are still of different opinions in regard to the classifi- 
cation of this group of organisms (see Chapter II). 
Foulerton and his associates have made an extensive study of this group, 
both saprophytic and parasitic varieties, and they agree with some others in cal- 
ling attention to the acid-fast character of some of the varieties and to the appar- 
ent relationship of the group to B. tuberculosis, B. mallei, and B. diphtheria. 
To us, however, the relationship does not seem to be close enough to place all of 
these organisms in one group. We have shown (Chapter II) that the apparent 
branching in B. diphtheria is not a true branching. If not classed with the true 
bacteria, they should either be put in a group by themselves or be classed with 
the cladothrix group since their apparent branching takes place in a manner 
similar to that described as occurring in the latter group. 
Foulerton considers all organisms in the group classed as higher bacteria as 
belonging to a single genus, streptothrix, which he places with the hyphomy- 
cetes, or mold fungi, because of their growth in branching threads from spore- 
like bodies. He says that streptothrix and actinomyces are absolutely synony- 
mous terms, and that the majority of pathologists consider them so. 
Wright and others do not agree with this view (see below). It seems to us 
that more minute work, both clinical and experimental, should be done on this 
group of infections before a classification can be accepted. 
Leptothrix Infections.-Leptothrix forms are frequently found in 
the human mouth {Leptothrix buccalis), and one or two writers have 
claimed that under certain conditions these may become pathogenic, 
but since no corroborative work has been done, and very little is known 
about the group, no opinion can be formed of the worth of these ob- 
servations. 
Cladothrix Infections.-The organisms found in the comparatively 
few cases which have been considered by their observers to be due to 
cladothrix have not been minutely, enough studied to decide definitely 
as to their true or false branching, the characteristic chosen to separate 
them from the nocardia; hence it is difficult to separate the two groups, 
but an attempt should be made, since the difference said to exist 
between them is an important one from a morphologic stand-point. 
Clinically, however, according to the reports, the cases cited are very 
similar to those said to be due to nocardia (streptothrix) and to actino- 
myces. 
Gasten found in a case of clinically typical actinomycosis, in which 
abscess cavities were found along the spinal column, not the usual 
actinomyces in the yellow, granular pus, but a fine mass of filaments. THE ACTINOMYCES 
477 
Cultures grew on all the ordinary media, best at incubator tempera- 
ture, but also at lower temperature on gelatin. The gelatin stick 
culture, which was especially characteristic, formed on the surface 
a whitish button; delicate threads stretched out in all directions from 
the point of inoculation. On agar and potato rumpled, folded films 
with white deposit forms on the surface, which contained spores. 
Animal inoculation gave positive results only in a few cases of intra- 
peritoneal injection of rabbits and guinea-pigs. Purulent nodules 
were found in the peritoneum. Gasten called the organism Cladothrix 
liquefaciens. 
Eppinger found in postmortem examination of a case of chronic 
cerebral abscess, which was the result of purulent meningitis, in the 
pus and abscess walls, etc., a delicate fungoid growth which he suc- 
ceeded in cultivating on various media. On sugar agar it formed 
yellow, rumpled colonies which finally developed into a skin. On 
potato it grew rapidly, but the colonies remained small, at first a white 
granular deposit, which afterward turned red, and on the twentieth 
day resembled a crystallized almond. It did not grow well on gelatin. 
In bouillon it formed on the surface a small white granule, which 
became deeper in the centre as it grew and sank to the bottom as a 
white deposit. The bouillon remained clear. 
Microscopically, the fungus consisted of fine threads without branches 
which exhibited distinct motility. No flagella were observed. It 
was judged to be a cladothrix, to which the name "asteroides" was 
given by the author. It proved to be quite pathogenic for rabbits 
and guinea-pigs, and produced an infection called pseudotuberculosis. 
Mice were not affected by inoculation. 
THE ACTINOMYCES. 
The little clumps produced by this group of parasites were first seen 
by Von Langenbeck in 1845 and the organisms were later discovered 
by Bollinger (1877) in the ox. They were given the name of actinomyces, 
or ray fungus, by the botonist Harz. They were reported in human 
beings by Israel in 1878. 
The characteristics of the microorganisms, first described minutely 
by Bostrom (1890) and by Wolf and Israel (1891), differed greatly 
and have led to confusion. Bostrom's organism grew best areobically 
and developed well at room temperature. He noted the intimate rela- 
tion of the organism with fragments of grain, and this led to the finding 
of similar microorganisms in the outer world on grains,, grasses, etc. 
There is no doubt that some suppurative processes have been due 
to organisms of these characteristics, but they do not seem to excite 
true actinomycosis. 
Wolf and Israel described a microorganism from two human cases, 
which differs from that described by Bostrom, but agrees with the 
microorganisms obtained by most of the more recent investigators 
from typical actinomycosis. It grew best under anaerobic conditions 478 
PATHOGENIC MICROORGANISMS 
and did not grow at room temperature. Its growth was much less 
luxuriant than Bostrom's microorganism. On the surface of anaerobic 
agar slant cultures on the third, fourth, and fifth day numerous minute 
isolated dew-drop-like colonies appeared, the largest pinhead in size. 
These gradually became larger and formed ball-like, irregularly rounded, 
elevated nodules varying in size up to that of a millet-seed, exceptionally 
attaining the size of a lentil or larger. As a rule, the colonies did not 
become confluent, and an apparently homogeneous layer of growth 
was seen to be made up of separate nodules if examined with a lens. 
In some instances the colonies presented a prominent centre with a 
lobulated margin and appeared as rosettes. A characteristic of the 
colonies was that they sent into the agar root-like projections. In 
aerobic agar slant cultures no growth or a slow and very feeble growth 
was obtained. In stab cultures the growth was sometimes limited to 
Fig. 166 
Smear from bouillon culture of actinomyces. X 1500 diameters. (From Wright.) 
the lower portion of the line of inoculation or was more vigorous there. 
In bouillon, after three to five days, growth appeared as small white 
flakes, partly floating and partly collected at the bottom of the tube. 
Growth occurred in bouillon under aerobic conditions, but was better 
under anaerobic conditions. The organisms here grow in branching 
and interlacing filaments, which later tend to break into segments 
(see Fig. 166). The microorganism in smear preparations from agar 
cultures appeared chiefly as short homogeneous, usually straight, 
but also comma-like or bowed rods, whose length and breadth varied. 
In many cultures short plump rods predominated, and in others longer, 
thicker, or thinner individuals were more numerous. The ends of the 
rods often showed oval or ball-like swellings. Swollen clubs were formed 
irregularly in the presence of blood or serous fluids. 
On intraperitoneal inoculation guinea-pigs and rabbits after four 
to seventeen weeks showed, after being killed, tumor growths mostly THE ACTINOMYCES 
479 
in the peritoneal cavity and in one instance in the spleen. Microscopic 
examination of the tumors showed in all cases but one the presence of 
typical actinomyces colonies, in most cases with typical "clubs." The 
general histological appearance of the tumors was like that of actino- 
myotic tisue. 
Wolf in a later paper reports that an animal inoculated in the peri- 
toneal cavity with a culture of the same organism had lived a year 
and a half. At the autopsy several tumors were found in the peritoneal 
cavity, and in the liver a large typical tumor in which were many 
colonies which by microscopic examination were shown to be typical 
club-bearing actinomyces colonies. 
Wright in 1905 made an extensive study of actinomycosis and added 
greatly to our knowledge of it. 
Naked-eye Appearance of Colonies of Parasite in Tissues.-In 
both man and animals they can be readily seen in the pus from the 
affected regions as small, white, yellowish or greenish granules of pin- 
head size (from 0.5 to 2 mm. in diameter). When pus has not formed 
they lie embedded in the granulation tissue. 
Fig. 167 
A typical " club''-bearing colony of actinomyces. X 325 diameters. (From Wright.) 
Microscopic Appearance.-Microscopically,, these bodies are seen 
to be made up of threads, which radiate from a centre and present 
bulbous, club-like terminations (Fig. 167). These club-like termi- 
nations are characteristic of the actinomyces. They are generally 
arranged in pairs, closely crowded together, and are very glistening 
in appearance. They are more common in bovine than in human 
lesions. They have been thought to be reproductive elements, but 
they are probably simply a reaction of the filament end to the host 
tissue. The threads which compose the central mass of the granules 
are from 0.3m to 0.5m in diameter. The threads show true branching 
and in the older colonies show a segmentation which gives them the 
appearance of chains of cocci. Sometimes the whole centre of the 480 
PATHOGENIC MICROORGANISMS 
colonies seems to be a mass of coccus-like bodies most of which are 
considered spores or conidia; the clubs are from 6/z to 8/z in diameter. 
The threads and spores are stained with the ordinary aniline colors, 
also by Gram's solution; when stained with gentian violet and by Gram's 
method the threads appear more distinct than when stained with 
methylene blue. The clubs usually lose their stain by Gram's method 
and take the contrast strain. 
Isolation of Actinomyces.-There are two cultural varieties, one 
grows aerobically and the other anaerobically. The anaerobic variety 
is grown with difficulty. A large number of solidified blood-serum 
or serum agar tubes are inoculated with the hope that one or two 
will develop a growth. The culture appears much like one of tubercle 
bacilli. It grows, however, into the medium, and takes on a yellowish 
hue. Wright recommends that granules, preferably obtained from 
closed lesions, are first thoroughly washed in sterile water or bouillon 
and then crushed between two sterile glass slides. In bovine cases 
make sure the granule has filamentous masses, for, if not, no culture 
will grow. The crushed granule is transferred to a tube of melted 
1 per cent, glucose agar at 40° C. The material is thoroughly distributed 
by shaking and the tube placed in the incubator. A number of granules 
after washing should be placed on the inside of a sterile test-tube and 
allowed to dry. In this way, should the material be contaminated, 
the drying of the granules for several weeks may kill off the other 
organisms. The tube should be examined daily. If a number of living 
filaments were added to the agar a large number of colonies will develop. 
These will be most numerous in the depth in a zone five to twelve 
millimeters below the surface (aerophiles). 
The cultures are quite resistant to outside influences; dried, they 
may be kept for a year or more; they are killed by an exposure of 
five minutes to a temperature of 75° C. 
Experimental Inoculation in Animals. -True progressive infection 
is rarely or never obtained by the injection of pure cultures in rabbits, 
guinea-pigs, or other small animals. In the bovines, however, the 
disease has been produced from cultures. The cultures form the char- 
acteristic " club"-bearing colonies in the tissues of the experimental 
animals. These colonies are either enclosed in small nodules of connec- 
tive tissue or are contained in suppurative foci within nodular tumors 
made up of connective tissues in varying stages of development. 
Wright does not accept the prevalent belief, based on the work of 
Bostrom, Gasperini, and others, that the specific infectious agent of 
actinomycosis is to be found among certain branching microorganisms, 
widely disseminated in the outer world. He thinks that these forms 
belong to a separate genus, Nocardia, and that those cases of undoubted 
infection by them should be called nocardiosis and not actinomycosis. 
The term actinomycosis should be used only for those inflammatory 
processes the lesions of which contain the characteristic granules or 
"drusen." That Nocardia ever forms these characteristic structures 
in lesions produced by them has not been convincingly shown. THE ACTINOMYCES 
481 
Occurrence.-Actinomycosis is quite prevalent among cattle, in 
which it occurs endemically; it is more rare among swine and horses. 
Many cases have in recent years been reported in man. The disease 
is rarely communicated from one animal to another and no case is 
known where a direct history of human contagion has been obtained. 
The cereal grains, which from their nature are capable of penetrating 
the tissues, have been found in centres of actinomycotic infection 
in the lower animals. The microorganism may also be introduced by 
means of carious teeth. Cutaneous infection has been produced by 
wood splinters, and infection of the lungs by aspiration of fragments 
of teeth containing the fungus. The presence of the microorganism 
in cereal grains, which was formerly accepted, is denied by Wright 
and therefore certainly placed in doubt. The further distribution of the 
fungus after it is introduced into the tissues is effected partly by its 
growth and partly by conveyance by means of the lymphatics and 
leukocytes. Not infrequently a mixed infection with the pyogenic 
cocci occurs in actinomycosis. 
Characteristics of Disease in Man and Animals.-In the earliest 
stages of its growth the parasite gives rise to a small granulation tumor, 
not unlike that produced by the tubercle bacillus, which contains, in 
addition to small round cells, epithelial elements and giant cells. After 
it reaches a certain size there is great proliferation of the surrounding 
connective tissue, and the growth may, particularly in the jaw, look 
like, and was long mistaken for, osteosarcoma. Finally, suppuration 
occurs, which, according to Israel, may be produced directly by the 
fungus itself. 
The course of the disease is very chronic. Usually the first sign is 
a point of infiltration about the lower jaw or lower on the neck. This 
almost painless swelling increases and finally softens in its centre. The 
necrotic tissue finally forces a passage externally or, passing downward, 
infects the pleura, lungs, mediastinum, or ribs. As a rule, the disease 
is not accompanied by fever. In cattle the disease is usually situated 
in some portion of the head, especially in the jaw, tongue, or tonsils, 
hence called lumpy jaw, wooden tongue, etc. Primary lung, intestinal, 
and skin lesions are not infrequent. These local lesions sometimes 
scatter and produce a general infection and the udder may be 
involved. 
Treatment.-In 1892 Nocard showed that cases in animals might 
be cured by iodide of potassium, calling attention to the fact that 
Thomassen had recommended this treatment in 1885. It is given in 
doses of 11 to 21 drams once a day. Salmon and Smith (U. S. Bureau 
of Animal Industry, Circular No. 9G) give directions as to its use. 
Mycetoma (Madura Foot).-This is a purulent inflammation of 
the foot occurring primarily in warm climates. The inflammation 
is accompanied by much irregular enlargement of the foot. Three 
varieties of this condition have been described based upon the color 
of the granules found in the diseased area: (1) white, (2) black, and 
(3) red. The white variety has been studied by Musgrave and Clegg 482 
PATHOGENIC MICROORGANISMS 
(1907), who have isolated an organism resembling somewhat actino- 
myces and somewhat the organism isolated by Wright (1898) from a 
black variety of the disease which is probably a true mold. 
NOCARDIA (STREPTOTHRIX) INFECTIONS. 
The most familiar name of this group of microorgansims is strepto- 
thrix, but this name had already been used for another genus; there- 
fore, according to the rules of nomenclature, nocardia, which name 
was proposed by Trevisan in 1889 for the organism discovered by 
Nocard in farcin des beufs of cattle, should be employed. Wright 
calls attention to the misuse of the term streptothrix, and gives the 
reasons for the employment of the term nocardia in its place. 
From widely scattered localities and at long intervals of time reports 
have been published describing unique cases of disease produced by 
varieties of microorganisms belonging to the genus nocardia. In some 
of these cases points of similarity can be recognized in the clinical 
symptoms and the gross pathologic lesions, while others differ widely 
in both respects. They have been found in brain abscess, cerebrospinal 
meningitis, pneumonic areas, and in other pathologic conditions. 
Eppinger injected cultures into guinea-pigs and rabbits, and observed 
that they caused typical pseudotuberculosis. Consolidation of portions 
of both lungs, thickening of the peritoneum, and scattered nodules 
resembling tubercles were noted by Flexner in a case of human infection 
as due to nocardia in which the pathologic picture of the disease re- 
sembled so nearly that of tuberculosis in human beings that the two 
diseases could be separated only by finding the causative microorganism 
in each case. But in no two cases reported up to the present time 
have the descriptions of the microorganisms found agreed in all par- 
ticulars. In some cases no attempt at cultivation was made. In other 
cases numerous and careful plants on various culture media failed 
to develop the specific organism. As late as the year 1904 Tuttle 
was able to find the reports of only twelve cases in which nocardia was 
found in sufficient abundance to have been an important, if not the 
principal, factor in producing disease. These cases were all fatal, 
and only once was the character of the disease recognized during life. 
As the clinical symptoms and the lesions in the human subject as well 
as in the animals experimentally inoculated with nocardia often resemble 
those of miliary tuberculosis, the question is naturally suggested whether 
cases of nocardia tuberculosis are not more numerous than the few 
reported cases would indicate. The almost universal prevalence*of 
genuine tuberculosis and the extreme gravity of the disease have so 
long occupied the attention and study of the medical profession that 
much is taken for granted, and in cases in which the symptoms and 
lesions resemble with some closeness those characteristic of the well- 
known disease they may easily be set down without question to the 
account of the tubercle bacillus. The cases of nocardiosis reported NOCARDIA INFECTIONS 
483 
which simulated tuberculosis have been fatal, and the lesions for the 
most part have been widely distributed, but in a number of cases old 
lesions have been found which suggest that the disease may have been 
localized for a longer or shorter time, and then, by some accident, 
may have become rapidly general. In this respect also these cases may 
resemble tuberculosis. Whether all cases of nocardiosis in the human 
subject are general and fatal or, as in tuberculosis and actinomycosis, 
whether there may be cases of localized disease which recover, are 
questions which have not yet been decided. The methods employed 
to demonstrate the presence of tubercle bacilli render nocardia more 
or less invisible. Again, unless the observer keeps in mind the possi- 
bility of nocardia infection, he may not appreciate the importance 
of finding slender threads with or without branches, and may consider 
them accidental bacilli, or varieties of leptothrix or non-pathogenic 
fungi. 
As the lungs have appeared to be the seat of the primary infection 
in most of the cases of human nocardiosis it is very desirable that ^11 
cases presenting the physical signs of tuberculosis, in which repeated 
examinations fail to discover the tubercle bacillus, should be system- 
atically examined for threads. In this way alone can the frequency of 
the disease be determined. Gram's method of staining or the Ziehl- 
Neelsen solution decolorized with aniline oil seem to be the most reliable 
agents for demonstrating these organisms. Varieties of nocardia are 
widely distributed and are not very infrequently met with, but as yet, 
with the exceptions mentioned above, very little is known about them. 
Tuttle's report of a case of general nocardia infection at the 
Presbyterian Hospital gives such a good clinical, bacteriologic, and 
pathologic picture of a case of this infection that a considerable portion 
of it is repeated here: 
Six days before her admission, to the hospital her illness began with a severe 
chill and fever and pain in her left side and back. The following day the pain 
in the side was worse and breathing was difficult. She began to cough and had 
some expectoration, but no blood was noticed in the sputa. At irregular inter- 
vals she had alternating hot and chilly sensations. 
On admission, the patient complained of pain in the left side of the chest, 
cough, fever, weakness, and prostration. Her temperature was 103° and her 
pulse and respirations were rapid. 
The history of the disease and the physical signs indicated an attack of acute 
lobar pneumonia, the area of consolidation being small and situated in the lower 
part of the left upper lobe in front. Frequent and violent coughing, with ahnost 
no expectoration, pain in the affected side and in the lumbar region, restlessness 
and sleeplessness, and involuntary urination were the symptoms noted during 
the first four days in the hospital. The pneumonic area increased somewhat 
and extended backward to the posterior axillary line, and the temperature was 
continuous at 103° to 103.5°. On the fifth day the temperature fell two degrees 
and signs of resolution appeared in the consolidated area. The apparent improve- 
ment, however, was of short duration. On the sixth day the temperature rose 
to 104.5°, and continued to rise each day, reaching 107.5° shortly before death, 
which occurred on the ninth day in the hospital and the fifteenth day of the 
disease. There were repeated attacks of profuse sweating. On the day before 
her death three indurated swellings beneath the skin were noticed. One. on the 484 
PATHOGENIC MICROORGANISMS 
left forearm, about the size of a walnut, apparently contained pus. Two, of 
smaller size, were situated in the right groin. 
Blood-cultures from a vein in the arm, taken on the sixth day, remained sterile. 
The leukocyte count on the seventh day was 36,000. 
Autopsy.-On the right arm, the left forearm, the abdominal wall, and on 
both thighs there are eight or ten slightly projecting, rounded, fluctuating, sub- 
cutaneous swellings from one-half inch to one inch in diameter. The skin over 
most of these nodules is unaltered, but over the larger ones there is a slight bluish 
Fig. 168 
Fig. 169 
Streptothrix from bouillon culture. (From 
Tuttle.) 
Young strep to thrix threads showing ter- 
minal buds. (From Tuttle.) 
Fig. 170 
Portion of kidney showing minute and large areas of infection. 
discoloration. The nodules are composed of bluish-gray, thick, mucilaginous 
matter, which is very tenacious and can be drawn out into long threads. The 
lower lobe is thickly studded with miliary tubercles, and scattered through the 
entire lung are suppurating foci. Liver and spleen normal. Kidneys: The 
description of one applies to both. The surface is everywhere and evenly dotted 
with minute white spots, which suggest septic emboli rather than tubercles. A 
few prominent white nodules from one-quarter inch to one-half inch in diameter, 
contain thick, tenacious matter (Fig. 170). Section shows that the entire 
substance of the kidney is densely studded with these minute white granules. NOCARDIA INFECTIONS 
485 
The gross pathological conditions were interpreted before nocardia was found 
as follows: An old tuberculous nodule in the right lung; acute miliary tuber- 
culosis in the right lung and peritoneum; acute lobar pneumonia, affecting the 
left lung; septic infarctions and pyemic abscesses of both lungs, heart muscle, 
both kidneys, pancreas, mesenteric lymph nodes, and subcutaneous connective 
tissue. The miliary tubercles of the right lung and peritoneum presented the 
characteristic appearance of genuine tuberculosis. They were minute, hard, 
gray, almost translucent nodules, while the granules in the kidneys were of 
an opaque white or yellowish-white color. 
Microscopic Examination.-Smears from the abscess beneath the 
skin and on the surface of the kidneys were stained with methyl blue, 
carbol-fuchsin, and by Gram's method. The smears resemble those 
made of tenacious sputum. There is a large amount of mucoid material 
containing a considerable number of leukocytes. Occasionally irregu- 
larly curved, thread-shaped microorganisms are found. They vary 
considerably in length and thickness, and broken and apparently 
degeneratinghhagments are seen. The more slender threads are evenly 
stained, butjsome fragmentation or beading of the protoplasm can 
generally beXobserved. The thicker threads and broken fragments 
show deeply stained globules and irregular bodies in a faintly visible 
rod or thread-shaped covering. Some branching threads are observed, 
but more commonly they are not branching. No other microorganisms 
are found in the smears. Sections from the lower lobe of the right 
lung, stained with hematoxylin and eosin, show in certain places the 
identical microscopic appearances which are considered characteristic 
of tuberculosis. Stained by Gram's method, with care not to decolorize 
too completely, threads like those described in the abscesses are found 
in great abundance, but rather faintly stained. No threads can be 
found within the typical tubercles with giant cells, but in the zones 
of small cells around them they are seen in great numbers, winding 
about among the cells and forming a sort of network. In the minute 
foci of small cells one or two fragments of threads are generally seen, 
and a moderate number in the small abscesses. In the areas of more 
diffuse infiltration these threads are abundant. No other microorgan- 
isms can be found except in the pneumonic area of the left lung, where 
some groups of cocci are seen. 
Culture Experiments.-Six tubes of Lbffler blood-serum were in- 
oculated from the kidneys and kept at 37° C. On the third day minute 
white colonies appeared in some of the tubes, and on the fifth day all 
the tubes showed from three to ten or twelve similar colonies in each. 
The colonies increased in size until some of them reached a diameter 
of one-eighth of an inch. The color, at first white, changed to yellowish- 
white and then to a decided pale yellow. The well-developed colonies 
cling firmly to the surface of the medium and were not easily detached 
or broken up. The growths in all of the tubes were absolutely pure, 
and consisted of branching threads like those found in the sections. 
Loffler's blood-serum seems to be the most suitable medium for 
cultures. The growth on this medium is more rapid and abundant 
than on any of the other media tried. 486 
PATHOGENIC MICROORGANISMS 
On plain agar and glycerin agar the growth is the same as on blood 
serum, but is less rapidly developed. 
In bouillon the growth is slow. If the tube is not disturbed or jarred, 
minute white tufts are seen clinging to the surface of the glass. But 
if the tube is shaken even slightly they sink slowly to the bottom, 
forming a white, fluffy layer. These growths when undisturbed resemble 
minute balls of thistle-down. The yellow color is not apparent even 
in the mass at the bottom of the tube. 
It is strictly aerobic. 
Morphology (Figs. 168 and 169).-On blood-serum the threads are 
comparatively thick and coarse, but those growing in bouillon are 
very slender and delicate. The main trunk also is often thicker than 
the branches. When unstained they are homogeneous gray threads, 
without any appearance of a central canal or double-contoured wall. 
There is never any segmentation of the threads. When properly 
stained there is always a distinct beading or fragmentation of the 
protoplasm, but overstaining with fuchsin produces rather coarse, 
evenly red rods. The branching is irregular and without symmetry, 
and the branches are placed at a wide angle, very nearly, and some- 
times quite, at right angles. This is best seen in specimens taken 
from liquid media. The irregularly stellate arrangement of the 
branches, which was observed by Eppinger in his original specimen, 
is often seen in young organisms floated out from a liquid medium. 
Spore Formation.-On examining the deep orange or red-colored 
growth upon potato, one is surprised to find that the threads have 
entirely disappeared and that the specimen consists of moderately 
large coccoid forms. These represent the spore form of the organism, 
and when planted upon blood-serum the branching threads again 
appear. The spores stain readily with carbol-fuchsin and are not 
easily decolorized. They are spherical, or nearly so, but often appear 
somewhat elongated, apparently from beginning germination. They 
are killed by exposure to moist heat of 65° to 70° C. for an hour, but 
are more resistant to dry heat. Drying destroys the threads after a 
comparatively short time, but the spores retain their vitality for an 
indefinite period. A dried-up potato culture retains its vitality at the 
end of almost four years. 
The identity of Tuttle's microorganism is not fully established. It 
is undoubtedly a nocardia, but it does not agree in all particulars with 
any of the varieties described. 
Animal Inoculations.-A number of rabbits and guinea-pigs were 
inoculated subcutaneously upon the abdomen and in the neighborhood 
of the cervical, axillary, and inguinal lymph nodes with colonies broken 
up in salt solution. Indurated swellings were produced at the point 
of inoculation and a number of abscesses resulted. The abscesses 
developed rapidly and some of them opened spontaneously, while 
others were incised. The material evacuated did not resemble ordinary 
pus, but was thick and mucilaginous and exceedingly tenacious, like 
that from the subcutaneous abscesses of the patient described above. NOCARDIA INFECTIONS 
487 
The microscopic appearance was the same, and the nocardia threads 
were found in considerable numbers. Several rabbits and guinea-pigs 
and two cats received peritoneal inoculations, but none of them showed 
any sign of infection. When rabbits were inoculated intravenously, 
a rapidly fatal general infection was produced, and the lesions were 
similar in kind and distribution to those described in the human subject. 
Other Cases Reported.-Ferre and Faguet found in Bordeaux, in a 
cerebral abscess in the centrum ovale, a branching fungus, colored by 
Gram, which corresponded to nocardia. It grew on agar in round, 
ochre-colored colonies; on potato there was little growth visible; slimy, 
tough colonies, which became gray and remained free from white 
dusting on the surface. Inoculations in rabbits and guinea-pigs were 
negative. 
Varieties of nocardia have been found in the human vagina. We 
have found a variety of nocardia in several cases of stillbirth with 
infection of the placenta with the same organism. 
Nocardia in Cases Simulating Actinomycosis or Tuberculosis.- 
Sabraces and Rimere found, in a case of cerebral abscess and a case 
of chronic lung disease with occurrence of subacute abscesses, fungi 
which differed from actinomyces. The organisms were contained 
in the lungs and pus, in the latter in pure culture. They grew best 
at 37° C. in the presence of oxygen. On agar plates round, wart-like 
colonies were found with yellowish under and whitish upper surface. 
Grew particularly well on fat and glycerin media; in milk a flesh- 
colored rim was developed; in gelatin agar a rough, brownish deposit, 
becoming black with age. Gelatin was liquefied. The culture had a 
strong odor of old mold. A yellowish pigment was usually produced 
which dissolved in ether; in an atmosphere of pure oxygen a brown 
pigment. Animal experiments gave positive results only when to a 
fourteen-day-old bouillon culture lactic acid was added; then pseudo- 
tuberculosis was produced. 
Numerous cases have since been observed in which nocardia proved 
to be the cause of chronic lung diseases, clinically suspected to be 
tuberculosis. 
Treatment.-Recently autogenous vaccines have been tried in certain 
cases, but it is yet too soon to determine with what result. 
References. 
Bostroem. Beitr. Path. Anat., etc., 1890, ix. 
Foulerton. The Strepotrichoses and Tuberculoses, Lancet, 1910, clxxviii, 551, 626, 
and 769 
Musgrave and Clegg. Phila. Jour. Sci., 1907, iii, 2, 477. 
Nocard. Ann. de 1'Inst. Past., 1888. 
Tuttle. Med. and Surg. Rep., Presbyterian Hosp., New York, 1904, vi, 147. 
Wright. Jour. Exp. Med., 1898, iii, 421, and Jour. Med. Res., 1905, viii, 349, and 
Osler's Modern Medicine, 1907, i, 327. CHAPTER XXXVI. 
FILTRABLE VIRUSES. DISEASES OF UNKNOWN ETIOLOGY. 
FILTRABLE VIRUSES. 
There exists a class of infectious diseases from which it has been 
quite impossible up to the present time to demonstrate visibly any 
individual microorganisms, although infective material from such 
diseases may, with certain precautions, be passed through stone filters 
of varying degrees of porosity (for types of filters, see p. 92); the 
filtrates will contain the virus and be capable of reproducing the disease 
with all its characteristics when inoculated into a susceptible animal. 
Examined microscopically, even with the highest powers, the filtrate 
is limpid, and, except in a few diseases which will be described in detail 
later on, not a sign of characteristic particulate matter can be seen. 
Such a filtrate, therefore, contains either ultramicroscopic organisms or 
small organisms with refraction and staining powers so faint that they 
cannot be demonstrated by our present methods. 
Certain precautions must be observed in such filtrations. In the 
first place the filter must be shown by actual test to be free from infec- 
tion. Any and all of the test organisms must be absolutely retained 
and none pass into the filtrate. A few minute species of germs have 
been found to pass certain grades of filters, especially under pressure. 
Thus the bacillus of guinea-pig pneumonia, which is 0.5^ x 0.7/q 
passes Berkefeld V (Wherry); a spirillum isolated by von Esmarch 
passes, according to him the Berkefelds, Chamberland F, and other 
filters; a minute water flagellate was found by Borrel to pass through 
the coarser filters. Recently, Wolbach and Binger have shown that two 
spirochetes (new species) found in pond water may pass through Berke- 
felds V, N, and W, by pressure and by suction. It is seen, therefore, 
that certain known organisms within the limits of visibility may pass 
even some of the finer filters. The filtration must be completed within 
a moderate time, because even bacteria as large as the typhoid bacillus 
may, in media containing a certain amount of albuminous material, 
grow in time through the filter. The material to be filtered should, 
furthermore, be greatly diluted and first filtered through filter paper 
in order to avoid the clogging action of extraneous material. 
If after all the proper precautions have been taken the filtrate is 
pathogenic it must be shown that the symptoms are due to a micro- 
organism and not to a toxin. This may be decided by inoculating a 
series of animals successively with the filtrate obtained from a pre- 
viouslv so inoculated animal. DISEASES PRODUCED BY FILTRABLE AGENTS 
489 
From our present knowledge, filtrable agents may be divided into 
two groups: (1) those which have not yet been morphologically demon- 
strated (ultramicroscopic?); (2) those which are shown to be within 
the limits of visibility. A third group may be made of the diseases 
produced by viruses of questionable filtrability. 
GROUP I.-DISEASES PRODUCED BY FILTRABLE AGENTS OF 
UNKNOWN MORPHOLOGY. 
Foot and Mouth Disease.-This is probably the first disease shown 
to be produced by a filtrable agent. It is a highly infectious disease of 
cattle. Other domestic animals, as well as man, may also be attacked. 
In man it usually occurs among people handling infected cattle and by 
drinking the milk of infected animals. The disease is characterized by 
the appearance of vesicles in the mouth and around the coronet of the 
foot as well as between the toes. Lbffler and Frosch, in 1898, discovered 
that, after diluting the contents of an unbroken vesicle with twenty to 
forty times its volume of water and passing the resulting fluid through 
a Berkefeld filter, the filtrate contains the virus. This virus remains 
infectious for some time. One attack of the disease usually produces 
immunity. Lbffler claims to have produced a serum which has immuniz- 
ing properties. 
Mosaic Disease of Tobacco.-This is the second disease in which 
a filtrable virus was demonstrated. The disease causes the young 
tobacco leaves to become devoid of chlorophyll in spots which enlarge, 
turn brown, and the underlying tissue becomes necrotic. Beijerinck 
in 1899 showed that the filtrate from a porcelain filter promptly repro- 
duced the disease on tobacco leaves. 
Cattle Plague (Rinderpest).-This fatal European and African 
disease of cattle is characterized by inflammation of the intestinal 
mucous membrane. The blood is infectious and filtrates of it through 
Berkefelds and Chamberland F (Nicolle and Adil-Bey, 1902) produce 
the disease. No organism can be seen. Immunity follows one attack. 
It can also be produced by a subcutaneous inoculation of bile from an 
infected animal. 
Yellow Fever.-The undiluted serum from cases of this disease has 
been shown by the American commission (1901) and others (see p. 590) 
to pass the Berkefelds and the Chamberland B filters as clear filtrates, 
and in this form to be infectious; therefore some forms at least of the 
■specific organism are probably ultramicroscopic. 
Rabies.-No bacteria have been discovered that are considered as 
factors. The probability of the Negri bodies being protozoa and the 
exciting factor is considered in Chapter XLV. The virus of rabies 
has been shown to be filtrable (Remlinger, 1903). Poor and Steinhardt 
(1913) showed that gland virus is more filtrable than that from the 
brain. It may pass through the coarser Chamberland filters. 
Hog Cholera.-De Schweinitz, Dorset, Bolton, and McBryde demon- 
strated in 1905 that the blood of hogs suffering from hog cholera con- 490 
PATHOGENIC MICROORGANISMS 
tains a filtrable virus which is capable of producing the disease on 
inoculation into healthy hogs. This virus passes Chamberland B and 
F filters. It leaves the body in the urine and probably enteres another 
animal through contaminated food. King, Baeslack, and Hoffman 
found in 1913 a short motile spirochete (Spirocheta suis) in the blood 
in a series of cases of hog cholera. They think this organism may be 
a stage of the specific organism which may produce filtrable granules. 
This disease is a very fatal and contagious disease of swine char- 
acterized by fever and ulcerative enteritis. Immunity follows one 
attack. An immune serum is produced. Animals are also immunized 
by a mixture of infected blood and immune serum (sensitized virus). 
The hog cholera bacillus was earlier supposed to be the cause of this 
disease. 
South African Horse Sickness.-This is a warm-weather disease, 
more common in animals that do not pass the night under cover. The 
horses are uneasy, have difficulty in breathing, and a reddish froth 
exudes from their mouths. The temperature rises in the day time, but 
has a tendency to drop at night. In severe cases an edematous swelling 
of the head and neck may appear. MacFadyen succeeded in passing 
blood serum of a diseased horse (diluted) through the Berkefelds and a 
Chamberland F, not through a Chamberland B filter. 
Chicken Sarcoma.-In 1910 Rous discovered a spindle-celled tumor 
in chickens, which he has reproduced in other chickens by transplanta- 
tion, by inoculation of dried tumor tissue, and by the inoculation of 
Berkefeld filtrates from emulsions of tumor cells. 
Novy's Rat Virus.-Novy has found an extremely virulent filtrable 
virus from a disease of rats, which passes all filters. He has not yet 
published full reports. 
GROUP II.-DISEASES PRODUCED BY FILTRABLE AGENTS 
SHOWN TO BE VISIBLE. 
Contagious Pleuropneumonia of Cattle.-This malady affects bovines, 
but not other species. Typically, there is an inflammation of the lungs 
and the pleura which is invasive and causes necrosis of the diseased 
parts. Nocard and Roux succeeded in cultivating the organism in 
collodion sacs placed in the peritoneal cavity of rabbits, using a mixture 
of serum and bouillon. After two weeks a very faint turbidity appeared 
in the sacs; coincidently the fluid became infective. Nocard and Roux 
described the organisms as minute spheres and spirals just within the 
limits of visibility. They showed (1899) that the organisms passed the 
Berkefelds and a Chamberland F, but not a Chamberland B filter. 
Immunity is produced by a single attack. It has also been produced 
artificially by the inoculation of cultures or virulent exudates. 
Epidemic Poliomyelitis.-This is a disease which affects chiefly the 
central nervous system. It occurs usually in children, and appears 
sporadically and in epidemics in many countries. In New York, in 
1907, there was an epidemic of over 2000 cases and in Texas, in 1912, DISEASES PRODUCED BY FILTRABLE AGENTS 
491 
there was a large outbreak. In nature, the virus enters probably 
through the upper air passages. The chief symptoms of the disease 
are fever, with or without sore throat, followed in a few days by paresis 
and paralysis. The mortality is low. There is usually permanent 
injury to parts of the motor areas of the nervous system, with resulting 
deformity. The principal microscopic changes are a marked exudation 
of polynuclear leukocytes into the lymph spaces and the cerebrospinal 
fluid. The changes are usually specially marked in the anterior com- 
missure and the cornea of the cervical and lumbar regions of the cord, 
but the whole nervous system is more or less affected. The bloodvessels 
may become thrombosed and ruptured. The disease seems to be closely 
related to, if not identical, with certain diseases manifested by acute 
encephalitis and bulbar paralysis, e. g., Landry's paralysis. That the 
disease also bears a marked resemblance to rabies is quite apparent. 
The finding of the virus in the salivary glands and the demonstration 
of its filtrability give added evidence of its similarity to rabies. 
Until recently, nothing was definitely known of the etiology. Now we 
have a series of studies which seem to have practically cleared up the 
subject. Landsteiner and Popper (1909) reported the transmission of 
acute poliomyelitis to apes. They inoculated the spinal cord intraperi- 
toneally and produced typical symptoms and lesions, but did not succeed 
in transmitting from ape to ape, probably because they used a mild 
case. Flexner (1909) and Lewis transmitted the disease from monkey 
to monkey by means of intracerebral inoculations. Landsteiner and 
Levaditi (1909) also transmitted it from monkey to monkey. They 
found that the virus lives some time outside of the body; that the 
degenerated nerve cells are taken up by phagocytes, and there is an 
analogy between the lesions of poliomyelitis and those produced by 
rabies. They demonstrated that the virus is filtrable. Leiner and 
Wiesner transmitted the disease from monkey to monkey, found young 
animals more susceptible than older ones, and spinal fluids, blood, and 
spleen negative. Flexner transmitted the disease by inoculating into 
the blood or peritoneal cavity, also by the subcutaneous method, and 
independently found the virus to be filtrable. Landsteiner and Leva- 
diti found the virus in the salivary glands and suggested the saliva, 
moist or dry, as a source of contagion. 
Soon after this (1911) Noguchi and Flexner announced that they 
had obtained cultures in media similar to that used by Noguchi in 
cultivating spirochetes (see p. 103). In such media, in about five days 
the pieces of tissue employed become surrounded by an opalescent 
haze which increases for about five days more, and a sediment gradually 
forms. Giemsa's stain shows the presence of minute globoid bodies 
(0.15 to 0.3 diam.) in pairs, short chains, and masses. Cultures were 
also obtained from the filtered virus. Monkeys inoculated with these 
cultures for a variable number of culture generations may die with 
typical lesions of the disease. The authors consider these bodies the 
cause of the disease. They further report that the cultures are fil- 
trable. This work awaits corroboration. Levaditi states that he cannot 492 
PATHOGENIC MICROORGANISMS 
obtain the results of Noguchi, but that he obtains evidence of growth 
by the living tissue method. Rabbits have also been found to be 
somewhat susceptible to the virus (Kraus and Menicke, Marks). 
Marks states that the disease in rabbits does not resemble that in man. 
Immunity.-One attack seems to give certain immunity. Flexner 
and Amoss state that, experimentally, the virus inoculated into the 
blood is capable of being neutralized by infraspinous injections of 
immune serum. 
Relapsing Fever.-Todd and Wolbach have found (1914) that 
Spirocheta duttoni may be forced through even the finer grades of 
Berkefeld filters by a pressure of from fifty to ninety pounds to the 
square inch. 
GROUP III.-DISEASES PRODUCED BY VIRUSES OF QUESTIONABLE 
FILTRABILITY. 
Smallpox and Related Diseases.--The details of the disease smallpox 
are considered in a later chapter. In 1908 Casagrandi reported that 
the virus was filtrable under pressure through coarser Berkefeld filters. 
Vaccine virus had already been reported as filtrable by some investi- 
gators, and as non-filtrable by others. Our results agree with those 
of the latter observers. The opinion as to the filtrability of the other 
"pox" diseases is not unanimous. 
Dengue.-Ashburn and Craig claim to have reproduced dengue in 
susceptible individuals by a similar procedure to that employed in 
yellow fever. The virus passes a Berkefeld filter. The intermediary 
host in natural infection is claimed by them to be Culex fatigans. 
Measles.-This very definite exanthematous disease still remains 
among those of unknown etiology. Both cell inclusions (Field, Ewing, 
and others) and bacilli have been reported as having an etiologic relation- 
ship. The reports have not been corroborated. In 1905, Hektoen 
produced measles in two human cases by the inoculation of blood 
drawn from an infected case at an early stage of the disease. Anderson 
and Goldberger in 1911 inoculated monkeys with measles blood and 
demonstrated the virus in the blood of the inoculated animals and in 
the secretion of the upper air passages. They also demonstrated the 
period of infectivity of the blood. They reported positive results with 
Berkefeld filtrates. 
Scarlet Fever.-Scarlet fever is an acute febrile, highly infectious dis- 
ease, characterized by a diffuse punctuate erythematous skin eruption, 
accompanied by catarrhal, croupous, or gangrenous inflammation of 
the upper respiratory tract and by manifestations of general systematic 
infection. The disease was probably known long before the Christian 
era, but the present name does not appear until the time of Sydenham 
(1685), who differentiated the disease from measles. It is very generally 
disseminated, but is much more common in temperate climates than 
in the tropics. The specific exciting factor is thought by many to be a 
streptococcus, of the Streptococcus pyogenes type,, but the evidence in 
favor of this view is very slight (see Streptococcus pyogenes}. DISEASES PRODUCED BY VIRUSES 
493 
Mallory (1914) found certain bodies which he describes as follows: 
"They occur in and between the epithelial cells of the epidermis and 
free in the superficial lymph vessels and spaces of the corium. The 
great majority of the bodies vary from 2ju to 7/z in diameter, and 
stain delicately but sharply with methylene blue. They form a series 
of bodies, including the formation of definite rosettes with numerous 
segments, which are closely analogous to the series seen in the asexual 
development (schizogony) of the malarial parasites, but in addition 
there are certain coarsely reticulated forms which may represent stages 
in sporogony or be due to degeneration of the other forms." He gave 
the name Cyclasterion scarlatinale to these bodies in consequence of the 
frequent wheel and star shapes of the rosette. 
Field and others have not been able to corroborate these findings. 
The reports that a filtrable virus is found lack corroboration. 
Trachoma.-This condition has already been considered in a previous 
chapter. Bertarelli reported that he was able to produce a specific 
filtrate. Nicolle also reported positive results with filtrates. The 
descriptions of the disease, however, are too vague to allow an opinion 
as to the truth of these reports. 
Typhus Fever.-The occurrence of this infectious disease in epidemic 
form has disappeared from civilized lands. The fact that the body 
louse may transmit the disease helps explain why it has always been 
classed as a filth disease. It still occurs endemically in parts of Europe 
and North and South America. In Mexico it occurs in epidemics where 
it is known as Tabardillo. In New York, it occurs occasionally in 
mild form under the name of Brill's disease. Brill thought it was a 
new disease, but Anderson and Goldberger have shown that typhus 
fever and Brill's disease are the same. Nicoll and Krumwiede observed 
four cases which clinically verified these findings. The disease is 
characterized by high temperature and a petechial rash. 
Nicolle (1909) showed that the old world typhus can be transmitted 
to the chimpanzee and from this to the macacus with typical eruption 
in each case. He also showed that the disease is transmitted by the 
ordinary body louse (Pediculus vestimenti). Anderson and Goldberger 
(1909) were the first to transmit typhus fever of Mexico (tabardillo) 
to monkeys. They were able to transmit directly from human beings 
to the macacus and capuchin and from monkey to monkey. Ricketts 
and Walker (1910) also found that the macacus was directly susceptible 
to the disease. They based their diagnosis chiefly upon a rather 
indefinite fever and in most cases somewhat distinct symptoms of 
illness. They also found that the monkey may pass through an attack 
of typhus so mild that it cannot be recognized clinically, but it results 
in immunity. The immunity test is a reliable proof of the previous 
occurrence or non-occurrence of typhus at least within a period of one 
month. They found that typhus was transmitted to the monkey by 
the bite of the louse. They further state that in stained preparations 
of blood of patients taken from the seventh to the twelfth days of the 
disease thev invariably found a few short bacilli similar to those which 494 
PATHOGENIC MICROORGANISMS 
belonged to the hemorrhagic septicemia group. In moist preparations 
they saw similar forms in all cases. No motility was observed. No 
cultures could be obtained. They examined the dejecta of many lice 
and found similar bodies in large numbers in infected lice and occasion- 
ally in non-infected lice. Pediculus capitus may also transmit the 
disease. 
Recently, Plotz reported the isolation in pure cultures of a gram 
positive pleomorphic anaerobic bacillus from the blood of six typhus 
cases and six cases of Brill's disease. He states that he obtained com- 
plement fixation with the blood of typhoid patients. Attempts at 
filtration by Ricketts and Wilder and Anderson and Goldberger were 
at first negative. But Nicolle, Connor and Wilder showed that filtered 
blood inoculated into monkeys made them immune. The corroboration 
of these findings by the others makes greater the possibility of the 
virus having a filtrable stage. 
Several other diseases of less importance have been listed as belonging 
to those produced by a filtrable agent. A short review of the subject 
up to 1912 is given by Wolbach. 
OTHER DISEASES OF UNKNOWN ETIOLOGY. 
Rocky Mountain Spotted Fever.-This is an acute infectious disease 
characterized by fever and a more or less hemorrhagic eruption. Some 
years ago, Wilson and Chowning thought they found a protozoan in 
the blood similar to Babesia, a Texas fever. Their findings have not 
been corroborated. Their investigations proved, however, that rabbits 
are susceptible to the disease and that a tick of the genus Dermacentor 
probably carries the infection. Then Ricketts and Gomez made some 
very interesting studies on the disease. They found that guinea-pigs 
and monkeys are susceptible as well as rabbits, and they further found 
that in guinea-pigs and monkeys an attack of spotted fever produces 
a strong active inherited immunity characterized by a serum with 
high protective but low curative power, and that the production of the 
serum in the horse with the use of serovaccination in man may give 
practical results. They found a moderate number of diplococcoid 
bodies in the blood of infected guinea-pigs and monkeys, and fewer in 
man. They found that the virus is transmitted by the infected female 
tick to her young through the eggs. If the larvae from these eggs are 
allowed to feed upon normal guinea-pigs, these animals come down 
with the disease. Immense numbers of these apparent organisms are 
found in affected eggs and none were found at first in normal eggs. 
Afterwards Ricketts found a few, but he thought these might be an 
avirulent species of the same organism. The salivary glands, alimentary 
sac and ovaries of infected female ticks are swarming with these bodies, 
while normal ticks seem to have none. Lastly, Ricketts found that 
these bodies agglutinate with specific serum, 1 to 300 dilution. 
Mumps.-Comparatively few studies have been made of this infec- 
tious disease, probably because of its low mortality. By puncturing OTHER DISEASES OF UNKNOWN ETIOLOGY 
495 
the parotid gland, Laveran and Catrin obtained a diplococcus which 
stains easily, is Gram-negative, and grows on ordinary culture media. 
No satisfactory specific studies have been made of this organism. 
Pellagra.-This disease has been much studied recently. The theory 
that it is due to the ingestion of damaged corn has received a check from 
the work of Siler, Garrison and MacNeal (1913) who state that they 
were unable to get evidence to support this theory. They consider the 
disease a specific infection caused by unknown means. 
References. • 
Anderson and Goldberger, Public Health Report, 1910, 1912 and 1913. Jour. Amer. 
Med. Assoc., 1911 and 1912. 
Ashburn and Craig. Jour. Inf. Dis., 1907, iv, 440. 
Ewing. Jour. Inf. Dis., 1909, vi, 1. 
Field. Jour. Exp. Med., 1903, vii, 343. 
Flexner and Lewis. Jour. Amer. Med. Assoc., 1909, liii; and Jour. Exp. Med., 1909, xii. 
Flexner. Huxley Lecture, Lancet, 1912, ii, 1271. 
Flexner and Noguchi. Jour. exp. Med., 1913, xviii. 
Flexner and Co-workers. Jour. Exp. Med., 1913 and 1914. 
Hektoen. Jour. Ind. Dis., 1903, ii. 
King, Baeslack and Hoffman. Jour. Inf. Dis., 1913, xii, 39 and 206. 
Kraus u. Meinicke. Deut. Med. Woch., 1909, xxxv. 
Lbffler and Frosch. Cent. f. Bakt., 1898. 
Landsteiner and Levaditi. Compt. Rendus Soc. de Biol., 1909. 
Mallory. Jour. Med. Res., 1904, x, 483. * 
Marks. Jour. Exp. Med., 1911. 
Nicolle. Compt. rend. Acad. Sci., 1909 and 1911; Assoc, de 1'Inst. Past., 1910, 1911, 
and 1912. 
Nicoll, Krumwiede, Pratt and Bullowa. Jour. Amer. Med. AsSoc., 1912, lix, 521. 
Plotz. Jour. Amer. Med. Assoc., 1914, Ixii. 
Poor and Steinhardt. Jour. Inf. Dis., 1913, xii, 202. 
Read, Carrol, Agramonte and Lazear. Philadelphia Med. Jour., 1900. 
Remlinger. Ann. de 1'Inst. Pasteur, 1903, 834. 
Ricketts and Wilder. Jour. Amer. Med. Assoc., 1910, lx, 309. 
Rous and Murphy. Jour. Exp. Med., 1911, xiii. 
Wilder. Jour. Inf. Dis., 1911, 9. 
Wolbach. Jour. Med. Res., 1912, xxviii, 1. CHAPTER XXXVII. 
FLAGELLATA. 
The flagellates that are pathogenic to man belong chiefly to the 
genera Trypanosoma and Leishmania, but certain other genera are 
formed occasionally in man which may be mentioned because of the 
possibility of their becoming pathogenic. According to the classifica- 
tion of Calkins (1908) the flagellates parasitic in man are from three 
orders, the Monadida, the Heteromastigida, and the Polymastigida] 
according to that of Doflein and Koehler (1913), they are from two, 
Protomonadina (one to three flagella), and Polymastigina (four to eight 
flagella). Six families, according to the morphology of the flagella, are 
distinguished, three of which contain forms parasitic for man, namely, 
Cercomonadidce (one flagellum and no blepharoplast), T rypanosomeda? 
(one flagellum, a blepharoplast, and an undulating membrane), and 
Bodonidoe (two flagella). 
.Under the Trypanosomedoe, which include most of the pathogenic 
forms, are placed the genera Leptomonas, Herpetomonas, Leishmania, 
Trypanosoma, and Schizoiypanum and Endotrypanum. Under the 
Bodonidce are placed the genera Bodo and Prowazekia, and in the order 
Polymatigina the genera Trichomonas and Lamblia have been found in 
human beings. 
Hartmann puts the Trypanosomata, with other blood-parasites, in an order, 
the Binucleata, and makes the Spirocheta an appendix of this order. According 
to this arrangement the Hemosporidia are taken from, the Sporozoa and placed 
with the Trypanosomata in this order, the malarial organisms supposedly lose 
through their parasitism many of the characteristics ascribed to this order. 
Material and Methods for Study.-A number of flagellates (Bodo, for in- 
stance, see p. 528) are found in the large intestine of the lower animals. The 
toad, the grass lizard, and the guinea-pig may contain some interesting forms. 
As these forms are easily obtained and remain alive a long time outside of the 
body, they are well fitted for class study. 
The feces are obtained by pressing lightly over the anus of the animal, or 
if the whole intestinal tract is to be examined, by sacrificing the animal and 
dissecting out the parts wanted. The material is placed in a clean watch- 
glass and thinned if necessary with physiologic salt solution. Hanging drops 
may be made in physiologic salt solution or in such a solution made a little 
thick by the addition of gelatin in order to retard the motion of the flagellates 
somewhat so they may be better studied. 
Permanent preparations may be made according to directions given on p. 
73. As most of the pathogenic members of this group may be difficult to 
obtain in the living condition at any stated time, they must be studied by 
students principally in stained smears and sections. 
If one can obtain rats infected with Tr. lewisi, others with one or more path- 
ogenic forms; still others with Spirocheta obermeieri, the infecting organisms 
can be kept alive by frequent reinoculation of the heart's blood, subcutaneously LEPTOMONAS, HERPETOMONAS, AND CRITHIDIA 
497 
or intraperitoneally into the fresh animal, or cultures may be carried on (see 
below). But this is an expensive and tiresome work in those laboratories where 
such work is not being carried on, and generally one must rely on the permanent 
preparation. In the development in the second host one must also study the 
stained specimens in the great majority of instances. 
The fresh specimens of blood are obtained from the tail tip of the rat, or 
the ear of the dog; they may be examined after dilution with physiologic salt 
solution in the hanging drop, or in a drop spread under a cover-glass and ringed 
with vaselin. For permanent preparations films of the blood are spread, fixed, 
and stained in the usual way; Giemsa's method of staining (p. 81) is very 
satisfactory. 
For section work of the various organs the fixatives and methods given on 
page 84 may be used. Special methods are given under each organism. 
Artificial Cultures of Blood-flagellates (see p. 106).-These, according 
to Novy and MacNeal, may be made on a culture medium consisting 
of a mixture of ordinary nutrient agar with variable amounts of fresh 
defibrinated rabbit or rat blood. The best all around results are ob- 
tained with equal parts of blood and agar. The agar is melted and cooled 
to 50° C., then the blood is added and thoroughly mixed. The tubes 
are inclined until the medium stiffens, when they should be inoculated 
at once with blood or other infected material containing living try- 
panosomes. The surface of the medium should be very moist, so water 
of condensation may form. Generally evidence of growth may be 
observed in three or four days. 
The members of this genus are round or oval flagellates with a long 
anterior flagellum and a more or less pointed posterior extremity which 
is sometimes ameboid. The vesicular nucleus is situated anteriorly, 
and passing through the organisms from flagellum to posterior extremity 
is an axial elastic fibril (Fig. 171). Division into two daughter forms 
has been observed. 
A number of cercomonada, none of them well studied, have been 
observed in different animals as well as in man. They are very numerous 
in stagnant water. 
Cercomonas hominis (Davaine, 1854) was observed in the dejections 
of a cholera patient by Davaine. The body is 10^ to 12/x long and 
pear-shaped, pointed posteriorly. The flagellum is twice as long as 
the body. Davaine also reported a smaller form in the stools of a 
typhoid patient. Other observers have noticed similar forms in human 
stools, some associated with "Amoeba coli." Similar forms have been 
seen also in an echinococcus cyst of the liver, in the sputum from a case 
of lung gangrene, in the exudate of a hydropneumothorax, and a few 
times in the urine. They are all probably harmless invaders. 
CERCOMONAS. 
LEPTOMONAS, HERPETOMONAS, AND CRITHIDIA. 
Certain flagellates found in the digestive tract of mosquitoes, flies, 
and other insects are very similar to trypanosomes. Among them 498 
PATHOGENIC MICROORGANISMS 
several species have been recognized, but they need to be more fully 
studied in order to determine their definite relationship to each other 
and to the genus trypanosoma. Leptomonas is described as having 
a single flagellum directed forward and arising near a blepharoplast 
situated in the anterior part of the cell. Herpetomonas is distinguished 
from leptomonas by a flagellum containing two filaments and by a 
delicate filament extending from the blepharoplast toward the posterior 
end. Crithidia has a rudimentary undulating membrane. The dis- 
tinctions between these three genera and the genus Trypanosoma which 
have been recognized are: (1) the former contain no undulating mem- 
brane or only a rudimentary one, and (2) their centrosome or blepharo- 
plast usually lies at the side of, or anterior to, the nucleus instead of 
posterior to it, as in Trypanosoma (Fig. 171). 
Fig. 171 
Schematic drawings of flagellates belonging to the trypanosomidee, showing differential 
points: a, cercomonas; b, leptomonas and Leishmania; c, herpetomonas; d, crithidia; 
e, trypanosoma. 
These distinctions, Novy claims, may disappear in the cultural 
forms of the three genera, when trypanosomes may show a rudimentary 
undulating membrane and an anterior blepharoplast. His caution 
in regard to confusing these insect flagellates with developmental stages 
of vertebrate blood parasites should be remembered. 
The Leishman-Donovan bodies found in kala-azar are closely related 
to the genus Leptomonas. They are considered a separate genus, 
Leishmania. 
LEISHMANIA (LEISHMAN-DONOVAN BODIES AND ALLIES). 
Certain fevers of severe malarial-like types known in different sec- 
tions of the tropics by different names (dum-dum fever, cachexial 
malaria, Kala-azar) have been shown to have a causal relationship 
by the finding of similar protozodn-like bodies in the lesions. These 
bodies were first minutely described by Leishman in 1903 as being LEISHMANIA 
499 
present in certain cells in the spleen of cases occurring in India, called 
by him dum-dum fever. He considered them as possibly trypano- 
somes, but did not name them. Later in the same year Donovan de- 
scribed similar bodies in cases of what he called malarial cachexia. The 
bodies were first called the Leishman-Donovan bodies; then Laveran 
and Mesnil who examined Donovan's preparations and considered 
the organisms similar to those causing Texas fever in cattle, called them 
Piroplasma donovani. Ross, however, thought they constituted a dis- 
tinct genus which he called Leishmania. This genus is now accepted, 
hence they are known as Leishmania donovani. Rogers and Patton 
Fig. 172 
Leishmania tropica in a case of tropical ulcer. Smear preparation from the lesion 
stained with Wright's Romanowsky blood-staining fluid. The ring-like bodies with white 
central portions and containing a larger and a smaller dark mass are the microorganisms. 
The dark masses in the bodies are stained a magenta, while the peripheral portions of the 
bodies in typical instances are stained a pale robin's egg blue. The very large dark masses 
are nuclei of cells of the lesion. X 1500 approximately. (After Wright.) 
place them with the genus Herpetomonas, but until we know more of 
the limits of variation of all these forms it seems best to make them a 
separate genus. They have since been found in different parts of India, 
in China, Tunis, Algiers, Arabia, Egypt, South Africa, Italy, Greece, 
and Portugal. Wright in this country has reported in a case of tropical 
ulcer, or Delhi boil, from an Armenian immigrant, bodies which, accord- 
ing to his excellent photographs (Fig. 172) and description, must 
be identical with, or very closely related to, Leishman's bodies. On 
account of the different pathologic conditions in which they are found, 
however, they are classed as a different species, Leishmania tropica 
Wright. The form found in infantile splenomegaly is considered another 500 
PA T HOGE NIC MICROORGANISMS 
species, with the name Leishmania infantum Nieolle. Darling described 
an organism resembling that of Kala-azar found in a fatal disease of 
tropical America. Though the organism, he says, resembles Donovani, 
he thinks it has enough points of difference to be placed in a different 
Fig. 173 
Cultural forms of L. infantum, showing the flagellate type. (After Nieolle.) 
genus; therefore he gives it the name Histoplasma capsulatum, and calls 
the disease histoplasmosis. He says it differs from Donovani in the 
form and arrangement of its chromatin nucleus and in not possessing a 
chromatin rod. It has a refractile achromatic capsule. LEISHMAN IA 
501 
Morphology.-The bodies as seen in the cells of the host are circular 
to elliptical in shape, from to 4p. in diameter, and contain two nuclei, 
a large oval one at one part of the periphery and a small circular or 
rod-shaped one (blepharoplast) near or at the opposite part of the peri- 
phery. This smaller body stains more intensely than the larger one, 
while the cytoplasm of the parasite stains very dimly, sometimes show- 
ing only a faint peripheral rim. Near the rod-shaped nucleus may 
sometimes be seen a minute granule or rod which is the rudiment of 
the flagellum. Any nuclear and cytoplasmic staining methods will 
bring out these points in Zenker-fixed material. Smears stain well by 
Wright or the Nocht-Romanowsky methods. It was not until these 
organisms were cultivated outside of the body that their relationship 
to the flagellates was established (Fig. 173 and Plate IV, I, Fig. 1, b). 
Site in Body.-The bodies have been found in large endothelioid cells 
in the spleen, liver, bone-marrow, lymph nodes, kidney, lungs, testes, 
skin, muscles, intestinal ulcers and in the leukocytes in the peripheral 
blood. In this last situation they are only found in appreciable numbers 
in advanced cases. 
The large cells containing the parasites are supposed by Christophers 
to be the endothelial cells from the finest capillaries. Donovan states 
that he found small forms in the polynuclear leukocytes of the peripheral 
circulation when the temperature was above 103°. Marshall found 
them chiefly in the mononuclears. Rogers was able to demonstrate 
them more easily in the peripheral circulation by centrifuging the blood 
and examining the leukocytic layer. Nicolle and Conte and others 
obtained them in the mononuclears found in the fluid of blisters pro- 
duced experimentally on the skin. 
Cultivation.-Rogers has grown abundant pure cultures of the bodies 
in a slightly acid citrated blood-medium at 20° to 22° C. Nicolle and 
later Novy have shown that L. infantum is pathogenic for dogs and 
that cultures may be obtained with comparative ease from the infected 
animals (Fig. 173). Nicolle has also cultivated L. tropicum. 
In these cultures the rounded organism elongates, the flagellum 
develops, and the Leishman-Donovan body becomes a flagellate, like 
the Leptomonas (Fig. 171). 
Insect Carriers.-Rogers and Patton have shown that the bed-bug 
may transmit the disease, and Patton has demonstrated the develop- 
ment of the organism up to the fully flagellated stage in the gut of this 
insect. The Sargents have shown that L. infantum may be transmitted 
by the dog flea. 
Effect on Human Host.-The pathologic changes are those following the degen- 
erations, subsequent to the growth of the organisms in the large mononuclear 
cells. The symptoms, in the cases of general infection are: (1) very much 
enlarged spleen and less enlarged liver; (2) progressive anemia with peculiar 
dark, earthy pallor of skin (Kala-azar), progressive emaciation, and muscular 
atrophy; (3) long-continued, irregularly remittent, and intermittent fever 
(97° to 104°); (4) hemorrhages, such as epistaxis, bleeding from gums into 
subcutaneous tissue, producing purpuric eruption; (5) transitory edemas of 
various regions. There are often complications, such as congestion of lungs, 502 
PATHOGENIC MICROORGANISMS 
dysentery, and cancrum oris. The blood shows practically no loss of red-blood 
cells, but a diminution of hemogloblin; there is a decrease in the leukocytes, 
principally polynuclears, giving a relative increase of mononuclears. 
Negative points which help in the diagnosis are: absence of malaria, no 
typhoid or Malta fever reaction, resistance to medication, quinine, as a rule 
having no effect, though in early cases, and with large continued doses a few 
good results have been reported. Puncture of spleen or liver with the finding 
of Leishman-Donovan bodies makes the diagnosis certain. Sometimes the bodies 
may be found in the peripheral blood. 
The duration of the disease is from a few months to several years. The 
percentage of deaths in systemic infections is great; in some forms of the disease, 
at the height of an epidemic, it may reach 98 per cent. Strangers from non- 
tropical countries are specially susceptible. The infection in children known as 
splenomegaly is similar to that in the adult. 
The local disease known as Delhi or Aleppo boil or tropical ulcer is a compara- 
tively non-dangerous circumscribed chronic ulcer in which the endothelial cells 
contain the organisms in large numbers (Fig. 172). Recovery is followed by 
marked immunity. 
Complement-fixation.-Makkas and Pappassoterion state that a specific 
antigen gives positive results with both Kala-azar and syphilis, but a syphilitic 
or a non-specific antigen gives positive results only with syphilis, therefore both 
should be used. 
Prophylaxis.-Segregation and perfect cleanliness, especially in regard 
to bedbugs and fleas, are recommended as the best means of eradicat- 
ing the disease. 
' References. 
*S. T. Darling. The Morphology of the Parasite (Histoplasma capsulatum), etc., Jour. 
Exper. Med., 1909, xi, 515. 
Doflein u. Koehler. M. Kolle and Wassermann, 1913. Sec. Ed., Jena. 
M. Mayer. Leishmania. M. Kolle and Wassermann, 1913. Sec. Ed., Jena. 
Sergent. Ed. and et. Bull. Soc. Path. Exp., 1912, v, 595. 
Makkas and Pappassoterion. Arch. d. Med., 1911. 
Nicolle. Le Kala-azar infantile. Ann. Inst. Pasteur, 1909, xxiii, 361 and 441. 
Novy, MacNeal, and Torry. Jour. Inf. Dis., 1907, iv, 223. 
W. S. Patton. Scientific Memoirs by Officers of Medical and Sanitary Departments 
of Government of India, new series, No. 31. 
Wenyon. Jour. Trop. Med., London, 1912, iii, 13. 
Wright. Jour. Med. Research, 1903, x, 472. CHAPTER XXXVIII. 
TRYPANOSOMA. 
Pathogenic Forms.-Very many species of trypanosoma have been 
described, and the number reported as distinctly pathogenic is increas- 
ing. Two of the latter are known to be pathogenic for man; a closely 
related form that was described by Chagas in 1909 is made a new 
genus. The accompanying table gives a list of the better-known patho- 
genic forms for mammals with their chief differential characteristics. 
They are divided by Laveran into three groups according to the 
different characters of the flagellum. Their general characteristics 
and broader classifications are given in Part I. (See also Plate IV, I, 
Fig. 2). 
Historical Note.-The first species of trypanosome studied with any degree 
of fulness is the comparatively non-virulent T. lewisi. It was probably first 
seen in the blood of the rat in 1845, but was not well described until 1879, 
when Lewis studied it more fully. Since then it has been studied by many 
observers. It is found in the blood of from 2 to 3 per cent, of wild rats through- 
out the world. 
The first of the more pathogenic trypanosomes was discovered by Evans 
in the blood of East Indian horses suffering from surra, but it was not well 
studied until 1893, when Lingard's important work on surra led, in a way, to 
all the subsequent work on diseases caused by trypanosomes. The next year 
a trypanosome was discovered by Bruce in the blood of horses and cattle 
suffering from nagana in Zululand and other parts of Africa. Bruce further 
demonstrated the important fact that the disease was transmitted by the 
bites of flies, the tsetse flies {Glossina). Announcements of other pathogenic 
trypanosomes in different parts of the tropics quickly followed. In 1896 Rouget 
found that dourine, a disease of equines in Algiers and South Africa, was caused 
by a trypanosome (T. equiperdum). Then the South African disease of horses, 
called mal de Caderas, was shown by Voges to be due to a similar flagellate, 
while in 1902, Theiler found a variety of trypanosome in the blood of cattle in 
the Transvaal suffering from the disease called galziekte, or gall-sickness. The 
number of trypanosomes found in the tropics is constantly increasing-both 
pathogenic and non-pathogenic forms. 
Man was thought to be comparatively immune to trypanosomes until the 
important discovery was made that trypanosomes are the specific cause of a 
definite disease known as sleeping sickness, which occurs chiefly in the African 
negro. 
In 1898 Nepvieu reported having found trypanosomes in the blood of 6 out 
of more than 200 cases of human beings examined for malarial organisms and 
in a seventh case which was apparently in good health. 
The eighth case is reported by Dutton in 1901. 
The tenth and eleventh cases were published by Manson in 1902. Broden 
published two more cases, and Baker three. 
In 1904 Castellani stated that the cause of sleeping sickness of the negro is 
a trypanosome. He found trypanosomes in the centrifugalized cerebrospinal 
fluid of 20 out of 34 cases of this disease. His work has been fully corroborated. 504 
PA TIIOGENIC MICROORGANISMS 
Groups. 
Sub- 
groups. 
Species. 
Date dis- 
covered. 
Where first 
found. 
Size in microns. 
Other 
characteristics. 
Pathogenesis. 
Invertebrate host. 
Length. 
Breadth. 
Vertebrate 
host. 
Name of 
disease. 
Laboratory 
animals. 
Name. 
Place of 
development. 
Max. 
Min. 
Max. 
Min. 
1 
T. lewisi Kent. 
T. evansi Steele 
1881 
1885 
Throughout 
world. 
India. 
38 
34 
10 
18 
3.0 
2.0 
1.5 
1.5 
Very motile, nu- 
cleus at anterior 
and middle third, 
blepharoplast 
rod-shaped. 
Rats. 
Equidae 
cattle 
camels, etc. 
Trypano- 
somiasis. 
Surra. 
Rat slight. 
Acute. 
Rat fleas 
(CtenophthaL 
mus) and lice 
(Hfematopi- 
nus). 
Stomoxyx 
(fly). 
Intestines. 
Unknown. 
always free. 
2 
3 
T. brucei Plimmer and 
Bradford. 
T. hippicum Darling. 
T. equiperdum Doflein. 
1899 
1910 
1901 
Zululand. 
Central 
America. 
North Africa. 
34 
28 
35 
18 
18 
16 
2.5 
3.0 
3.0 
1.5 
1.5 
1.5 
Post, end usually 
bluntly rounded. 
Equidae 
cattle. 
Equidae. 
Equidae. 
Nagana. 
Trypano- 
somiasis. 
Dourine. 
Acute. 
Acute. 
Acute. 
Glossina. 
Transmitted by 
coitus. 
Gut and sali- 
vary glands. 
Unknown. 
S 
4 
T. equinum Voges. 
1901 
South 
America. 
25 
22 
2.0 
15 
Kinetonucleus 
very minute. 
Equidae 
cattle. 
Mal de 
caderas. 
Acute. 
Hydrocherus 
Capybara. 
Unknown. 
I. Flagel 
5 
6 
7 
T. vivax Ziemann. 
T. capr® Kleine. 
T. transvaaliense Laveran. 
T. theileri Laveran. 
T. (Schizotrypanum) cruzi 
Chagas. 
1905 
1910 
1903 
1902 
1909 
Cameroon. 
German East 
Africa. 
Transvaal. 
German East 
Africa. 
Brazil. 
31 
32 
50 
70 
Aver 
16 
18 
18 
25 
age 
3.0 
4.25 
6.0 
5.0 
20 
2 
1.75 
4 
2 
Very motile, club 
shaped. 
Heavier build than 
vivax. 
Blepharoplast 
near nucleus. 
Very large. 
Dividing forms 
not seen in blood, 
blepharoplast 
large 
Equidae 
cattle. 
Equidae 
cattle. 
Bovidae 
slight. 
Equidae 
cattle. 
Man 
monkey. 
Trypano- 
somiasis. 
Trypano- 
somiasis. 
Trypano- 
somiasis. 
Sleeping- 
sickness. 
Trypano- 
somiasis. 
Slight. 
Glossina. 
Glossina. 
Conorhinus. 
Proboscis. 
Proboscis. 
Unknown. 
Gut and sali- 
vary glands. 
Differential Characteristics of More Important Trypanosomes, Pathogenic for Mammals (Modified from York and Blacklock). PATHOGENIC FORMS OF TRYPANOSOMA 
505 
II. Flagellum sometimes free II. Flagellum never 
and sometimes not. free. 
O CO 00 
T. congolense Borden. 1904 Congo. 19 8 Aver- 1.5 Equid® Trypano- Acute. Glossina. 
age, ruminants somiasis. 
T. nanum Laveran. 1905 Soudan. 16 10 Aver- 1.5 Probably identi- Equid® Trypano- Acute. Glossina. 
age, cal with Congo- ruminants somiasis. 
lense. 
T. montgomeryi Laveran. 1909 North East 20 10 Aver- 3.3 Heavier buildthan Trypano- Slight. 
Rhodesia. age, congolense. somiasis. 
T. simi® Bruce. 1912 Nyasaland. 23 12 .. Monkeys. Trypano- Acute for mon- Glossina. 
somiasis. keys and pigs. 1 
T. gambiense Dutton. 1902 1 Gambia. 39 13 .. Man. Sleeping Acute. Glossina 
sickness 
or try- 
panoso- 
miasis. 
T. pecaudi Laveran. i 1907 Senegal. 35 14 .. Posterior nuclear j Sleeping Acute. Glossina. 
forms. । sickness 
or try- 
panoso- 
; miasis. 
T. rhodesiense Stephens and 1910 North East 39 12 .. .. Posterior nuclear Man. Sleeping Very virulent. Glossina. 
Fantham. | Rhodesia. forms. sickness 
or try- 
panoso- 
miasis. 
T. Uganda Stephen and | 1913 Uganda. 38 13 .. .. Posterior nuclear Sleeping Acute. Glossina. 
Blacklock. forms. sickness 
or try- 
panoso- 
Proboscis. 
Proboscis. 
Unknown. 
Proboscis. 
Gut and sali- 
vary glands. 
Gut and pro- 
boscis. 
Gut and sali- 
vary glands. 
Gut and sali- 
vary glands. 
miasis. 
T. equi Blacklock and Yorke 1913 North Africa. 36 14 .. .. Posterior nuclear Equidae. Sleeping Acute. Transmitted by coitus 
forms. sickness y 
or try- 
panoso- 
miasis. 506 
PATHOGENIC MICROORGANISMS 
The trypanosomes found in these cases resemble each other; they arc, therefore, 
included under the same name, Trypanosoma gambiense Dutton. A similar 
form found by Stephens and Fantham in 1911 in cases of sleeping sickness in 
Rhodesia has been given another specific name, 7'. rhodesiense, chiefly because 
of the characteristic action in experimental animals (see table). 
Chagas, in 1909, states that a trypanosome which he had discovered in a small 
monkey (Callethrix hapalepeneceUata) is the cause of human infection in Rio de 
Janeiro. Because of its ability to grow in the tissues of infected animals 
it is classified by some as a new genus-schizotrypanum. It is carried 
by a hemiptera, genus Conorhinus. The flagellate is small with a large blephar- 
oplast (kinetonucleus). It grows on blood-agar readily and infects laboratory 
animals easily. Chagas reports developmental forms in the monkey's lung and 
in the gut of the fly. 
Fig. 174 
Agglutination of Trypanosoma lewisi. (Levaran and MesniL) 
Comparative Characteristics of the Different Species.-The form 
changes of the same species in the same host are so varied that few 
have been found absolutely characteristic of a single species, and, as 
physiologic properties alone are not considered final in species classi- 
fication, we cannot be sure that all of the organisms in this group des- 
cribed as separate species are so until more of the complete life histories 
are known. Until then each new form found with distinct physiologic 
properties, though apparently morphologically similar to others, may 
expediently be considered a new species. 
As an example of the difficulty of deciding whether or not a trypanosome 
found is a new species, the history of T. rhodesiense may be taken. All of the 
points brought forward by Stephens and Fantham, the discoverers, as evidence 
of a new species have been combatted by others; and its relation to T. gambiense 
on one side and T. brucei on the other is an open question. At present the points 
are in favor of its being a distinct species. MOTILITY 
507 
Morphology.-Size.-The variations recorded in the dimensions of the 
species we are considering may be seen by glancing at the above table. 
The trypanosomes pathogenic for man (T. gambiense and T. rhodesiense} 
have the smallest average size of the group. With the exception of 
T. theileri and T. transvaaliense, which are much larger than any other 
of these forms, the variations in size of the different species are not so 
marked as they are in the same species under different conditions. 
Shape.-In shape, though all follow the type, each species varies 
greatly according to conditions of growth and multiplication. At times 
they may be slender and worm-like, at others they may be so short and 
thick as to be almost round. T. lewisi has the posterior (aflagellar) 
end often thinner and more pointed than the other species. T. evansi 
is generally a little longer and thinner than T. lewisi, while T. brncei 
has a more rounded aflagellar end than either, and is generally broader, 
more club-shaped (Plate IV, Fig. i, 2). 
The cytoplasm differs slightly in the different forms. T. lewisi is 
relatively free from chromatoid granules, while T. brucei has usually 
many. Myoneme fibrils have been demonstrated in some species and 
probably all contain them. An oval vacuole has been seen in some 
species. 
The nuclear apparatus is essentially similar in all forms. The two 
nuclei (tropho- and kinetonucleus) vary somewhat in position and 
size in the different species and at different stages in the same species. 
In T. theileri and in young forms of T. leivisi, both nuclei lie close 
together near the centre of the organism. In T. lewisi the tropho- 
nucleus is situated more anteriorly than in the other species. In T. 
rhodes lense the nucleus is often posterior to the centre, especially in 
experimental animals. 
Many variations from the type forms are seen. Some are no doubt degen- 
eration and involution forms. Three forms, however, which are more or less 
constantly seen in all the species have been interpreted as definite phases in 
the life cycle. These forms were first described by Schaudinn in T. noctuoe, 
and were interpreted by him and since then by others as male, female, and 
indifferent form. The male cells are smaller, more hyaline, and more free from 
granules than the female. The nucleus of each sexual cell rids itself of male and 
female chromatin, respectively. The indifferent cell, on the contrary, has a com- 
plete nucleus. Opinions differ as to Schaudinn's interpretation being the correct 
one. More research is needed before we can arrive at a definite conclusion. 
Motility.-The first thing noticed on examining a fresh hanging 
drop of blood at a magnification of 100 to 300 diameters is active 
movements of the red-blood corpuscles in certain areas, and, on care- 
fully focusing over one of these areas, the rapidly wriggling worm-like 
organism may be seen. As the movements become slower, the flagellum 
may be seen swaying from side to side and the wave-like movements 
of the undulating membrane are quite discernible. Movement is 
twofold: (1) progression with an auger-like motion effected by the 
undulating membrane assisted by the flagellum; (2) contractions of 
the body assisted no doubt by myoneme-like structures. Relatively, 508 
PATHOGENIC MICROORGANISMS 
T. lewisi is most active and T. brucei least. Motility soon ceases 
outside of the body, continuing longer if the organism has been kept 
in the ice-box than at higher temperatures. Aflagell ar forms, sometimes 
ameboid, have been frequently described in the blood of mammals. 
Reproduction.-The usual method of multiplication is binary longi- 
tudinal fission. In several species a rosette-like segmentation has also 
been observed. Longitudinal fission begins usually with division of 
the kinetonucleus, then of the trophonucleus and cytoplasm; but 
this order of division seems to be quite variable. The flagellum often 
appears to be dividing first, and probably division always starts with 
the centrosome-like basal granule of the flagellum. In many cases a 
new flagellum seems to be formed instead of division of the old one. 
The details of division have not been frequently studied, but it is 
probable that both nuclei divide by a primitive mitosis. During division 
the kinetonucleus generally moves near the trophonucleus. Generally 
the fission is equal, but occasionally the daughter trypanosomes may 
be quite unequal in size. This is notably the case in division of T. 
lewisi where the cytoplasm may divide so unequally that the process 
may be compared to budding. The resulting small parasites have at 
first no undulating membrane, hence they resemble somewhat Lepto- 
monas. These young forms may divide several times in succession, 
producing smaller and smaller fusiform parasites. As a result, some 
forms are so small that they can only be seen when agglomerated or 
in motion (Schaudinn). The question as to whether trypanosomes 
undergo phases of development in their invertebrate hosts has been 
widely studied. Prowazek has described sexual forms of 7'. lewisi in 
the body of the louse. The subject, however, requires further research 
for convincing evidence. 
Insect Carriers.-The trypanosomiasis of vertebrates are trans- 
mitted by blood-sucking insects (dourine is possibly an exception). 
Bruce (1894) first showed that 7'. brucei was conveyed by the 
fly Glossina morsitans. Since then other varieties of flies also have 
been shown to spread the disease (table, pp. 504 and 505). Among 
them Glossina palpalis (Fig. 175) is supposed to be the chief agent in 
transmitting human trypanosomiasis. These flies bite by day and in 
full moonlight. The infective period of the insects after they have 
bitten a sick animal is variable. Bruce found living trypanosomes in 
the proboscides of the flies up to forty-eight hours. Up to one hundred 
and eighteen hours he found them in the flies' stomachs, after one hun- 
dred and forty hours he found the stomachs empty, and what appeared 
to be dead parasites in the excreta. Klein and others have since found 
that after a certain number of days (usually from twelve to twenty-five 
days) a small percentage of the biting flies, which were fed sufficiently 
on an infected animal become infective again. This is due to the fact 
that the trypanosomes .have developed in the gut and have passed to 
the salivary glands of the fly. They remain in the glands probably 
during the life of the fly, and continue to be infective (Bruce, Hammer- 
ton, Bateman, Mackie, and others.) EFFECT ON VERTEBRATE HOST 
509 
Dourine in horses is usually communicated during the act of coitus. 
Surra may be transmitted to dogs in eating infected animals. The 
rat trypanosome is conveyed by contaminative infection through the 
dejecta of the fleas or lice when biting a new host. 
Cultivation.-Novy and MacNeal were the first (1903) to cultivate 
trypanosomes in the test-tube. They have grown T. lewisi through 
many culture generations extending over several years. At the last 
tests made the parasites were as virulent as at the beginning. The 
culture medium used in their work was the condensation fluid from 
slant tubes of ordinary nutrient agar containing variable amounts of 
fresh defibrinated or laked rabbit or rat blood. The best results were 
obtained with a mixture of equal parts of blood and agar. At room 
temperature the growth is slower but surer than in the thermostat. 
A culture at room temperature retains its vitality for months; thus 
in one case the trypanosomes were alive after three hundred and six 
days. Novy and MacNeal also cultivated in vitro T. bmcei, T. evansi, 
and various bird trypanosomes. The latter they found especially easy 
to cultivate, while the former are much more exacting in their require- 
ments than is the T. lewisi. They require two parts of blood to one 
of agar, and growth is best at 28° C. The primary cultures are not 
transferred until three weeks. These are not virulent for animals, but 
subcultures regain their virulence. Mice and rats die in six to four- 
teen days after inoculation. Guinea-pigs have a relapse in two to ten 
weeks. 
The great majority of trypanosomes experimented with have been 
found by various workers to be cultivatable, with more or less ease. 
Trypanosoma gambiense, however, seems to be more difficult to cultivate 
(Thompson and Sinton). 
Effect on Vertebrate Host (Pathogenesis) .-Lower Animals.-- 
Many of the lower vetebrates have become, through mutual toleration, 
natural hosts of the trypanosome. It is probable that each pathogenic 
trypanosome has an indigenous wild animal as natural host and that 
in this way the supply to strange mammals coming into the vicinity 
is kept up. These strange animals, being unaccustomed to the native 
trypanosomes, often succumb to the infection. 
Symptoms.-In general the descriptions given of the symptomatology of 
trypanosomiasis in various animals show a great similarity, though there is much 
variation in individual cases. The average clinical picture, according to Mus- 
grave and Clegg, is as follows: After an incubation period which varies in the 
same class of animals and in those of different species, as well as with the con- 
ditions of infection, and during which the animal remains perfectly well, the 
first symptom'to be noticed is a rise of temperature. For some days a remit- 
tent or intermittent fever may be the only evidence of illness. Later on the 
animal becomes somewhat stupid; watery, catarrhal discharges from the 
nose and eyes appear; the hair becomes roughened and falls out in places and 
the peripheral lymph nodes are enlarged. Finally the catarrhal discharges 
become more profuse and the secretions more tenacious and even purulent; 
marked emaciation develops; edema of the genitals and dependent parts 
appears; a staggering gait, particularly of the hind parts, comes on, in some 
forms passing on to paralysis. This is followed by death. There may be various 510 
PATHOGENIC MICROORGANISMS 
ecchymoses and skin eruptions. Parasites are found in the blood more or 
less regularly after the appearance of the fever. They arc often more numerous 
in the enlarged lymph nodes and in the bloody edematous areas than in the 
general circulation. 
The autopsy shows general anemia, an enlarged spleen with hypertro- 
phied follicles, more or less gelatinous material in the adipose tissue, the liver 
slightly enlarged, a small amount of serous exudate in serous cavities, edem- 
atous condition, and small hemorrhages in various tissues. There is a relative 
increase of the mononuclears in the blood. 
The duration varies from a few days to many months. The prognosis seems 
to be influenced to a certain extent by the species of host. It is probably always 
fatal in horses. Some cattle recover. The cause of death is pcssibly a toxic 
substance, though no definite toxin has been isolated. Mechanical disturbances 
(emboli, etc.) also probably play a part in producing death. 
Man.-Sleeping sickness, or human trypanosomiasis, is an endemic 
disease in certain regions of equatorial Africa. Neither age nor sex 
are predisposing factors, but occupation and social position seem to 
have a marked influence, the great majority of cases occurring among 
very poor field workers. As these workers are all negroes, the question 
of the relative influence of race cannot be determined. The white 
race, however, is not immune, as has been frequently shown. 
Fig. 175 
Glossina palpalis, carrier of the human trypanosomiasis caused by T. gambiense. X 4. 
(Kolle and Wassermann.) 
Symptoms.-The course of the disease is very insidious, as the trypano- 
somes may exist in the blood for a long time before entering and growing in 
the cerebrospinal fluid and causing the characteristic symptoms. Therefore, 
the symptoms may be divided into two stages. In the first stage there is only 
an irregular fever with enlargement of the peripheral lymph nodes. In the second 
stage the fever becomes hectic, the pulse is constantly increased; there are neu- METHODS OF EXAMINATION 
511 
ralgic pains, partial edemas and erythemas, trembling of the muscles, gradually 
increasing weakness, emaciation, and lethargy. The somnolence increases until 
a comatose condition is developed and death occurs. In the second stage try- 
panosomes are always found in the cerebrospinal fluid. Throughout the disease 
they are usually found in small numbers in the blood. 
Duration.-The first stage may last for several years; the second, from 
four to eight months. The percentage of deaths in cases reaching the second 
stage is 100. Whether some in the first stage recover is not yet certain. 
T. gambiense, the trypanosome first shown to be pathogenic for human 
beings, is irregularly pathogenic for some monkeys (Macacus rhesus and others), 
for dogs, cats, and rats. It is less pathogenic for mice, guinea-pigs, rabbits, 
horses, baboons, cattle, and swine. In fact it has a very wide range of patho- 
genicity. 
Flies (Gl. palpalis) have been found to be infected with this trypanosome in 
areas which have had no human population for several years, a longer time than 
the life of a fly. And since trypanosomes are not known to be hereditarily 
transmitted, it is concluded that certain mammals are harboring the trypano- 
somes, thus acting as reservoirs. 
T, rhodesiense is more pathogenic for experimental animals than is T. gambiense. 
Pathological Changes.-Congestion of the meninges; increased quantity 
of cerebrospinal fluid; hypertrophy of spleen, liver, and lymphatic ganglia; 
diminished hemoglobin and number of red cells; number of leukocytes about 
normal, but a relative increase of eosinophiles, mast cells, and lymphocytes. 
Enlargement of the superficial lymph nodes has been noted as an early 
symptom and has thus been made use of in diagnosis. Dutton and Todd 
found that 91 per cent, of natives in the Congo Free State, who had posterior 
cervical glands enlarged, showed trypanosomes in the punctured gland juice. 
Diagnosis of Trypanosomiasis in General.-This should be made 
as early as possible in order to prevent the spread of the disease. An 
early positive diagnosis can only be made by the determination of 
the peripheral infection. This is done in two ways: first, by micro- 
scopic examination of freshly drawn blood, cerebrospinal fluid, or tissue 
from enlarged peripheral lymph nodes; second, by animal inoculation 
of the blood or other tissue. In the microscopic examination it may 
be necessary to examine the blood of the suspected animal for sev- 
eral days in succession. The parasites are rarely absent in the early 
stages in domestic animals for more than a few days at a time, while 
in man the time may be much longer. 
Methods of Examination.-Blood.-If the direct examination of 
the blood is negative, 10 c.c. should be withdrawn from the vein, and 
after adding a tenth of its volume of citrate of sodium it should be 
centrifuged for ten minutes, and the sediment examined in hanging 
drop and in smear. The great majority of the parasites will be found 
collected with the white cells in the thin white layers which may easily 
be removed with a fine pipette. The parasites are readily detected 
with the low power of the microscope (X 100) by areas of irregularly 
moving cells. If only a small amount of blood can be obtained, the 
tiny tubes recommended by Wright in his opsonin work (p. 210) may be 
used. 
Cerebrospinal Fluid.-Ten c.c. of the fluid withdrawn by lumbar 
puncture should be centrifuged for fifteen minutes and the deposit 
should be examined under 150 to 200 diameter magnification. 512 
PATHOGENIC MICROORGANISMS 
Cultures sometimes give positive results, especially from infections 
in lower animals and in infections with T. rhodesiense. 
The Inoculation Test.-If the trypanosomes cannot be found by 
the above methods, animal inoculation should always be made. 
Monkeys, if possible, should be used, or if monkeys cannot be ob- 
tained, dogs or rats may be used. A few drops to 1 c.c. of the blood 
or other tissue from the suspected animal should be inoculated intra- 
peritoneally or subcutaneously. 
Smears may be stained by any modification of the Romanowsky 
method. Giemsa's method (p. 81) gives good results (Plate IV, Fig. 1,2). 
Prophylaxis.-The disease is readily controlled by preventive meas- 
ures. There should be strict quarantine regulations governing the 
importation of animals. When the disease has once appeared, the 
following general measures should be taken: 1. Suspected animals 
should be isolated. 2. All infected animals should be destroyed. 
3. As far as possible, all biting insects should be destroyed, and the 
land in the neighborhood of human habitation should be suitably cul- 
tivated. 4. The bodies of infected animals should be protected from 
biting insects for at least twenty-four hours after death. 5. Susceptible 
animals should, if possible, be made immune. 6. All means possible 
should be used to exterminate the reservoirs. 
Treatment.-The whole question of treatment is still in the experi- 
mental stage. The chronic course of the disease with relapses often 
after long intervals makes it impossible, especially in cases of human 
trypanosomiasis, to come quickly to a conclusion in regard to the effi- 
ciency of any drug. Many drugs have been found to possess trypano- 
cidal properties. 
Atoxyl (p-amino-phenyl-arsenic acid, 
/OH \ 
/\AS =Z o 
'x/l nh2 \}N2 / 
introduced by Thomas (1905) and used first by Thomas and Breinl 
in treatment of experimental trypanosomiasis, has proved to be the 
drug above all others to have a beneficial effect in the different forms 
of this disease. Much experimental work has been done on the dif- 
ferent phases of treatment by atoxyl and its allies, in the course of which 
some very interesting facts relating to chemotherapeutics have been 
demonstrated. Ehrlich has added to his "side-chain theory," while 
others have advanced quite different views in regard to the action of 
this group. The chief facts are the following: 
1. Atoxyl does not act in vitro unless mixed with an oxidizing sub- 
stance. In vivo atoxyl combines with a blood-constituent to act upon 
the trypanosomes. 
2. After the first few treatments with any of these drugs trypano- 
somes may become resistant to the drug. This resistance is more or 
less specific for all members of the group to which the drug used belongs. COMPLEMENT-FIXA TION 
513 
There are a few exceptions, e. g., an atoxyl-resisting strain may still 
be influenced by arseno-phenyl-glycine or by orsudan. 
This acquired resistance lasts for some time in the species of animal 
used, but may be quickly lost if the resisting trypanosomes are inocu- 
lated into another species. Passage through an insect carrier may 
cause a drug-fest trypanosome to become again susceptible to the drug. 
3. The time of the reappearance of parasites after a discontinuation 
of treatment is more or less regular. With T. gambiense, in rats and 
monkeys, the period is generally 50 to 60 days. With T. brucei in rats, 
guinea-pigs, and dogs, the time is only 11 to 25 days. 
4. In order to pronounce an animal cured a long period must elapse, 
since relapses may occur at a very late date (226 days in rats infected 
with T. brucei and treated with atoxyl). 
In human trypanosomiasis favorable reports from atoxyl treatment 
still continue to come, though the percentage of cures claimed by Koch 
is probably not reached. Just now good reports are being received 
from the use of another arsenic compound, introduced by Ehrlich, 
namely, arseno-phenyl-glycine. 
Serum Therapy.-Various normal sera from different animals have 
been tried with practically no success. A few have prolonged life. 
Thus Laveran and Mesnil state that human serum injected in sufficient 
quantities shows manifest action on the disease, and that sometimes 
cure results in mice and rats. Further, by alternating human serum 
with arsenic they obtained better results still. Kanthack, Durham, 
and Blandford showed that animals recovering from trypanosoma 
infection were immune to further infection. Rabinowitsch and Kempner 
have made a very careful study of immune serum produced by T. 
lewisi. They have shown that an animal may be hyperimmunized 
and that then its serum, in comparatively large doses, inoculated into 
mice at the same time as the trypanosomes, or twenty-four hours before 
or after, allows no development of the organisms. Laveran and Mesnil 
state that the serum causes the rapid destruction of the organisms 
by the leukocytes, though MacNeal, on the other hand, states that the 
trypanosomes are destroyed by a cytolytic action of the serum. This 
immune serum also has a similar action on the trypanosoma of dourine. 
The serum of animals hyperimmunized against other varieties of 
trypanosoma is not as active as that obtained by the inoculation of 
T. lewisi. Koch suggested that immunity might be produced by the 
inoculation of attenuated parasites, and Novy and MacNeal have 
succeeded in attenuating cultures of T. brucei, and have obtained some 
success in protecting experimental animals against virulent cultures. 
Complement-fixation.-The earlier tests did not promise practical 
results. Recently, however, several investigators have given more 
favorable reports. In this country Mohler claims that the test is of 
great worth in diagnosing dourine, many cases of which have occurred 
in some of our western states (notably Iowa and Montana). Mohler, 
Eichhorn, Buck, and Traum state that they use a fresh antigen pre- 
pared from the spleens of rats dead after infection with surra (used 514 
PA THOGENIC MICROORGANISMS 
because it is easier to transmit than is dourine). The antigen is simply 
a filtered emulsion in salt solution of the infected spleen. The emulsion 
from each spleen is made up to 40 c.c. by addition of salt solution. A 
fresh antigen must be prepared and titered (see Part I) each day. The 
results have been controlled by autopsies of animals giving a positive 
reaction. 
References. 
Breinl and Nierenstein. Ann. of Trop Med. and Parasit., 1909, iii, 395. 
Bruce. "Trypanosomiasis" in Osler's Modern Medicine, 1907, i, 460. 
Bruce, Hammerton, Bateman and MacKie. Proc. Roy. Soc., 1911. 
Ehrlich. Ueber Partial funktionen der Zelle., Munch, med. Woch., 1909, v, 217. 
Hindle. Observations on the life history of T. lewisi in the rat louse. Assoc, of Trop. 
Med. and Parasit. 
Laveran et Mesnil. Trypanosomes and Trypanosomiases, Trans, by Nabarro, London, 
1907. 
MacNeal. Jour. Inf. Dis., 1904, i, 537. 
MacNeal and Novy. Contrib. to Med. Research, Vaughan Anniv., 1903, p. 645. 
MacNeal and Novy. Trypanosomes of Mosquitoes, Jour, of Infect. Diseases, 1907, 
iv, 223. 
Mayer, M. Trypanosomen als Krankheitserreger. In Kolle und Wassermann's 
Handbuch d. Path. Mikrcbrganismen, 1913, 2d ed., Jena. 
Mesnil et Kerandel. Sur I'action preventive et curative de 1'arsenophenylglycite 
dans les trypanosomiases experimentales et en particulier dans les infections a T. gambiense. 
Bull. d. 1. Soc. d. path, exot., 1909, ii, 402. 
Mohler, Eichhorn, and Buck. Jour. Agric. Res., 1913, i, 99. 
Musgrave and Clegg. Trypanosoma and Trypanosomiasis, etc., Manila, Bureau of 
Public Printing, 1903. 
Novy and MacNeal. Trypanosomes of Birds, etc., Jour. Infect. Diseases, 1904, i; 1905, 
ii, 256. 
Novy, Perkins, and Chambers. Jour. Inf. Dis., 1912, xi, 411. 
Nuttall. Parasitology, 1913, v, 275. 
Stephans and Fantham. Proc. Roy. Soc., 1910, Ixxxviii, 28. 
Thompson and Sinton. Amer. Trop. Med., 1912, vi, 351. 
Vianna. Mem. d. 1'Inst. Oswald. Orig., 1911, iii, 276. 
Woodcock. The Hemoflagellates and Allied Forms in Lankester's "A Treatise on 
Zoology," London, 1909. Part I, first fascicle, p. 193. 
Yorke, W., and Blacklock, B. The Differentiation of the more important mammalian 
trypanosomes. Amer. Trop. Med. and Parasit., 1914, viii, 1. C HAP T E R XXXIX. 
SPIROCHETA AND ALLIES. 
The genus Spirocheta was introduced by Ehrenberg in 1838, who 
differentiated it from spirillum by its flexibility. Schaudinn in 1905 
thought he saw an undulating membrane in Spirocheta refringens, so he 
added this characteristic to the genus and considers that thus its 
relationship to the flagellated protozoa, genus Trypanosoma, is indicated. 
Since the appearance of the work of Schaudinn and Hoffmann (1905) 
showing the etiologic relationship of a spirochete to syphilis, the Spiro- 
chetce have been brought into great prominence. 
Numerous spirochetes and spiral organisms have been described, 
some associated with Treponema (spironema) pallidum in syphilis, 
some in other lesions or in the normal secretions of both man and the 
lower animals; and still the question as to their classification is unsettled. 
The majority of observers, however, are willing to admit that the 
structure of many of the varieties classed with this group is more 
complicated than that of bacteria and that hence the group may be 
an intermediate one between protozoa and bacteria. 
The chief reasons given for considering spirochetes protozoa are: (1) the 
flexibility and the indications in many of longitudinal division and of undulat- 
ing membrane; (2) the demonstration of forms intermediate between the 
trypanosomes and the spirochetes (Sp. balbianii); (3) the spirochetal forms 
of certain trypanosomes (Tr. noduce); (4) stages of development in the louse 
and tick. 
In favor of the bacterial nature of spirochetes are: (1) the rigidity of some 
forms, the lack of undulating membrane in most and of definite nuclear appara- 
tus in all, and the evidence of transverse division in all and of flagella arising 
from the periplast in some; (2) the cultivation of certain forms (e. g., Sp. refrin- 
gens, by Levaditi; Sp. obermeieri, by Novy; several spirochetes by Noguchi) 
for many generations without development of trypanosome forms. 
So far the studies on this group show that the spirochetes and allies prob- 
ably occupy a position intermediate between the protozoa and the bacteria. 
We study them here because of the claims that they are closely related to 
the trypanosomes. 
It may be well to note briefly the chief characteristics of the more 
familiar non-pathogenic ones in order better to understand the relation- 
ships between them and the Treponema pallidum and other pathogenic 
forms. 
Material and Methods for Study.-The large Spirocheta balbianii is found 
in the stomach of oysters fresh from salt water. Smaller spirochetes are fre- 
quently found in human mouths. When fresh syphilitic or relapsing fever 
material can be obtained this should be examined. The Treponema pallidum 
(the spiral organism of syphilis), because of its low refractive index, is seen 516 
PATHOGENIC MICROORGANISMS 
when alive with difficulty by the ordinary microscope, but with the dark- 
stage illumination, especially if a drop of distilled water is added to the serum 
containing the organisms, it is seen distinctly and its motion and structure 
may be more easily studied. The fluid containing the organisms should be 
dropped on an ordinary glass slide, covered with a thin cover-glass, and well 
sealed with vaselin as most spirochetes are anaerobic. Material may be ob- 
tained from syphilitic lesions as follows: The lesion is first thoroughly washed 
and dried with distilled water and sterile gauze. Part of the base and margin 
is then scraped with a curette until the superficial tissue is removed and blood 
appears. The blood is wiped away with sterile gauze until clear serum begins 
to ooze. A drop of this serum is used for examination. 
Smears should be made as thin as possible and may be stained (1) by Giemsa 
according to the method on page 81 (Tr. pallidum stains reddish). A modi- 
fication of Giemsa, used by Schereschewsky (see References) has been highly 
recommended by various workers; (2) by Goldhorn's method (p. 82). 
Cultures.-Pure cultures have been obtained of the Spirocheta dentium 
in the following manner: Poured serum-agar plates are made of various dilu- 
tions of material from the mouth containing these spirochetes. After being- 
kept in the thermostat at 37° C. under anaerobic conditions for nine to twelve 
days the spirochetal colonies are fished and planted in agar tubes as stick 
cultures. 
Pure cultures of Spirocheta obermeieri by Novi and of Spirocheta refringens 
by Levaditi have been obtained by growing in collodion sacs. (For other culture 
experiments see below.) 
Spirocheta Balbianii (Certes). - This immense form, next largest 
known to the Spirocheta plicatilis Ehrenberg, may be found in the 
stomach of the oyster. It is important for study because it is appar- 
ently a transitional form. In fact, it is considered a trypanosome 
by Perrin and others. Miihlens gives its characteristics as follows: 
Length 26/z to 120^, in width to 3m- The body is flattened and pos- 
sesses an undulating membrane which is visible during life on some 
individuals. It has 4 to 8 flat, wide spiral coils. Its movements are 
lively, similar to those of trypanosomes, but more corkscrew-like. 
During motion its form is apparently easily changed. The rim of 
the undulating membrane does not end in a free flagellum, but one 
end of it seems to be attached to a triangular mass of chromatin (basal 
granule, blepharoplast?) which is a part of the central chromatin 
material. The nuclear material is arranged in a more or less spiral 
band along the entire centre of the organism. 
Before division this nuclear band, after passing through chroma- 
some-like changes, breaks up into pairs, and division takes place 
longitudinally between them. Division is often incomplete for a time, 
the two ends remaining attached. 
Spirocheta balanitidis.-This is a spirochete found by Simon in 
Balanitis circinata and regarded by some as the specific cause of this 
disease. Hoffmann and Prowazek describe it as a rather strongly re- 
fractive, actively motile, band-shaped organism, shorter and thicker 
than Spirocheta pallida, with 6 to 10 coils, staining bluish-red with 
Giemsa's method and exhibiting an undulating membrane and at 
either end a periplastic cilium. 
Miihlens thinks this may be identical with Spirocheta refringens. SPIROCHETES IN TUMORS 
517 
Levaditi has recently reported cultivating it (see below under Trepo- 
nema pallidum). 
The Mouth Spirochetes.-Three varieties of non-pathogenic forms 
are commonly found in normal mouths. 
1. Spirocheta Buccalis (Cohn).-Length, 10m to 20m; thickness, |m 
to 1/jl. It has 3 to 10 irregular flat coils. No true cilia have been 
demonstrated, but Schaudinn, Hoffman, and Prowazek say it has an 
undulating membrane. It stains violet with Giemsa. 
2. Spirocheta Dentium (Koch).-This is much smaller than the previous 
form. It is as thin as the pallidum and is somewhat similar to it in 
refraction, staining qualities, and in the fixity of its coils during motion. 
It is somewhat smaller and stains a little more easily with Loffler's 
flagella stain, and flagella have been demonstrated. Neither definite 
undulating membrane nor nuclear material has been seen. It is 4m to 
12m long, and has 4 to 20 regular spirals of about the same appearance 
as those of the pallidum. Pure cultures have been made from this 
spirochete as described above. 
3. A middle form between these two has been found in the mouth. 
This also is somewhat similar to the pallidum, but it is larger and has 
less regular spirals; moreover, it stains more intensely with the blue 
of Giemsa, only in poorly prepared specimens does it appear red. 
Spirocheta refringens is also found in the mouth, but it is espe- 
cially interesting from the fact that it is so often found associated 
with the Treponema pallidum in the various lesions of syphilis. It 
is not in such large numbers as the pallidum and probably bears 
the relation of a restricted secondary invader. It is generally longer 
than the pallidum (10m to 30m) and much thicker (|m to |m)- In life 
it is much more refractive. It has 3 to 15 irregular wide, flat spirals 
which change their shape during motion. Its movements are much 
more lively than those of pallidum. With Giemsa it stains quickly 
and easily, a blue to a blue-violet tone, according to the length of 
staining. Schaudinn states that it possesses an undulating mem- 
brane. Levaditi claims to have demonstrated terminal cilia for this 
organism and to have cultivated it in collodion sacs in the rabbit's 
peritoneum. 
Spirocheta Vincenti.-Accompanying the fusiform bacilli in Vincent's 
angina (see p. 319) are many spirochetes similar to the "middle form" 
found in the mouth. Whether they are identical with these spirochetes 
or whether they are a special variety (or, as some think, a second form 
of the fusiform bacillus) still remains to be determined. Their relation- 
ship to the disease is also uncertain. 
Spirochetes in Tumors.-Lowenthal, Borrel, and others found 
spirochetes in small numbers in certain mouse tumors. Ewing and 
Beebe found a few in some dog tumors and others have reported their 
occasional presence in both ulcerating and non-ulcerating human 
tumors, but apparently never in sufficient numbers to account for the 
tissue reaction. Gaylord, however, found that in repeated transplants 
of a mouse tumor, as the inoculated material became more virulent the 518 
PA THOGENIC MICROORGANISMS 
number of spirochetes greatly increased. Calkins studied the mor- 
phology of Gaylord's spirochete and decided it to be a distinct species. 
He has also found this species in primary as well as in transplanted 
tumors. It is much shorter and thicker than the pallidum, and has blunt 
ends. It closely resembles the spirochetes found comparatively fre- 
quently by Tizzer and others in apparently normal mice, though the 
possibility of infection in these cases was not ruled out. 
Miscellaneous Spirochetae.-Besides the spirochetes found in syphilis, 
in frambesia, and the spiral organisms causing African and European 
relapsing fevers, all of which will be described below, spirochetes have 
been found (1) in the normal intestinal tract of mosquitoes and human 
beings as well as in the diarrheal stools of the latter; (2) in the blood 
of mice, fowls (Sp. gallinarum causing relapsing fever in fowls and 
Sp. anserina found in a similar condition of geese); (3) in various ulcera- 
tive and gangrenous process of man. 
The fowl spirochetes have been most studied. Marchoux and Sal- 
imbeni were the first to show that the tick, Argos persicus, is a carrier 
of these spirochetes. The mechanism whereby the tick infects the fowl 
has been minutely worked out by Nuttall and his associates. 
Noguchi has cultivated several species of spirochetes by the same 
method he used for growing Tr. pallidum (see below). Four of these, he 
stated, are new species, Sp. macrodentium and Sp. microdentium from 
the mouth, Sp. phagedensis from human genitals, and Sp. calligyrum 
from condylomata. Noguchi also reports the cultivation in successive 
transplants of Sp. recurrentis and Sp. duttoni. 
Treponema (Spironema) Pallidum (Spirocheta pallida). - This 
organism is found in large numbers in syphilis, an infectious disease 
of human beings, characterized by its long course and by the definite 
stages of its clinical manifestations. 
Historical Note.-Notwithstanding the fact that syphilis is one of the oldest 
diseases known and studied, only recently has definite light been thrown upon 
its cause in the discovery of the Treponema pallidum (Schaudinn and Hoffmann). 
Before this it was thought that the bacillus described by Lustgarten (1884) 
and others as occurring in small numbers in the lesions of syphilis bore an etiologic 
relationship to the disease, but there were no evidences to support this view. 
Many other bacteria have been erroneously regarded as the probable cause of 
syphilis. 
From time to time various observers have described protozoan-like bodies 
in syphilitic lesions, but their observations have not been confirmed. 
Schaudinn announced early in 1905 that working with Hoffmann he found 
in the fresh exudates of chancre a spiral organism possessing characteristics 
similar to those of the spirochetes and he named it Spirocheta pallida. Later 
he concluded that this organism was individual enough (that is, it showed no 
undulating membrane, but possessed flagella) to be placed in a separate genus, 
so he called it Treponema pallidum. He thought that the organism was the cause 
of the disease. Since then there have been extensive studies on human syphilis 
and on experimental syphilis in lower animals with the result that the work of 
Schaudinn and Hoffmann has been abundantly corroborated and many new facts 
have been brought out. 
The Organism (Fig. 176.-The Treponema pallidum is a very delicate 
structure closely resembling in morphology and staining reactions CULTIVATION 
519 
the Spirocheta dentium. It is somewhat longer, 4m to 20m long (average 
10m), and thinner, |m to |m in diameter. It has three to twenty sharp, 
deep spirals. The relationship between the length and the depth of 
the spirals is different in the two species; in Treponema pallidum length 
is to depth as 1 is to 1-1.5 (Im long and Im to 1.5m deep), while in Spiro- 
cheta dentium the average relationship is 1:0.5 the spirals being more 
shallow. The angle of the spiral turn is very sharp in both forms (more 
than 90°). 
Fig.' 176 
Fig. 177 
The two spirochetes in the 
centre are Tr. pallidum; the 
three others, Sp. refringens. 
(Schaudinn and Hoffmann.) 
Treponema pallidum appearing as bright refrac- 
tive body on a dark field as shown by India ink or 
ultramicroscope. 
Flagella-like anterior and posterior prolongations are often seen in 
the pallidum. The double flagella occurring rarely at one end are 
interpreted by Schaudinn as beginning longitudinal division. Schau- 
dinn states that the division occurs very quickly (hence the reason 
why so few dividing forms are seen in stained preparations) and that 
it may be followed only by the most experienced observers during life. 
In the living condition the organism is not very refractive, so it is seen 
at first with difficulty. Its characteristic movements are rotation on its 
long axis, quivering movements up and down the spiral which is com- 
paratively rigid, slight forward and backward motion and bending of 
the entire body. By the use of the ultramicroscope the motility of the 
organism is clearly seen (Fig. 177). 
Cultivation.-In 1909 Levaditi and McIntosh obtained impure cul- 
tures of spirochetes in collodion sacs containing human serum and 
syphilitic material and placed in the peritoneal cavity of a monkey 
(Macacus cynomolyus). Schereschewsky reported that he had ob- 520 
PATHOGENIC MICROORGANISMS 
tained impure cultures of a spirochete from syphilitic lesions and 
blood in the following culture medium: horse serum sterilized by heat 
(58° to 60° C.) until it is of jelly-like consistency, and afterward autolysed 
at 37° for three days. A piece of tissue excised from the lesion (e. g., 
base of a papule or part of a lymph node) is inoculated into this medium, 
and grown at 37° C. The culture begins in three days, but the optimum 
is reached in 5 to 12 days. 
Miihlens (1909) and later Hoffmann (1911), reported that they had 
also obtained cultures of a pallidum-like spirochete from syphilitic 
lymph nodes, grown at first in Schereschewsky's medium and afterward 
transplanted to broth and grown anaerobically. Animal experiments 
are negative. 
Levaditi and Stanesco about the same time reported growing two 
species of spirochetes from a case of balanitis. One, a new one, which 
they found very like pallidum, but non-pathogenic for monkeys, and 
which they named Sp. gracilis; the other Sp. balanitidis. They em- 
ployed as media (1) collodion sacs in tubes of fluid horse serum; 
(2) horse or human serum heated to 75° C. These spirochetes were 
never obtained in pure culture. 
Noguchi (1911) obtained pure cultures from syphilitic lesions in the 
rabbit, and later from human beings. His culture medium contained 
in deep tubes was a mixture of 1 part of ascitic or hydrocele fluid and 
2 parts of 2 per cent, agar, in which was placed a small portion of sterile 
rabbit's kidney or other organ. The medium was covered by a deep 
layer of albolene. The spirochetes inoculated along a central stick 
grow out into the medium as a diffuse layer, while most contaminating 
bacteria remain in the line of inoculation. Subcultures made away 
from the contaminating growths, finally become pure. Noguchi states 
that he has obtained syphilitic papules by scarifying monkeys (cer- 
copithecus and macacus'), and that such monkeys' blood gives a positive 
Wassermann reaction. 
Pathogenesis.-So far as is known, syphilis in nature appears only 
in man. Since 1879, when Klebs stated that he had produced syphilis 
in monkeys by the inoculation of human virus, various experimenters 
have reported its transmissibility to these animals by direct inoculation. 
Most of the earlier reports did not state the exact identity of the 
animals employed nor did they give details of methods and results. 
Metchnikoff and Roux in 1903 produced a typical chancre on the genital 
mucosa of the young chimpanzee twenty-six days after inoculation. The 
essential lesion was followed by inguinal adenitis, and thirty days later by a 
generalized papular eruption. The virus was transferred in this case to lower 
monkeys. Most monkeys developed a primary lesion only., but some had 
abundant secondaries. 
Since the discovery of the Tr. pallidum, experiments on monkeys have been 
more numerous and have been followed by more helpful results. More has 
been learned about the course of the infection in man, the evidence in favor 
of the Tr. pallidum being the cause of the disease has been strengthened, and 
many interesting investigations in regard to immunity have been made. Usu- 
ally inoculations made by scarification on eyebrows or genitals are successful. 
The primary lesions are seen generally about thirty days after infection. In PATHOGENESIS 
521 
somewhat more than half the cases, after a slightly longer period, secondary 
symptoms (squamous papules on skin, and mucous patches in mouth) show them- 
selves. No tertiary symptoms have been observed. Rabbits were first suc- 
cessfully inoculated by Hansell, then by Bertarelli. The eye and the testes 
are favorable localities. Generalized syphilis has been produced in young 
rabbits by intracardiac inoculation (Uhlenhuth and Mulzer). 
Important features in regard to course of the infection have been sum- 
marized by Ewing as follows: "If the virus is applied to the broken epithe- 
lium, a chancre develops, but if similar virus is inoculated into the subcutaneous 
tissue an initial lesion does not follow, immunity does not develop, and the 
animals remain susceptible to subsequent inoculation of the epithelium. Yet 
in several instances Neisser was unable to produce chancres in monkeys which 
had previously received subcutaneous injections of syphilitic material, indicat- 
ing that immunity may sometimes appear after such subcutaneous injections. 
Possibly the leukocytes of the subcutaneous tissue destroy the virus before it 
can begin to multiply. Hence, small superficial wounds may be more dangerous in 
man than deep ones. Nevertheless, it is recorded by Jullien that two French sur- 
geons, accidentally inoculated by deep needle punctures, developed pronounced 
signs of constitutional syphilis, as attested by Fournier, but failed at any time 
to show signs of a chancre at the point of inoculation. It remains to be seen 
whether the observations of the clinicians or those of the experimental pathol- 
ogists represent the true laws of infection in syphilis. 
"In monkeys the virus exhibits a certain choice of epithelium for its entry. 
The abdominal skin resists the entry, the eyebrows and genitals are most 
readily inoculable in apes, and the palpebral borders in catharinians. The 
period of incubation varies from thirty days, on the average, in the chim- 
panzee, to twenty-three days in lower monkeys, but the shorter the incuba- 
tion, the shorter and less severe the subsequent disease. 
"That the virus circulates in the blood in certain stages of syphilis has been 
clearly shown experimentally. Although Neisser inoculated human subjects 
with the blood of florid syphilis without effect, a result which is now explicable, 
Hoffmann, in two of four experiments, produced syphilis in monkey (Macacus 
rhesus') by inoculating the skin with human blood drawn forty days and six 
months after the appearance of the chancre. The resulting primary lesions 
were typical, appearing after the usual incubation and showing a characteristic 
histologic structure and the presence of Tr. pallidum. 
" Syphilographers are agreed that tertiary lesions are not contagious. Ex- 
perimental studies have shown, however, that some tertiary lesions are capable 
of transmitting the disease. Salmon had negative results with an ulcerated 
gumma in the eighth year of the disease. Yet Neisser produced chancres and 
secondaries in a gibbon and in a macacus with the material from a non-ulcerated 
gumma (duration unknown), but the periods of incubation were very long, 
fifty-one and sixty-eight days. All tertiary lesions do not seem to contain the 
virus, as Neisser found the material from tubercserpiginous lesions non-infec- 
tious. It appears also that secondary infection and ulceration of tertiary lesions 
reduce their infectivity. None of these observations invalidates the clinical 
experience that tertiary lesions are practically harmless for the patient's neigh- 
bors, but they suggest greater caution in dealing with tertiary lesions. 
"According to Colles' law, a mother who gives birth to a syphilitic infant 
may not herself contract the disease, but thereafter remains immune to inocu- 
lation. This law may be explained by the infection by the embryo or ovum, and 
the transference of immunity to the mother by the blood or by some other 
method. The probable mode of origin of the maternal immunity is suggested 
by an observation of Buschke and Fischer who found spirochetes in the inguinal 
lymph nodes of such a case which remained entirely free from the symptoms 
of the disease. The observation, taken with the failure of subcutaneous and 
intraperitoneal inoculation to infect monkeys, may explain the workings of 
Colles' law. Levaditi and Sauvage claim to have shown that Tr. pallidum is 522 
PATHOGENIC MICROORGANISMS 
capable of invading the ovum. Finger and Landsteiner found the semen in one 
case of secondary lues infectious for apes, but in other cases their results were 
negative. It is, therefore, only necessary to suppose an occasional escape from 
the genital tract in order to complete the necessary conditions for the infection 
of the embryo with immunity in the mother. 
"Neisser endeavored to determine the degree and duration of the infec- 
tivity of the organs of monkeys and found that the virus persists especially 
in the blood-forming organs, spleen, lymph nodes, and marrow, while in the 
testicle also the virus is long preserved in active form. The other organs gave 
entirely negative results." 
The virus is not filtrable. It is readily destroyed by heat (52° C.). 
Luetin.-Noguchi found that an extract from his cultures, which he calls 
luetin, gives a characteristic cutaneous reaction in syphilic infection. In normal 
persons there is a slight local erythema, with possibly a small papule on the 
second day. In syphilitics there are three types of positive reaction: 
1. Papular type. Large indurated red papule which increases for about 
four days. 
2. Pustular type. The papule changes first into a vesicle and then into a 
pustule. 
3. Torpid type. After a ten-day latent period, a small papule forms which 
changes into a pustule. 
These observations of Noguchi have been corroborated by Cohan and 
Robinson. 
Syphilis in Man.-The course of the disease is divided into three stages: 
primary, secondary, and tertiary. The general character of the lesions 
in these stages is a more or less circumscribed formation of new tissue 
which is largely made up of small spheroidal cells alone or accompanied 
by fewer polyhedral cells, and occasional giant cells. 
The initial or primary lesion occurs in the form of a papule which develops 
into the so-called chancre, an ulcer with hardened base. Following this there 
is hyperplasia of the nearest lymph nodes. These lesions subside and six or 
seven weeks later the secondary lesions appear in various general eruptions on 
skin and mucous membranes and in other constitutional disturbances. The 
tertiary lesions which consist principally of the masses of new tissue called 
gummata are found throughout the viscera and in the periosteum. 
Schaudinn's spirochetes have been demonstrated in practically all 
the lesions of syphilis (they are most easily demonstrated in the pri- 
mary and secondary lesions), including the congenital types. 
Immunity.-Natural immunity in syphilis is very peculiar. After 
the development of the primary lesions, man is usually insusceptible 
to reinoculation during the active stage of the disease, but during all 
stages both man and monkey can, in some cases, be reinoculated. 
Reinoculation in the tertiary stage gives precocious lesions of the 
tertiary type, gummata, and tubercles. Neisser found reinoculation 
from twenty-four to one hundred and four days after. primary in- 
oculation in monkeys sometimes effective, more often negative. 
During the stage when the skin is refractory to inoculation secon- 
daries develop, showing that there is no complete immunity of the 
skin to the virus, since the Treponema is abundantly present in the 
lesions. Neisser suggests that cutaneous secondaries develop at 
periods of relative deficiency of immunity. He has shown that failure SPIROCHETES IN FRAMBESIA TROPICA 
523 
of reinoculation is not due to immunity to foreign infection and sus- 
ceptibility to auto-infection, since the patient's own virus in both man 
and monkey is ineffective. 
Passive Immunization.-Injection of large quantities of serum of 
syphilitics into chimpanzees has failed to produce definite immunity, 
although some of Neisser's animals after such treatment failed to take 
syphilis. The serum of a monkey cured of syphilis and subsequently 
injected over a period of fifteen months with the blood of a syphilitic 
subject in roseolar stage was without therapeutic effect. However, 
this serum dried and powdered prevented the chancre when placed 
on the site of inoculation one hour after the virus. 
The Wassermann Reaction.-Wassermann, Neisser, and Bruck were 
the first to apply the Bordet-Gengou phenomenon (see Part I) to the 
diagnosis of syphilis. According to most workers, enough work has 
been done since then to establish its value as a diagnostic test. Some 
interesting points have been brought out in connection with this study, 
and many different methods have been recommended. 
This test has been very widely used and positive results have been 
obtained in an immense number of cases (over 90 per cent, of cases with 
active general infection). In general paralysis and in the majority 
of cases of tabes, a positive reaction occurs which may be given also 
by the cerebrospinal fluid. On account of the difficult technique 
involved it can only be of use in the hands of experienced workers. 
Positive results have been reported in a number of other diseases as 
well as syphilis, but in many cases these results have not been generally 
accepted, and in other cases the diseases showing positive reaction 
as yaws, leprosy, dourine, etc., have been as a rule confined to the 
tropical countries, or else the positive reaction has been found only 
during a limited stage of the disease, as in scarlet fever, or the differ- 
ential diagnosis is otherwise marked, as with tuberculosis. The general 
opinion at the present time is that the test is of great practical value in 
the majority of cases of syphilis. It is used as a routine public health 
measure in New York City and several other centres. The reaction 
is practically always present during the secondary stage of the disease. 
The reaction gradually disappears when the disease becomes inactive 
or is cured. It may disappear before a cure is established, to reappear 
if an active process starts. Mercury treatment diminishes or annuls 
the reaction; while treatment with salvarsan may at first intensify it. 
The nature of the reaction and the technique of the test are considered 
in Part I. 
Spirochetes in Frambesia Tropica (Yaws).-Castellani in 1906 
announced that he had found in yaws a spiral organism which he 
called Spirocheta pertenuis. He determined that monkeys (macacus, 
sevznopithecns) are susceptible to inoculations with material from yaws 
patients apparently containing only this spirochete. Such material 
filtered is inert. He states that monkeys successfully inoculated with 
yaws do not become immune for syphilis, neither do those having had 
syphilis become immune for yaws. Castellani further states that specific 524 
PATHOGENIC MICROORGANISMS 
characteristics between the two diseases are also brought out by means 
of the Bordet-Gengou reaction. His work has been corroborated by 
several observers. Frambesia lesions similar to those produced by 
syphilis in the testicles of rabbits have been obtained by Nichols. 
Levaditi and Nallan-Larrier state that monkeys infected with syphilis 
are refractory to yaws, while those infected with yaws are susceptible 
to syphilis; therefore, they conclude that yaws is a mild form of 
syphilis. 
Spirocheta Obermeieri (Sp. recurrentis) and Allies.-These organisms 
are classed with the spirochetes as protozoa by Schaudinn, Hartmann, 
Miihlens, and others, but by Norris, Novy, and others they are still 
placed with the bacteria. Novy and Knapp have made extensive studies 
of Sp. obermeieri (the cause of relapsing fever in Europe) as well as of 
Sp. duttoni (the cause of tick fever), spirochetes from American 
relapsing fever, and Sp. gallinarum (fowl spirochete) and consider that 
they have demonstrated their bacterial nature and that many, if not 
all, spirochetes should be placed in this group. 
Fig. 178 
Fig.' 179 
Photograph of Sp. obermeieri showing 
terminal flagellum. X 3000. (After 
Novy.) 
Spirocheta obermeieri blood-smear. 
Fuchsin. X 1000 diam. (From 
Itzerott and Niemann.) 
Spirocheta (spirillum) obermeieri was first observed by Obermeier 
in 1873 in the blood of persons suffering from relapsing fever. It was 
found in large numbers during the height of the fever, it disappeared 
about the time of the crisis, and reappeared during the relapses. It 
was not found in other diseases. Obermeier considered it the cause of 
the disease, and his views were shown to be correct by the production of 
the disease in man and ape through experimental inoculation. 
Morphology.-The organisms are long, slender, flexible, spiral or 
wavy filaments, with pointed ends, from 16^ to 40^ in length and from 
one-quarter to one-third the thickness of the cholera spirillum (|p. to 
|/z). They stain somewhat faintly with watery solutions of the basic 
aniline dyes, better with Lbffler's or Kiihne's methylene-blue solu- BIOLOGIC CHARACTERS 
525 
tions, or with carbol-fuchsin; best with the Romanowsky method or its 
modifications. They are negative to Gram. Novy has demonstrated 
a terminal flagellum (Fig. 178). There are three to twelve wide, more 
or less irregular spirals. 
Biologic Characters.-In fresh preparations from the blood the spiro- 
chetes exhibit active progressive movements, accompanied by very 
rapid rotation in the long axis of the spiral filaments or by undulat- 
ing movements. They are found only in the blood or blood-organs, 
never in the secretions, and only during the fever, not in the intermis- 
sions, or at most singly at the beginning of, or for a short time after, an 
attack. 
When kept in blood-serum, or a 0.6 per cent, solution of sodium 
chloride, they continue to exhibit active movements for a considerable 
time. They may be preserved alive and active for many days in sealed 
tubes. They are killed quickly at 60° C., but they remain alive for 
some time at 0° C. Unsuccessful efforts to cultivate them in artificial 
culture media have been made from time to time. Koch has observed 
an increase in the length of the spirilla and the formation of a tangled 
mass of filaments. Novy finally succeeded in cultivating them in 
celloidin capsules placed in the peritoneum of rats. His culture 
remained virulent (with a slight loss) for many generations. 
Pathogenesis.-In man, whether the disease is acquired naturally or 
by artificial inoculation, the organism causes the following symptoms: 
After a short period of incubation the temperature rises rapidly, re- 
mains high for five to seven days, and then returns to normal by crisis. 
About seven days later there is another sudden rise of temperature, 
but this time the crisis occurs sooner. A second or third relapse may 
occur. The organisms increase in numbers rapidly in the blood from 
the beginning of the fever, large numbers often being found in every 
microscopic field. They began to disappear a short time before the 
crisis, and immediately after the crisis it is practically impossible to find 
them in the circulating blood. The mortality varies in different 
epidemics from 2 to 10 per cent. When monkeys are inoculated with 
human blood containing the spirilla, they become sick about three and 
a half days later, but show only the initial febrile attack or, at the most, 
an occasional short relapse. The organisms are found to have the 
same relation to the pyrexial periods as in man, Blood from one animal 
taken during the fever induces a similar febrile paroxysm when inoc- 
ulated into another animal. 
Metchnikoff showed that during the intermissions when the spirilla 
disappeared from the circulating blood they accumulated in the spleen 
and were ingested in large numbers by certain phagocytes and finally 
were destroyed. 
According to Lamb, a certain amount of immunity is conferred upon 
monkeys (Macacus radiatus) soon after an attack, but it disappears 
quickly. If the serum is removed during this time it is found to have 
some protective action when mixed with the blood-containing spirilla, 
and also to cause agglutination of the organisms. Novy (1906) showed 526 
PATHOGENIC MICROORGANISMS 
that a powerful specific germicidal body exists in the blood of rats 
during and after recovery, notably in the blood of hyperimmunized 
rats. An immunizing body probably distinct from this is also present. 
He also showed that passive immunity can be imparted by injections 
of recovered or hyperimmunized blood, that both active and passive 
immunity may last for months, and that the serum has both a prevent- 
ive and a curative action. Agglutinins are also present in such a serum. 
Breinl has shown that the immunity produced by Sp. obermeieri 
does not protect against Sp. duttoni, and vice versa. Neither does it 
protect against the Asiatic or American variety. 
The strain found in Bombay seems to be more virulent than that in 
Europe. 
Spirocheta Duttoni. The organism shown by Dutton (1905) to 
be the cause of African tick fever is very similar morphologically to 
Sp. obermeieri, but Novy, Frankel, and others have shown slight differ- 
ences which make them believe that it is another variety, if not another 
species of this group. Dutton demonstrated that this organism can 
be transferred to monkeys by the bites of young ticks (Ornithodorus 
moubata) at their first feed after hatching from infected parents. He 
accidentally demonstrated the fact that the disease can be inoculated 
into human beings through a cut surface, for after a wound received 
at autopsy he developed the disease which eventually caused his death. 
Koch corroborated the author's results. Leishman demonstrated 
that the second generation of ticks may also infect. He was unable to 
find spirochetes in tissues of ticks later than ten days after feeding, 
though young ticks from eggs hatched after this time were infective. 
He found, however, granules which he considers changed spirochetes. 
Hindle agrees with him. The fact that ticks may be infective long 
after the disappearance of typical spirochetes, if they are placed at 30° to 
35° C. is evidence of the infectious nature of these granules. Moller 
found that a tick may remain infective for a year and a half or more 
after its initial feed from an infected host. Moller also showed that 
the third generation of ticks might inherit infection. Sargeant and 
Foley and later Balfour have observed another type of relapsing fever 
in Africa, which they consider due to a different variety of spirochete 
(Sp. berberai). The filtrability of Sp. duttoni is mentioned on page 492. 
Spirocheta Carteri.-This spirochete was described by Carter in 
1877 as causing relapsing fever in Bombay. Monkeys were inoculated 
by Carter successfully with the human blood containing this spirochete. 
Spirochetes from Relapsing Fever in America.-Recently Darling 
has reported a study of the relapsing fever of Panama. He isolated 
the organisms in two cases and studied their characteristics. He 
finds they agree with those reported by Carlisle, Norris, and Novy 
for the organisms isolated by Norris, but they can only be differ- 
entiated from the other relapsing fever spirochetes by animal inocu- 
lations and by the disease in humans. Moreover, he finds that in all 
probability a polyvalent serum may be necessary for cure, since the 
serum from one strain did not protect against the other strain. INSECT CARRIERS 
527 
Insect Carriers.-The spirochetes are conveyed by the bites of arthro- 
pods. Many studies have been made on this subject since the work of 
MarchouX and Salimbeni on fowl spirochetes, and of Dutton and 
Todd on African tick fever. Nuttall and his associates have added 
much to our knowledge of ticks and other arthropods as vectors. That 
the bed-bug may occasionally be a conveyer was shown by Mackie 
and corroborated by Nuttall. Mackie later obtained stronger evidence 
that the body louse is the chief carrier. His observations have been 
corroborated by a number of observers. 
Nicolle and his colleagues found after twenty-four-hour spirochetes 
disappear from gut of the louse (P. vestimente and P. capitis) to reappear 
in from eight to twelve days and continue for eleven days and possibly 
longer. Monkeys inoculated with the contents of lice fifteen days after 
feeding, develop relapsing fever. 
References. 
Bertarelli. Centralbl. f. Bakt., 1906-1907, xli, 320, 639; xliii, 167, 238. 
Calkins. Jour. Infect. Diseases, 1907, iy. 
Castellani. Jour. Hygiene, 1907, vii, 558. 
Darling. Arch, of Int. Med., 1909, iv, 150. 
Ewing. New York State Jour. Med., 1907, vii, 177. (With good bibliography.) 
Flexner. Medical News, 1905, Ixxxvii, 1105. 
Hoffmann. Ztschr. f. Hygiene, 1911, Ixviii, 27. 
Levaditi and McIntosh, Ann. Inst. Past., 1907, p. 785. 
Levaditi and Naltan-Larrier. Ann. Inst. Past., 1908, xxii, 260. 
Hindle. Parasitology, 1911, iv, 133 and 183. 
Leishman. Jour. R. O. M. C., 1909, xii, 123, and Lancet, 1910, i. 1. 
Koch. Deut. med. Woch., 1905, and Berl. klin. Woch., 1906. 
Miihlens. Zeitschr. f. Hygiene, etc., 1907, vii, 405; Klin. Yahrb., 1910, 339; Kolle und 
Wassermann, 1913, Sec. Ed., Jena. 
Nichols. Jour. Exp. Med., 1910, xii, 616 and 1911, xiv, 196. 
Nichols and Fordyce. Jour. Amer. Med. Assoc., 1910, Iv, 1171. 
Noguchi. Jour. Exp. Med., 1909, xi, 84 and 392; Ibid., 1911 and 1912. 
Norris, Pappenheimer and Flournoy. Jour. Infect. Diseases, 1906, p. 527. 
Novy and Knapp. Jour. Infect. Diseases, 1906, iii, 291. 
Nuttall. Parasit., 1913, v, 262. 
Perrin. Arch. f. Protist., 1906, vii, 131. 
Schaudinn u. Hoffman. Arbeit a. d. Kaiserl. Gesundh., 1905, xxii. 
Schereschewsky. Centralbl. f. Bakt., e c. Orig. Abt. I. 1908, xlv, 91. 
Sobernheim. In Kolle u. Wassermann, 1913, Sec. Ed., Jena. 
Swift. The Jour, of Cutaneous Dis., 1909, July, and the Arch, of Int. Med<, 1909, 
iv, 376 and 494. 
Uhlenhuth and Mulzer. Berliner klin. Woch., 1910-1911. CHAPTER XL. 
BODO. POLYMASTIGIDA. 
BODO LACERTA (GRASSI). 
Bodo lacertee is frequently found in the intestinal contents of most 
of the higher animals, hence it is easily obtained for class study. A 
species of the Bodo has been observed in human urine. (Bodo urin- 
arius), but it is probably a harmless invader. 
Bodo lacertoe is wedge-shaped, the posterior part of the body being 
turned a half to a whole spiral on itself. It possesses two character- 
istic flagella, equal in thickness but unequal in length. In motion the 
longer one is directed forward, while the shorter is carried backward, 
functioning as a rudder, or a towing flagellum. Both flagella spring 
from basal granules which are well demonstrated by the iron hema- 
toxylin stain. They are situated in the extreme anterior part of the 
body and are attached to the nucleus by a delicate fibril (Fig. 180, 
b, c, and Plate IV, Fig. 1,3). The movement of the organism is character 
istic, it consists in a rapid irregular swimming in various directions with 
the anterior flagellum moving from side to side. The body itself shows 
a slightly sinuous motion. 
There are, two types of nuclei seen. First, the typical vesicular 
nucleus most frequently seen among the flagellates. This is round 
and has a definite membrane about which chromatin is arranged in 
irregular masses. In the centre, or eccentrically placed, is a compact 
karyosome. Iron hematoxylin preparations bring out an achromatic 
network between the chromatin masses and the karyosome. In the 
living condition the nucleus appears as a greenish glistening refractive 
vesicle. 
The second type of nuclear apparatus is seen in smaller organisms. 
This is a similar nucleus except that it is smaller and more compact; 
posterior to this is another nuclear-like body, varying much in shape 
and arrangement of chromatin (Fig. 180, c). This is the sexual 
chromidia. 
The cytoplasm appears in iron hematoxylin stained specimens as 
finely reticular. It contains many deeply stained granules. There is 
no mouth opening. Food is taken in by osmosis. 
Tn propagation, the two types just described develop differently. 
The first or ordinary type forms round division cysts. The flagella 
disappear and a delicate cyst membrane is formed. The increase in 
the size of the nucleus and the subsequent division may be followed 
in life. It lasts about twenty minutes. After a single or, more seldom, BODO LACERTA 
529 
a double division of the cell, the daughter cells, while still within the 
cyst, form their flagella, become very motile, finally break the cyst wall 
and swim out. 
The second type increases, in the free living condition by longi- 
tudinal division. The basal granules divide, the principal nucleus 
divides by mitotic division, the chromidia by amitosis. This all can 
be seen in hematoxylin preparations. Sexual division in this species 
occurs in cysts by autogamy. It is not easily followed in life be- 
cause of the high refraction of the cyst. The changes must therefore 
be studied in specimens stained with iron hematoxylin. 
Fig. ISO 
Bodo lacerlue (Grassi): a living; b and c stained; b type without, c type with chromidium 
(1000 : 1). (After v. Prowazek, from Kisskalt and Hartmann.) 
They are shortly as follows: The nucleus becomes larger and about 
its membrane appear small spheres of chromatin which finally leave 
the nucleus and gather together, forming the so-called chromidial or 
sexual nucleus, while the original or somatic nucleus gradually degene- 
rates. The new nucleus divides amitotically into two daughter nuclei; 
from these two smaller parts are then separated, as reduction nuclei? 
which also degenerate. The remaining parts of the two nuclei increase 
in size and then fuse to form a new nucleus. The organism may then 
leave the cyst or the cyst may become a lasting cyst and serve to infect 
a new host. 
Besides this method of fructification by autogamy in a cyst, is seen, 
34 530 
PATHOGENIC MICROORGANISMS 
though seldom, a copulation between two individuals of different sizes 
which afterward become encysted and divide into two to sixteen 
daughter flagellates. 
POLYMASTIGIDA. 
The order polymastigida consists of flagellates having several 
flagella projecting from different parts of the body. -The majority 
of the forms known are parasitic in certain fish. 
Trichomonas Vaginalis.-Donne in 1837 described a form which he 
found in the human vagina, and which he therefore called Tricho- 
monas vaginalis. It has been found by other. observers to be a 
frequent habitant of the vagina at all ages. It is also found occa- 
sionally in the acid urine of both sexes. The mode of infection of the 
female is unknown. The body of the parasite at rest is pear-shaped, 
but during action its ameboid movements cause it to assume various 
shapes. The size varies from 12/z to 25m long and 8/x to 15m wide. 
Fig. 181 
Fig. 182 
Trichomonas vaginalis. (Blochmann.) 
Lamblia intestinalis. (Schewiakoff.) 
The protoplasm is finely granular, excepting for two rows of larger 
granules which begin on either side of the nucleus and converge pos- 
teriorly. From the anterior part project three to four flagella, which 
seem to begin at a basal thickening near to, or connected with, the 
more or less oval, indistinctly vesicular nucleus. From the origin of 
the flagella an undulating membrane extends backward. The body 
also seems to possess a certain linear structure connected with the 
membrane. Contractile vacuoles have not been seen. 
Trichomonas hominis Davaine.-This form, found frequently in the 
human alimentary canal, is very similar to the Trichomonas vaginalis, 
but it is smaller and more pear-shaped. It has been found often in 
acute diarrheas, but no causal relation between it and the pathologic 
process has been shown. 
A similar form has been seen a few times in lung gangrene, aspira- 
tion pneumonia, and bronchiectasis. 
Lamblia intestinalis (Lambl, 1859), a flagellate belonging to this 
group, parasitic in the small intestines of mice, rats, rabbits, dogs, 
cats, and sheep, has also been found occasionally in the human in- LAMBLIA INTESTINALIS 
531 
testines. It is beet-shaped, bilaterally symmetrical, 10/z to 21/z long 
and 5/x to 12^ wide, possessing flagella 9/x to 14^ long. Anteriorly, 
this species has a characteristic concavity, the rim of which seems to 
be contractile, forming a sucking apparatus. The eight flagella of 
the organism are arranged in pairs: one anteriorly, two laterally, 
and one posteriorly. The nucleus is situated anteriorly and has a 
central constriction. The protoplasm of the body is thick and hya- 
line. Contractile vacuoles have not been seen. Schaudinn (1906) 
observed encystment, copulation, and complicated nuclear changes 
in this organism. 
Infection follows the ingestion of the cysts with unclean food. 
The parasites fasten themselves to the free surfaces of the epithelial 
cells by their sucking apparatus, but seem to exert no harmful nflu- 
ence on their hosts. They have been found most frequently in poor 
children who play often in dirt containing the cysts. Repeated small 
doses of calomel will cause their disappearance from the feces. CHAPTER XLL 
AMEBA. 
Several authors have reported the finding of amebae in man, especially 
in so-called tropical, ulcerative, or amebic dysentery, but as the first 
descriptions were incomplete and the laws of nomenclature were not 
strictly followed there resulted many synonyms for the same species 
and many species bearing the same name. 
At present the four that were considered as distinct species have now 
become three according to some authors, and only two of those de- 
scribed, from a practical stand-point, need be considered. These are 
Entameba histolytica, the form described by Schaudinn from tropical 
dysentery and considered by him the cause of that disease, and Entameba 
coli, the kind found in normal human intestines by Schaudinn and 
thought by him to be harmless. 
The discussion in regard to the "parasitic" and the "non-parasitic" forms is 
still going on. Some investigators say that certain amebae are strict parasites 
of the intestinal tract of mammals; that is, that these amebae cannot be culti- 
vated on artificial culture media, and that they are so different from the "sapro- 
phitic" forms, i. e., those that arc cultivated (sic), that they must constitute 
a different genus. 
Williams, Calkins, and others, on the contrary, believe that not enough 
study on the "cultural forms" has been done to draw definite conclusions as 
to their relationship to those seen in preparations from amebic dysentery. 
Historical Note.-Stiles has given a detailed history of the generic name 
Ameba and of the specific one Ameba proteus, and, finally, of the naming of 
the intestinal amebae. He shows why the name Entameba should be given to 
the genus described by Lambl and Lbsch. 
This article illustrates very forcibly the absurdity of bringing forth new 
names for organisms only half studied and of claiming that such organisms 
belong to new genera. 
The first report on intestinal amebae of man was made by Lambl in 1860 who 
announced the presence of ameboid forms in the intestinal mucus of a child 
who had died from enteritis. Supposedly the same forms were more fully 
described by Losch in 1875 under the name Ameba coli; Losch found his organ- 
isms in stools of a patient suffering from chronic dysentery and he succeeded 
by rectal injections in producing superficial ulceration in the large intestines of 
dogs. He therefore claimed that this organism is the cause of dysentery. His 
work was corroborated by many observers. In the meantime, amebae were 
found in diseases other than dysentery, and Grassi in 1879 reported them 
in healthy intestines. The work of Kartulis (1886), however, helped largely to 
establish the fact that amebae play an important part in the etiology of dysentery 
in Egypt. He was the first to find the organism in abscess of the liver in tropical 
dysentery. In our own country among the most important workers in this field 
are Councilman and Lafleur (1891). They conclude that amebic dysentery 
should be regarded etiologically, clinically, and anatomically as a distinct disease. 
They disapprove, however, of the name Ameba coli and propose the name 
Ameba dysenteric for the pathogenic form; but as they do not show in any way 
than by its pathogenesis that the species they describe is a new one, their name, SITES OF AM E BYE IN THE HUMAN BODY 
533 
according to the rules of zoological nomenclature, cannot be accepted. Harris' 
work, too, is important in showing an etiologic relationship between ameba; and 
a certain form of dysentery, but neither did he describe the morphology of his 
organism minutely enough to identify it with Schaudinn's histolytica which 
is described below. Casagrandi and Barbagallo in 1897 were the first to claim 
that the amebae so far described in man show differences enough from the 
fresh-water amebae to belong to a new genus. They therefore created the 
genus Entameba and gave the specific name Entameba hominis to amebae of 
the Ameba coli type. Schaudinn and Stiles agree with them as to the generic 
name, but consider that the correct specific name is complicated by the fact 
that there are different species in this group. Many observers (Kartulis, 
Councilman and Lafleur, Quincke and Roos, Kruse and Pasquale), have con- 
sidered that there are different varieties in the human intestines, but they 
have given no morphologic differences distinct enough to classify such varieties. 
Schaudinn is the first who bases upon a definite morphology his claim (1903) 
that at least one species among them is pathogenic and one non-pathogenic. 
The latter, which he found in normal human intestines, he says resembles those 
already described as Ameba coli; therefore he gives it the name Entameba coli; 
while the former, which he found exclusively in ulcerative tropical dysentery, 
he calls Entameba histolytica. 
The different views upon the relationship to disease of ameba; found in the 
human intestines may be summarized as follows: 
1. That the amebae in man have no pathogenic properties, hence are not 
the cause of amebic dysentery. (Cunningham, Grassi, Celli and Fiocca, Casa- 
grandi and Barbagallo, and others.) 
2. That any intestinal ameba may become pathogenic and cause the specific 
malady known as amebic dysentery. (Musgrave, Clegg, and others.) 
3. That amebae are able to keep up a preexisting inflammation. This was 
the original view advanced by Lbsch when he described the most commonly 
cited form, Ameba coli, and several authors have followed Lbsch in this opinion. 
4. That more than one species of amebae are found in man, at least one 
pathogenic, and one non-pathogenic. (Kartulis, Councilman and Lafleur, 
Quincke and Roos, Strong, Schaudinn, Craig, and others.) 
The study of bacillary dysentery by Shiga, Kruse, Flexner, and others (see 
under bacillary dysentery) has demonstrated that there are at least two forms 
of dysentery, one produced by amebae and the other by bacilli, and from the 
work on the former it now seems certain that it is produced by a specific form 
of amebae. 
Amebae have been reported in teeth cement and in carious teeth as well as 
in abscesses of the jaw. Flexner in 1892 described an amebic organism in the 
latter condition, and considered it identical with the organism described by 
Lbsch, Councilman and Lafleur as Ameba coli. In the same year Kartulis 
described similar organisms found in similar lesions. Gross and Sternberg 
found them in tartar of teeth. Smith has recently found amebae in practically 
all of a series of cases of Rigg's disease. Prowazek (1904) described a mouth 
ameba as a distinct morphologic species under the name of Entameba buccalis. 
Successful cultures have been made of amebae obtained from the intestines 
of man and other animals, as well as from certain fruits and vegetables. 
Musgrave and Clegg (1904) studied amebae in the Philippines by the cul- 
ture method and came to the conclusion that forms obtained from various 
sources were probably all a single species. 
Williams (1911) obtained pure cultures on sterile "tissue media'" of amebae 
isolated from the intestines of mammals, including one from a case of amebic 
dysentery. 
Sites of Amebae in the Human Body.-Intestines and neighboring 
tissues; abdominal cavity; abscess of liver, lung, pleura, brain, and 
mouth; necrosis of jaw-bone; urine; tartar of teeth. 534 
PATHOGENIC MICROORGANISMS 
Materials and Methods for Study.-Human cases of dysentery showing 
amebae in stools is so seldom on hand in the northern part of this country that 
they cannot be counted on. The non-pathogenic form in human intestines might 
be obtained after administration of a saline cathartic, but generally one must 
depend upon saprophytic forms for work with students, or upon cultures ob- 
tained from cases of amebic dysentery. Material rich in saprophytic forms may 
be obtained from an infusion in water of lettuce, cabbage, potato skins, or other 
vegetable material. Such an infusion should be made a week or two before 
it is needed, when it will be found that the pellicle which forms contains many 
varieties of protozoa and bacteria, among which are generally large numbers 
of ameboid forms. Often one may get good material from the feces of many of 
the lower animals, such as the lizard, toad, or guinea-pig. 
If one has material containing human intestinal amebae, kittens or puppies 
may be fed with the cysts in order to obtain a new supply. The amebae should be 
examined in both the fresh and fixed condition. Cultures may also be made as 
described below. 
Examination of the Fresh Material.-The study of the living amebae is ex- 
tremely important. This may be done by making a hanging drop or hanging 
mass (p. 72) from fluid containing amebae. The size, kind of motion, frequency 
of pulsation of contractile vacuole, and as much of the cell contents as possible 
should be noted. 
The stools should be examined on the warm stage as soon as possible after 
their passage (not later than two hours), and should be kept at blood heat 
until examined. A platinum loopful of material should be taken from the slimy 
masses in the thinner part of the feces, diluted with physiologic salt solution, 
covered with a cover-glass, and examined under moderate magnification. 
Harris found that a drop of a watery solution of toluidine blue added to a 
small particle of the feces stains the entoplasm of the amebae at once and the 
ectoplasm a few minutes later. The amebae seem to be quickly killed and 
often when natural forms are beautifully preserved the coverslips, after being 
washed in water and mounted in Farrant's medium, may be preserved for 
months, but after a time the stain completely fades. 
Permanent Preparations.-Thin films are made on glass slides or cover- 
glasses, and immediately, before they are allowed to dry, they are placed in 
the fixing solution. Cover-glasses may float, film down, on the surface of the 
fixative. Among the best fixatives are: Hot sublimate alcohol (50° C.), Zenker's 
fluid, hot Hermann's fluid, or methyl alcohol (p. 84). 
Stains.-Among the many good staining methods the following may be 
mentioned: 
1. Thin Delafield's hematoxylin, from one-half hour to several hours, then 
washed in water. (If overstained, the preparation may be differentiated in 
acid alcohol, controlling under the microscope, then washed in water.) The film 
or section is then passed successively through 70 to 95 per cent, and 100 per 
cent, alcohol, absolute alcohol + xylol, xylol, cedar oil, or Canada balsam. 
2. Heidenhain's iron hematoxylin (see p. 84). The smear is put from dis- 
tilled water into the iron-alum mordant for 4 to 12 hours, or overnight; well 
washed in distilled water; in stain from 2 to 24 hours, excess washed out in the 
iron mordant, controlled under the microscope (as decolorization occurs very 
quickly) until the nucleus is sharply differentiated; the chromatin of the 
nucleus must be a deep blue-black, and the cytoplasm a light gray; then a 
thorough washing in tap water and passage through the alcohols and xylol, 
and in Canada balsam, or cedar oil for mounting. 
3. After fixation in methyl alcohol one may use Giemsa's staining method 
(see p. 81), or a modification of the method suggested by Van Gieson for staining 
the Negri bodies in smears (see p. 83). 
Masses containing amebae, as mucous flakes or portions of the intestinal or 
liver abscess wall in amebic dysentery, or pieces of decaying vegetables may 
be fixed in toto in hot sublimate alcohol for one-half hour, washed in iodin 
alcohol for 24 hours, passed through the different strength alcohols and em- 
bedded in paraffin (see p. 85) for section-cutting if desired. MORPHOLOGY 
535 
Mallory and Wright recommend the following method for tissues: 
1. Harden in alcohol. 2. Stain sections in a saturated aqueous solution of 
thionin three to five minutes. 3. Differentiate in a 2 per cent, aqueous solution 
of oxalic acid for one-half to one minute. 4. Wash in water. 5. Dehydrate in 
alcohol. 6. Clear in oleum origani cretici. 7. Wash off with xylol. 8. Xylol 
balsam. 
Mallory's eosin methylene-blue method is also very good for sections (p. 83). 
Cultures of Amebae.-Pure mixed cultures may be made in the following way: 
From the material containing amebae a small loopful is removed with a platinum 
wire and isolated spots are touched over the surface of the following media poured 
in sterile Petri dishes: Agar 1.0, tap water 90.0, ordinary nutrient broth 10.0, 
mixed and sterilized in the regular way. It should be slightly alkaline to phen- 
olphthalein (1 per cent.). If necessary, feces contents may be thinned with phy- 
siologic salt solution before planting. In one to several days at 25° C. the amebae 
with the accompanying bacteria may overgrow the entire plate. We have found 
that amebae will grow as well upon nutrient agar-better with certain bacteria- 
as on the special media just mentioned. Klatsch preparations may be made of 
these cultures, or small pieces of agar and culture may be embedded entire. 
From such a culture the "pure mixed" cultures of Frosch may be made as 
follows: The amebae which have crept out to the periphery of the growth are 
taken out with their accompanying bacteria and transplanted. Usually one or 
two organisms favorable for the growth of the amebae accompany them and in 
this way one may finally get the amebae growing with one definite bacterium. We 
have isolated from a culture a single ameba unaccompanied by bacteria by the 
following simple method: Under the low-power lens with a fine platinum loop 
an isolated ameba is drawn to the edge of the agar plate. When it is well sepa- 
rated a disk of agar containing it is cut out following the margin of the objective 
and is transferred to a fresh agar plate. A very small quantity of a desired bact- 
erum is now added to the disk near the ameba, and a "pure mixed" culture 
results. 
Pure Cultures. - Certain varieties of amebae from the intestines of 
mammals grow without other organisms, i. e., grow in pure cultures, 
when inoculated on a piece of fresh sterile brain, kidney or liver placed 
upon Musgrave and Clegg's agar or on nutrient agar. They grow abun- 
dantly on such media at temperatures varying from 22° C. to 38° C. 
(Williams.) 
Morphology.-The morphologic characteristics of the ameboid stage, 
as described by various observers, seem not to have been minutely 
enough studied to be depended upon in differentiating the species. 
Moreover, descriptions have differed markedly. While Schaudinn 
and others, especially Craig of the more recent writers, say that it is 
easy to differentiate between the ameboid forms of histolytica and coli, 
Musgrave and Clegg, Strong, Williams and others say the points of 
difference are not marked 
The observations of Schaudinn and others may be summarized 
as follows: (1) Ent. coli is, on the whole, smaller than Ent. histolytica; 
(2) its ectoplasm is so small in amount and so slightly differentiated 
that it is only seen when the organism puts forth pseudopods, while 
the cortical zone of the Ent. histolytica is wider and is distinctly differ- 
entiated from the entoplasm; (3) the pseudopods of the former are 
small rounded delicate, and not highly refractive, those of the latter are 
larger, finger-shaped, firmer, and more highly refractive, thus indicating 536 
PATHOGENIC MICROORGANISMS 
the power of the organism to penetrate its host's tissues; (4) the nucleus 
of Ent. coll is very distinct in life as well as in stained spreads, due to a 
definite membrane, a more distinct karyosome, and much chromatin 
which is distributed throughout the nucleus with more of a collection 
about the periphery; the nucleus of Ent. histolytica on the other hand, 
is seen with difficulty during life, and stains faintly, owing to its delicate 
membrane, its small amount of chromatin, and small karyosome; the 
chromatin is collected about the karyosome and the periphery of the 
nucleus; the nucleus, moreover, is much more variable in shape, in the 
active organism than is that of the Ent. coli; (5) the entoplasm of 
Ent. coli is less granular and vacuolated and contains fewer red-blood 
cells than that of Ent. histolytica which sometimes shows immense 
numbers of these blood cells (Plate IV, Fig. II, 1-5). 
The above points of difference cited for organisms in the ame- 
boid stage may hold for forms living in the human intestines; but we 
have found that organisms from widely different sources (e. g., intes- 
tines of guinea-pigs and of dogs from New York and of humans from 
the Philippines) when grown with a favorable bacterium in the ther- 
mostat at body temperature may show appearances similar to each 
other and similar also to those described by Schaudinn for Ent. histo- 
lytica. More corroborative work, therefore, seems to be needed before 
we accept the above observations as being the whole truth. 
Reproduction.-In the vegetative stage probably all these forms divide 
by a primitive mitosis, though Schaudinn, Craig, and others saw only 
amitosis. All of our culture forms divide by mitosis and many observers 
have recently reported similar division in related forms. Schaudinn and 
others state that Ent. coli in the vegetative stage may divide by break- 
ing up (schizogony) into, at the most, eight daughter cells. In the 
latter instance according to these authors the nucleus undergoes a 
somewhat complicated process of division. But recently Walker, 
Hartmann, and Williams have presented evidence to show that this 
is probably not true. The nuclei continue to divide by binary mitosis. 
The vegetative stage of each intestinal organism takes place in the 
upper part of the intestines; as the feces become thicker, most of the 
vegetative forms die off, while some pass on to permanent cyst for- 
mation. As with many coccidia, parasitic amebae may pass through 
a long period of vegetative life before entering upon another phase 
wherein forms are produced capable of infecting a new host. The 
length of this period depends upon a number of circumstances. 
Under conditions favorable for the growth of the amebae, as in cases of 
diarrhea, the vegetative phase is considerably lengthened, while in 
healthy intestines, as the amebae pass down with the thickening feces, 
the infecting cysts are more or less quickly formed. 
The statement made by Schaudinn that Ent. histolytica during the 
vegetative stage may multiply by budding as well as by binary fission, 
is now considered incorrect. Unequal division, however, probably 
frequently occurs. 
It is now thought by Walker, Hartmann, Craig, and others that Ent. PATHOGENICITY 
537 
tetragena, described by Viereck, Hartmann, and Craig as a distinct 
species, is merely a phase of Ent. histolytica, though Hartmann calls 
the species tetragena and drops the name histolytica. 
The observations of Schaudinn on the sexual phenomena of Enta- 
meboe, though earlier corroborated by Hartmann, Craig, and others, 
are now considered incorrect by these same observers. The subject 
awaits further investigation. 
Viability.-The pathogenic amebae are apt to lose their motility 
very quickly above or below body heat, while the saprophytic forms 
remain motile at higher or lower degrees. Though the former lose 
their motility, they are not all killed by cold. They may still be infec- 
tive after freezing. Musgrave kept an encysted culture from a dysen- 
teric stool at -12° C. for 45 days and found it still viable at the end of 
that time. 
A temperature of 60° C. for one hour usually kills encysted cul- 
tures of amebae, according to Strong, but considerable variation has 
been noted in the degree of temperature necessary to destroy differ- 
ent strains.1 
Enemata of quinine sulphate and saturated solution of boric acid 
do not affect amebae in the intestinal canal, though quinine sulphate 
added to the stools invariably kills them in ten minutes. 
They are also destroyed in stools by weak solution of hydrogen 
dioxide, potassium permanganate, toluidine blue, and dilute acids. 
Luttle found that totto hydrochloric acid and silver nitrate 
check motility, but do not destroy parasites except after prolonged 
contact. Musgrave and Clegg found that in cultures treated with 
1 : 2500 solution of quinine hydrochlorate the parasite quickly encysts, 
and in from five to eight minutes may break up and disappear; ten 
minutes later cultures made produced no growth of amebae, while the 
bacteria grew well. 
Pathogenicity.-Lower Animals.--Just how pathogenic Ent. coli is 
for lower animals cannot be determined, as we have before stated, 
until a more minute study of the intestinal amebae is made. 
In regard to amebae from tropical dysentery (presumably Ent. histo- 
lytica), they have been shown to be pathogenic to young cats, dogs, and 
monkeys. The infection may take place in two ways: (1) By feeding 
material containing the cysts; (2) by rectal inoculations of the vege- 
tative forms. The best work done on dogs is by Harris in 1901, who 
found that puppies were particularly susceptible after rectal injections 
of fresh material from human dysentery cases. Morphine was adminis- 
tered before the injection in order to retard peristalsis. The disease 
developed in two or three days and lasted from four to sixteen days. 
The chief symptoms were a bloody diarrhea and progressive emaciation. 
The lesions observed in the intestines on postmortem examinations were a 
swollen and congested mucosa, over which were scattered numerous small 
ulcers. In two cases there were liver abscesses. 
1 An air-dried agar plate culture of ''Ameba coli" given us by Dr. Calkins who obtained 
it from the Philippines was viable after three years at room temperature. 538 
PATHOGENIC MICROORGANISMS 
Microscopically, the mucosa first showed slight exudative and productive 
inflammation, followed by necrosis and desquamation of the epithelial cells 
and their basement membrane. At the same time the interglandular tissues 
beneath became swollen and small hemorrhages occurred. Great numbers of 
macrophages collected. Ulceration proceeded from above downward. Many 
amebae were first seen in and between the epithelial cells, then in the connective 
tissue at the base or sides of the ulcers. Necrotic and suppurative processes 
producing varying degrees of suppurative inflammation may complicate the 
lesions. 
The abscesses which form in the liver contain degenerated liver cells, poly- 
nuclear leukocytes, red-blood cells, and groups of small amebae. 
As controls Harris tried rectal injections of various bacteria, in- 
cluding the Shiga bacillus. All gave negative results, however, and 
he considered that the amebae showed their specific action very plainly. 
Though he did not describe the morphology of the organism from 
his cases with enough minuteness to identify it with Schaudinn's 
histolytica, he gave enough points to make the inference strong that 
it is the same species. Whether Entameba coli would produce similar 
dysentery in young dogs is yet to be proved. As stated above, Schau- 
dinn found that he could produce the typical disease by feeding young- 
cats with cysts of Ent. histolytica, but could not get the same results 
by feeding the vegetative forms. 
Musgrave and Clegg injected "pure mixed cultures" of material 
from cases of clinical amebic dysentery as well as similar cultures 
of amebae from various sources into monkeys and produced dysen- 
tery. Musgrave fed monkeys with encysted amebae in bacterial cul- 
tures and obtained, in a small percentage of the cases, dysenteric 
stools and ulcerations in which amebae were found without their 
accompanying bacteria. Kartulis, Kruse and Pasquale, and Strong 
injected into the rectum the contents of liver abscesses containing 
apparently only the amebae and produced typical dysentery, 'with 
lesions similar to those seen in man. 
Strong states that the lower monkeys and the orang-outang in the 
Philippines contract the disease naturally. 
In Man.-According to Craig, about 50 per cent, of human beings 
harbor harmless amebae in their intestines. Schaudinn states that he 
found this form of ameba in one-half the cases examined in East 
Prussia, one-fifth of those in Berlin, and 256 times in 385 examinations 
in Austria. 
Recently Walker and Sellards have carried on an extensive study of 
the pathogenicity of the different forms in human beings--sixty 
Philippine prisoners-which they divided into three groups: 
1. Twenty men fed with cultures of amebae isolated from stools or Manilla 
water. The same ameba was recovered in cultures from several of the cases. 
No lasting parasitism occurred, neither did any case of dysentery result. 
2. Twenty men were fed stools containing Ent. coli. No cultures were 
recovered; no cases of dysentery occurred, but 17 cases were parasitized. 
3. Twenty men were fed stools containing Ent. histolytica. No cultures were 
recovered; four cases of dysentery occurred, and 17 cases were parasitized. TISSUE CHANGES 
539 
They conclude from these studies that they have furnished the proof of the 
pathogenicity of one form {Ent. histolytica), the non-pathogenic but parasitic 
nature of the other form {Ent. coli), and the saprophitic nature of cultural forms. 
These experiments are open to a number of criticisms, chief of which are: 
Probable immunity of the men, the use of only Musgrave and Clegg's culture 
medium for their cultures, and their cultivation only at room temperature. 
The disease produced by pathogenic amebae in man is known as 
amebic dysentery (amebic colitis, amebic enteritis, amebiasis entam- 
ebiasis). 
Incidence.-The disease occurs endemically in tropical countries. 
It is particularly prevalent in Egypt, India, and the Philippine Islands. 
It occurs frequently in parts of South America and southern United 
States. In northern United States few cases are reported, though 
Patterson, who in 1909 described three cases (without a description of 
the amebae present), and who calls attention to fifteen cases reported 
as endemic in New York City, since 1893, states that this disease is 
probably more widespread than is generally thought, and that if it 
were searched for more carefully more cases would be recognized. 
Patterson adds to his report a bibliography of cases reported as orig- 
inating in North America. Sporadic cases are found in Russia, Ger- 
many, Austria, Italy, and Greece. An occasional small epidemic may 
occur in the milder climates. Where it is endemic, the largest number 
of cases occur after the heavy rains have begun in early summer. 
Males are more frequently attacked, because more exposed to infec- 
tion. It may occur at all ages, but young adults seem most susceptible. 
The foreign white race seems to be more susceptible than natives. 
Unhygienic surroundings are generally a predisposing factor, but in 
the Philippines individuals of all classes are likely to be attacked who 
do not take continuous and extraordinary precautions in regard to 
their drinking water. 
Symptoms.-The symptoms may be mild or severe. The disease usually 
runs an irregular course marked by periods of intermission and exacerbation. 
It may begin acutely with slight fever, griping, tenesmus, and frequent stools. 
Occasionally, however,, the outset is gradual, lasting from a few days to sev- 
eral weeks. The disease is generally chronic, extending over a period of a 
few weeks or of many years. In the mild form which is usual in children, 
the general condition may be remarkably good, the only symptoms worth 
mentioning being the increased number of stools-two to six in twenty-four 
hours-which contain few to many amebae. In the severe forms there is a loss 
of appetite, great emaciation, some fever, acceleration of the pulse, sweating, 
abdominal pains, and a decided increase of the number of stools-twenty to 
sixty daily. The stools are more fluid and slimy and may be bloody. They 
contain amebae in varying numbers. In very severe forms the stools are watery, 
filled with blood, mucus, and sometimes sloughs. They vary in numbers from 
twenty to fifty in twenty-four hours and may contain many amebae. 
The milder forms may change suddenly to the severest, and the severest 
may suddenly become better and completely recover. 
Tissue Changes.-The lesions are chiefly in the large intestines. The walls 
are thickened in chronic cases, especially the submucosa. There are raised 
hemispheric areas of hemorrhagic catarrh and of ulceration. The whole of 
the large intestines may be affected or only more or less circumscribed areas. 540 
PATHOGENIC MICROORGANISMS 
The amebae pass between the epithelial cells, generally through small erosions, 
and they finally reach the submucosa by the lymph channels. Here reproduction 
takes place and the irritation to the tissue causes edema and infiltration of small 
spheroidal cells. This produces small elevations into the lumen of the intestines. 
The epithelium over these raised areas is finally eroded and then bacteria and 
intestinal contents help form the succeeding ulcers. The erosions or ulcerations 
have congested undermined margins, and yellowish-red bases. They vary in 
size from 2 mm. to about 2 cm. They are round, oval, or irregular in outline. 
The ulceration usually extends only to the submucosa, but may expose the 
peritoneum, and large sloughs may be cast off into the lumen of the intestines. 
Generally the slow inflammatory process in the submucosa leads to great thick- 
ening of the intestinal wall. 
The processes may be modified in various ways by the action of other micro- 
organisms, especially the bacteria in the feces. Healing takes place by the 
formation of connective tissue in the floors and by a gradual covering over with 
epithelium. In extensive lesions, scars may form. 
Peritonitis may occur with the production of an opaque gelatinous fibrinous 
fluid in which the amebae may be found. 
Abscesses may form in the liver (about 20 per cent, of all cases), less often 
in the lungs, and only occasionally in the brain and spleen. Amebae may reach 
the liver through lymph channels, portal vein, ancl peritoneal cavity. The 
other organs are only slightly changed. 
Source of Amebae.-Nothing can yet be said about the exact source 
of Schaudinn's pathogenic variety, as so few have identified the 
organism. Strong states that in Manila the greatest source of infection 
from amebae is the water supply, that amebae were cultivated from 
the water in large numbers in 1902, but no attempt was made to demon- 
strate their pathogenicity. In 1904, however, Musgrave produced 
dysentery in a monkey with a culture of a water ameba, though in a 
few experiments he was unable to infect cats from the amebae obtained 
from this monkey. Practically, it is proved that people in Manila 
avoid being infected with amebae if they do not drink local water, 
unless sterilized. Fresh vegetables as well as certain fruits may be 
sources of infection. Of course if pathogenic amebae are strict para- 
sites, the source would be only those substances contaminated with 
the host's excretions. 
As dilute acids quickly kill the motile amebae, it is probable that 
many of those ingested in this form are destroyed in the stomach. 
Immunity to the disease may exist. It is supposed that the amebae 
as they die produce toxic substances which call forth antibodies, 
but this has not yet been determined. The necrosis produced in the 
liver abscesses when bacteria are absent is an indication of the pro- 
duction of necrogenic substances (D. Wills). 
Prognosis.-The percentage of deaths in the severe cases is quite 
large, especially if accompanied by abscess of the liver. Probably 
25 per cent, of all cases are fatal. When treatment is begun early the 
prognosis is better. 
Treatment.-Emetin has been found to be practically a specific 
curative treatment when amebte are in the vegetative stage, but it 
does not affect cysts. Ipecacuanha is highly recommended by Manson, 
Dock, and others, especially since the introduction of salol-coated pills AMEBAE IN OTHER DISEASES 
541 
which allow the remedy to reach the intestines before it is absorbed, 
so that large doses may be given, without inducing marked nausea 
and vomiting. Besides rest and diet, high enema of bisulphate of 
quinine have been recommended. Harris has gotten good results from 
hydrogen dioxide enemata diluted from 4 to 8 times with water. About 
a quart is injected twice daily for a week, then the amount is gradually 
decreased. 
Points in Diagnosis of Amebae Found in Man.-Examination of 
stools should be made as quickly as possible after they have been 
passed and they should be free from urine. The amebae should be 
seen motile because, after encystment or death, it is often difficult to 
distinguish them from other intestinal contents. Bloody mucus or 
small pieces of necrotic tissue should be examined first as they often 
contain large numbers of amebae. 
If the movements are solid a dose of salts should be given and the 
fluid part of the resulting stools examined. 
Craig differentiates living pathogenic forms from non-pathogenic 
varieties by the former's (1) larger size, (2) greenish color, (3) dis- 
tinct hyaline, refractive ectoplasm, (4) faint nucleus, (5) many vacuoles 
and red-blood cells, (6) marked motility. 
An absolute diagnosis of liver abscesses can often only be made by an 
exploratory puncture and the finding of the amebae. If this is done, 
the surgeon should be at hand to operate if necessary. 
Ent. buccalis is usually found in the thick group of leukocytes and 
microorganisms collected between the teeth. The amebae are distin- 
guished from the leukocytes and cell detritus by (1) their large size, 
(2) their light, highly refractive greenish appearance, (3) their glisten- 
ing red color in contrast to the yellow-red of the leukocytes when 
hanging drops are stained with enough of a concentrated solution of 
neutral red to make them appear pink. 
Differential Diagnosis between Amebic and Bacillary Dysentery.- 
In amebic dysentery (1) the disease is generally chronic; (2) dysentery 
bacilli are usually not found in feces; (3) no severe toxic symptoms 
present; (4) abscess of liver frequent sequela; (5) lesion is in cecum and 
descending colon, not in small intestines. 
In bacillary dysentery, the finding of the bacilli, and a positive 
agglutination test, together with the clinical symptoms of intoxication 
make a certain diagnosis. 
AMEBAE IN OTHER DISEASES. 
Baelz found a very large ameba in the bloody urine and in the vagina 
of a twenty-three-year-old Japanese who was suffering from tuber- 
culosis of the lung. Jurgens, Kartulis, and Posner also reported finding 
similar amebae in cases of cystitis and bloody urine. 
In the ascitic fluid of a man who had carcinoma of the stomach 
Leyden found motile cellular elements which Schaudinn first pro- 
nounced independent organisms belonging to the rhizopoda and named 542 
PATHOGENIC MICROORGANISMS 
by him Leydenia gemmipara (Fig. 183). Similar organisms were found 
in the ascitic fluid of a girl who had an abdominal tumor. The organisms 
remained motile in the ascitic fluid seven days after removal. The 
organism possesses a pulsating vacuole and one vesicular nucleus; 
it divides directly and by budding. The individuals seem readily to 
fuse (plastogamy). Schaudinn later (1903) considered this organism 
a phase in the rhizopod chlamydophrys, and decided that it had no 
pathogenic action. 
Fig. 183 
Leydenia gemmipara (a phase of chlamydophrys stercorea. A, single ameba; B, plas- 
modia and budding; n, nucleus; n', nucleus dividing; cv, contractile vacuole; v, vacuole; 
er, red-blood cell; Kn, buds; Ka, ameba developed from bud. 
References. 
Calkins. The Pathogenic Rhizopoda, in Protozoology, New York and Philadelphia, 
1909. 
Craig. Studies upon the Ameba in the Intestines of Man, Jour. Inf. Dis., 1908, v, 324. 
Also Arch. Int. Med., 1914, xiii, 917, and Jour. Inf. Dis., 1913, xiii, 30. 
Councilman and Lafleur. John Hopkins Hospital Report, 1891, ii, 395. 
Dock. Jour. Amer. Med. Assoc., 1902, iv, 15. 
Harris. On the Alterations Produced in the Large Intestines of Dogs by the Ameba 
Coli, etc., Philadelphia, 1901; also Amebic Dysentery, Amer. Jour. Med. Sci., 1905. 
James. Ann. Trop. Med. and Parasit., 1914, viii, 133. 
Kartulis, in Kolle and Wassermann's Handbuch d. path. Mikroorg., 1913, Sec. Ed., 
Jena. 
Musgrave and Clegg. Amebas: Their Cultivation and Etiological Significance, Manila 
Bureau of Public Printing, 1904. 
Patterson, H. Endemic Dysentery in New York, with a Review of its Distribution 
in North America, Amer. Jour. Med. Sci., 1909, cxxxviii, 198. 
Schaudinn. Arbeiten a. d. Kaiserl. Gesundh.-amte, 1903, xix, 547. 
Stiles. Report of the Committee on the Relation of Protozoa to Disease, Amer. Public 
Health Assoc., 1904. 
Strong. Amebic Dysentery, in Osler's Modern Medicine, Philadelphia, 1907, vol. i. 
Walker and Seilards. Experimental Entamebic Dysentery. Philip. Jour. Sci. B., 1913, 
viii, 253. 
Williams and Gurley. Coll, studies from Res. Lab. Health Department, New York 
City, 1910, 
Williams. Jour. Med. Res., 1911, xx, 263. 
Williams and Calkins. Jour. Med. Res., 1913, xxiv, 43 CHAPTER XLII. 
SPOROZOA. CILIATA. 
The forms in the group sporozoa, which are parasitic in man, or 
which are of some medical interest, are, Coccidia, Nosevia, Sarcocystis, 
Babesia, and Plasmodium malariae and its allies. Babesia and the 
malarial organisms will be considered in separate chapters. 
Only one genus among the ciliates is pathogenic for man. 
The general characteristics have already been described in Part I.. 
EIMERIA STIEDA (COCCIDIUM CUNICULI). 
The Coccidium cuniculi is a sporozoan parasite of the rabbit first 
described by Lindemann in 1865. Young rabbits are especially sus- 
ceptible, and extensive epidemics may occur in breeding houses. 
Material and Methods for Study.-Rabbits infected with Coccidium cuniculi 
are often found, and the whole course of the infection may be followed with more 
or less ease. 
A certain amount of development may be watched in hanging drops of salt solu- 
tion emulsions. Sections and smears are prepared as described in Chapter III. 
The cysts are stained with difficulty. It is recommended that a thin solution 
of Delafield's or Grenacher's hematoxylin be used for twenty-four hours followed 
by eosin. Heidenhain's iron hematoxylin stain (p. 84) followed by Bordeaux red 
is especially good for sections. 
The symptoms of the disease are fever, diarrhea, yellowish mucous 
discharge from the nose and mouth, and progressive wasting. The 
liver is much enlarged and shows throughout its substance variously 
sized gray-white tubercles, generally surrounded by a capsule, and 
containing a slimy mass of degenerated host cells, in which are embedded 
the parasites. The parasites are also found in the feces and in the 
epithelial cells of the intestines, gall-ducts, and liver. The acute stage 
of the disease lasts about three weeks. The contents of the coccidial 
tumors in animals that have withstood the infection may later be 
emptied, leaving only a mass of cicatricial tissue. In such animals 
the oocysts may remain for a long time in the gall-bladder and intestines, 
and by passing out gradually with the feces may provide a source 
of infection for other animals. The infection is carried by food soiled 
with cyst-containing feces. The cysts pass with the food into the 
stomach, where the cyst wall and the spore sac are destroyed and the 
sporozoites are set free. The motile sporozoites pass through the 
ductus choledochus into the liver, some probably passing into the 
intestines and infecting the cells directly, a later infection of the in- 
testines occurring from forms developed in the liver. The organism 544 
PATHOGENIC MICROORGANISMS 
develops within the epithelial cells of the liver and gall-ducts until 
the cells are finally broken down and tissue cysts are formed, within 
which, after more or less complicated changes, cysts of the parasite 
are again formed. The life cycle of Eimeria Schubergi, a similar cocci- 
dium from the intestines of a myriapod, is shown on Plate IV, Fig. in, 
a, 1-16. 
Fig. 184 
a b c d e f g h i 
Showing spore formation in Coccidium cuniculi from the liver of the rabbit: a and b, 
young stage in the epithelial cells of the gall-ducts (the small oval is the cell nucleus); 
c, d, and e, the fertilized oocyst; in d the protoplasm is beginning to shrink away from the 
cyst wall, and in e it has contracted into a spherical form; f, segmentation into four sporo- 
blasts; g, elongation of the sporoblasts to orm spores; h, four complete spores in the oocyst; 
i, single spore more highly magnified, showing the two sporozoites and a small quantity of 
residual protoplasm. The life cycle has been fully worked out by Simon. (After Balbiani, 
from Doflein.) 
A few cases of human infection of the liver with the Coccidium 
cuniculi have been reported. The Coccidium hominis Rivolta, found 
a few times in the human intestines, as well as similar coccidia, found 
in the intestines of lower animals, may belong to the same species. 
Coccidium bigeminum (Stiles) is found in the feces of dogs, cats, 
polecats, and possibly human beings. The organism is characterized 
by the division of the oocyst into two united cysts, containing four 
spores. The size is 8/z to 15ju. The life cycle is not well known. 
Rhinosporidium kinealyi is the name given by Minchin and Fantham 
(1905) to a probable sporozoan found in the nasal mucous membrane 
of certain cases from India that were troubled with hemorrhagic nasal 
polyps. Nais reported four similar cases and Beattie another in 1906. 
MYXOSPORIDIA. 
The Myxosporidia belong to one of the most populous and abundant 
groups of the sporozoa, showing great structural variation as well as 
divergence in mode of life. Nevertheless the members have, as a 
group, the following well-marked characteristics: The trophozoite is 
ameboid; spore formation begins at an early period and proceeds 
continuously during the growth of the trophozoite; the spores are 
produced endogenously-i. e., within the protoplasm of the tropho- 
zoite, and each spore always possesses one or more very distinctive 
structures, "the polar capsules" (Fig. 185, c, d). 
The myxosporidia are habitants of fishes, reptiles, arthropods, 
and some other classes of animals. They infest especially arthropods, 
causing often most virulent epidemics. The most interesting member SARCOSPORIDIA 
545 
of this group is Nosema bombycis, the cause of silkworm disease 
(Pebrine). The organism forms many small spores each with one 
polar capsule. The spores which are carried by the food into the 
intestinal canal of the caterpillar, pass through the walls of the intes- 
tines, and infect all organs. Spores found in the ovary may be carried 
over to the newly hatched silkworms, thus causing a further dissemi- 
nation of the disease. 
Fig. 185 
Nosema bombycis: 1 to 5, spore formation; 6, infected follicle of testicle; 7, spores; a, b, 
fresh; c, d, treated with nitric acid. The acid causes them to swell up and increase in size 
by at least a half, at the same time making the polar capsule distinct. In d the filament 
is extruded. (After Balbiani.) 
The other member of this group, of interest here, is Nosevia lophii 
Doflein. Its interest lies in the fact that it has been found to infect 
only the ganglion cells of the sea-devil, thus apparently resembling 
in its parasitic nature the organism causing hydrophobia. 
SARCOSPORIDIA. 
This order is very little known but, considering the fact that through eating 
uncooked infected meat, it may be found in man, though rarely, its chief char- 
acteristics should be noted here. 
The Sarcosporidia are parasites of the striped muscles or connective tissue 
of some of the warm-blooded vertebrates (various birds and mammals). They 
are found in the adult state in elongated sacs known as "Rainey's" or 
"Miescher's Tubes" (Fig. 186). 
The trophozoite is a motionless elongated body, limited by a cuticle grow- 
ing into a complicated structure. Spore-formation begins at an early stage 
and proceeds during the growth of the trophozoite (Neosporidia) which may 
become very large. The spores, which are many, are minute sickle-shaped 
or spindle-shaped mononucleate bodies with a delicate envelope and at one 
pole an oval striated body which represents the polar capsule found in the 
myxosporidia (Fig. 187). 
In some cases the cyst wall calcifies and the contents of the cyst degenerate, 
with apparently no harm to the host; in other cases the cysts burst and their 
contents spread into the surrounding tissue, producing abscesses and tumors 
as with many myxosporidia and sometimes causing the death of the host. 546 
PATHOGENIC MICROORGANISMS 
The symptoms of sarcosporidiosis in the pig are paralysis of the hind ex- 
tremities, a skin eruption, and general systemic symptoms, such as increased 
temperature and pulse. 
In sheep especially, the disease often causes fatal epidemics. In the mouse, 
Sarcocystis muris is a deadly parasite. Theobald Smith showed that gray and 
white mice may become infected with Sar. muris by eating infected mouse 
flesh containing motile sporozoites. 
Laveran and Mesnil claim to have extracted a toxin {Sarcocystin) by means 
of glycerin or salt solution, which they have found extremely toxic for experi- 
mental animals. (0.0001 gm. kills 1 kgm. of rabbit.) The dried and powdered 
extracts are also virulent. These extracts will remain virulent for a long time 
in the ice-box, but will withstand heating above 60° for any time. 
Fig. 186 
Fig. 187 
Sarcocystis tenella from the striped 
muscle of a swine. A full-grown cyst, 
showing radiately striped membrane, which 
is broken on the right side. (Bertram.) 
Sarcocyslis miescheri; a, small cells from 
a cell group; b, loosening of the protoplasm 
from the cell wall; c, d, sickle-shaped 
bodies (sporozoites) formed from the small 
cells. (From Wasielewski.) (Manz.) 
Darling (1909) describes a case of human sarcosporidiosis occurring in 
Panama, from which he studied the organism and came to the conclusion that 
it was probably a different species from the one already described as occurring 
in man. He gives'a good historical review. Later he decides that morpho- 
logically his human sarcosporidia are identical with Sarcocystis muris. 
CILIATA. 
Balantidium coli (Malmsten, 1857). The body of this infusorium 
is egg-shaped, with a funnel-shaped mouth opening. The surface 
of the body is covered with a pellicula, under which is a distinct ecto- 
plasmatic sheath containing rows of basal granules from which the 
short, fine cilia arise. 
The cloudy entoplasm contains fat and starch granules and may 
contain many red-blood cells and other food particles from the host. CI LI AT A 
547 
Two contractile vacuoles have been seen. Posteriorly there is a small 
prominence marking the place where excreta are expelled. The 
chromatic macronucleus is bean-shaped, and the vesicular micronucleus 
is nearly spherical (Plate IV, Fig. iv, a-d). 
Division is transverse, the macronucleus dividing by simple constric- 
tion and the micronucleus by mitosis. Conjugation has been observed. 
Spherical cysts surrounded by a thick membrane are formed. 
Balantidium coli has been found in the large intestines of human 
beings and of swine-probably two distinct varieties. The variety 
occurring in human beings has been found in above 60 cases, principally 
in Sweden, but also in Russia, Scandinavia, Finland, China, Italy, 
Germany, the Philippines, and the United States. Most of these cases 
were suffering from severe chronic intestinal catarrh, often accompanied 
by bloody diarrhea. A number of observers (Strong, Brooks, and 
others) think the balantidium the primary cause of the catarrh, while 
others believe it to be a harmless inhabitant of the intestines, or at least 
only a secondary excitant (Opie, Malmsten, Doflein, and others). 
Schaudinn has described two additional species of balantidium found 
in the human intestines, which he has called, respectively, Balantidium 
minutum and Nyctootherus faba, probably both non-pathogenic. 
References. 
Darling. The Archives of Internal Medicine, 1909, and Jour. Exp. Med., 1910, xii, 19. 
Jollos. In Kolle und Wassermann, 1913, Sec. Ed., Jena. 
Laveran and Mesnil. Compt. rend. soc. Biol., 1899. 
Th. Smith. Jour. Exp. Med., 1901, vi, 1, and Jour. Med. Res., 1905, xiii, 429. CHAPTER XLIIL 
THE MALARIAL ORGANISMS. BABESIA. 
Introduction.-The malarial organisms are a group of protozoan 
parasites found to be the cause of a definite group of specific infec- 
tious fevers in man, called by the somewhat misleading term malaria, 
a term which signifies "bad air." 
They are classed as sporozoa, order hemosporidia, and are consid- 
ered by the majority of observers as forming one genus, plasmodium 
or hemameba. 
So far as is known, the only natural means by which the malarial 
organisms are transmitted to man is mosquitoes, genus anopheles. A 
part of the life cycle of the organisms is carried on in these mosquitoes. 
Experimentally the disease has been produced by direct inoculation of 
infected human blood into humans. Accidentally it has been conveyed 
in transfused blood. The parasites develop in man within the red-blood 
corpuscles which they finally destroy, thus producing the anemia and 
pigment granules peculiar to malarial fevers. 
Historical Note.-The fevers caused by these organisms were recognized 
and studied as early as 400 b. c., but it was not until 1880 that the true nature 
of the dancing pigment which had been observed long before was determined. 
At that time Laveran announced that he had discovered a parasite in the blood 
which he claimed was the cause of the disease and he published a good descrip- 
tion of several of the stages in the life of the organism. The public remained 
at first almost entirely unconvinced of the parasitic nature of these bodies. 
Many still believed that the bacillus described shortly before by Klebs was the 
cause of the fevers. Among others, Marchiafava and Celli in Italy believed that 
Laveran's organisms represented areas of degeneration within the red-blood 
cells, though Laveran himself demonstrated the organisms to them. When they 
began, however, to study the fresh tissue themselves they changed their opinion 
and later they published a number of valuable contributions on this subject. 
They gave the organism described by them the inappropriate name, Plasmodium 
malaria. Laveran's researches were later confirmed by many other observers. 
In 1885 Golgi showed that quartan fever depends upon a specific form of 
the parasite, and that the malarial paroxysm always coincides with the sporu- 
lation or segmentation of a group of parasites. Thus, in a single infection with 
the quartan variety a paroxysm occurs every fourth day, with a triple infection 
on successive days, segmentation with its accompanying paroxysms occurs 
daily. Golgi and others soon showed that tertian fever and estivo-autumnal 
fevers were each due to a distinct variety of the hemameba. These varieties 
are at present regarded by some as distinct genera, by others as species, belonging 
to a single genus. Councilman first called attention to the diagnostic value 
of the different forms which appear in the blood. 
Though it had been thought for nearly two thousand years that malaria is 
transmitted by insects, the question was not definitely settled until Ross in 
1896 clearly demonstrated that the hematozoa of birds were transmitted by a 
certain species of mosquito. These investigations of Ross were soon confirmed 
by Grassi, Bignami, and others. Manson proved (on his son) that mosquitoes ASEXUAL CYCLE OCCURRING IN THE BLOOD OF MAN 
549 
carried the infection to man. MacCallum's observations on the sexual forms 
of halteridium were a great advance, and Bignami, Grassi, and others soon 
proved that all varieties of malarial fevers are transmitted from man to man 
by mosquitoes of the genus Anopheles. Grassi worked out the complete life 
cycle of the pernicious type (estivo-autumnal), while Schaudinn (1901) did the 
same for the tertian form. In the later classifications the genus name Plasmo- 
dium has been changed to Hamameba. 
Materials and Methods for Study. -If a case of malaria is at hand the organism 
may be examined alive under the microscope by allowing a cover-glass to drop 
gently upon a drop of fresh blood placed upon a clean glass slide. For finer 
differential points, however, smears should be made (see p. 73), which may 
be stained by any one of several methods such as that of Jenner, Giemsa, or 
Wright. (See p. 81 for details of staining.) 
The Parasite.-Three distinct species of malarial organisms in man 
have been described: Hamameba vivax (causing tertian fever), Hama- 
meba malaria (causing quartan fever), and Hamameba falciparum 
(Laverania malaria, Hamomenas pracox, causing estivo-autumnal fever). 
The last species has been divided by certain authors into two varieties, 
a quotidian and a tertian, but it is the present opinion that only one 
species is definitely known. See table of the chief differences between 
these forms (also see Plate VII). 
Each of these species undergoes the two phases of development 
already alluded to, one within the red-blood cells of human beings 
(the asexual phase); the other within the digestive tract of the mosquito 
(the sexual phase). The form changes which each parasite undergoes 
in humans and which the benign tertian undergoes (which may be 
considered a type for all) in both hosts are shown on Plate VII. Briefly, 
they may be described as follows: 
The Asexual Cycle (Schizogony) Occurring in the Blood of Man.- 
The young form is often difficult to find in fresh blood. A pale area 
is seen on an otherwise unaltered red corpuscle, situated usually 
eccentrically, about one-tenth the size of the red corpuscle or about 
one-fourth its diameter, when at rest presenting a rounded appearance, 
but usually actively ameboid, throwing out distinct pseudopodia 
never remaining long in the same focal plane, frequently dipping, so to 
speak, into the substance of the corpuscle. It is often called the hyaline 
form because it is free from pigment, but it is not hyaline in the proper 
sense of the term. It is also called the ring form, because of its resem- 
blance to a ring in stained preparations; but it is never a true ring. 
The ring appearance is produced by the formation of a large food 
vacuole. The young organism passes from the surface to the interior 
of the red corpuscles and grows there at the latter's expense. 
Powley claims that the organisms never enter the red-blood cells. 
They only feed from the surface. 
The forms intermediate between this and the segmentation stage ap- 
pear in the fresh blood simply as larger parasites, which are readily 
found on account of the reddish-brown pigment granules that they 
contain. These granules begin to appear several1 hours after the or- 
ganism has infected the red-blood cell. At this time the organism is 
1 See table for number of hours in each species. 550 
PATHOGENIC MICROORGANISMS 
usually actively ameboid and the granules have a lively dancing 
motion, due to protoplasmic currents in the parasite. The infected 
corpuscle is swollen and paler. In the estivo-autumnal form the infected 
red-blood cells are smaller than normal. 
When the parasite has approached nearly to its full growth, it 
occupies the greater portion of the corpuscle, which is now more 
difficult to make out. The pigment is still more evident, so that this 
form is therefore most readily found. At this stage amoboid move- 
ments are not so active. When full growth is reached, segmentation 
occurs. The forms up to the period of segmentation are called schizonts. 
The morphologic changes which have been going on in the parasite 
preparatory to segmentation are best studied in properly stained smear 
preparations. In the living organism, they become presently suffi- 
ciently distinct to be followed; the pigment gathers more or less centrally 
into a compact mass, and a peripheral notching indicates that the 
parasite is preparing to divide into a number of segments called 
merozoites; the number of these segments varies in the different species. 
(See table.) Suddenly the segments separate as small spheroidal 
bodies, the young parasites. A corpuscular remnant and the pigment 
float away and are ultimately ingested by phagocytic cells. The 
young parasites attach themselves to red corpuscles as before and the 
human cycle is repeated. 
In a suitably stained preparation the young parasite (see Plate VII), 
appears to be a disk consisting of a central pale, unstained area, known 
Partly schematic. Drawn and rearranged by Williams. Giemsa's stain. 
The asexual forms show cycle of the organism in the red-blood cells of the human host. 
They show schematically the time of fever and the day of segmentation. 
Tertian type: 
Fig. 1.-Segmented organism. 
Fig. 2.-Young ring form in cell and a young form on surface. 
Fig. 3.-Growing schizont; irregular form due to great motility; beginning pigment 
formation; red-blood cell becoming paler. 
Fig. 4.-Larger schizont with dividing nucleus. Red-blood cells pale and stippled. 
Fig. 5.-Nucleus divided into four clumps. 
Fig. 6.-Further division of chromatin and formation of irregular rosette. Pigment 
finely granular in centre. 
Fig. 7.-Segmentation. Note eighteen merozoites (usually sixteen). 
Quartan type. Shows following differences from tertian. Slightly larger, fewer seg- 
ments (usually eight), and more regular. Pigment coarse. Red-blood cells unaltered. 
Segmentation every seventy-two hours. 
Estivo-autumnal type. Shows following differential points: Merozoites smaller and 
more numerous (thirty-two ?); organism less motile with less pigment. Red-blood cells 
smaller and greenish (in fresh cells color). 
Sexual Forms. Show cycle of development in mosquito. 
Fig. 1 (A to E).-Male (J) and female (?) forms of tertian type formed in human 
blood; F, flagellation of male type in stomach of mosquito; G, H, changes in female type 
and fertilization in stomach of mosquito. 
Fig. 2.-Development of sporocyst within mosquito. Liberation of sporozoites, which 
find their way to the salivary gland. 
Fig. 3.-Sexual forms of estivo-autumnal type found in human blood, showing develop- 
ment of sickle-shaped bodies. 
EXPLANATION OF PLATE VII. PLATE VII 
ASEXUAL FORMS OF MALARIAL PARASITES 
(SCHIZOGONY) 
TERTIAN TYPE 
QUARTAN TYPE 
/ESTIVO-AUTUMNAL TYPE 
A. W. WILLIAMS, DEL. ASEXUAL CYCLE OCCURRING IN THE BLOOD OF MAN 
551 
Name of or- 
ganism. 
Size of parasite 
up to segmen- 
tation (schiz- 
ont). 
Motion of young 
schizont in cor- 
puscle. 
Time of appear- 
ance of melanin 
granules and 
their arrange- 
ment. 
Shape of seg- 
menting para- 
site, number 
of segments, 
and site of 
segmentation. 
Asexual cycle 
complete in 
Sexual forms. 
Incubation 
period. 
Effect on 
human host 
tissues. 
Remarks. 
H. v i v a x 
(parasite of 
tertian 
fever). 
I/2 to slightly 
larger than 
normal red- 
blood cell (may 
occasionally be 
almost twice 
size). 
Markedly ac- 
tive. 
6 hours. First 
scattered then 
gathered in 
centre. Fine- 
ly granular. 
Actively danc- 
ing. 
Irregular mul- 
berry. 
12-24 (aver- 
age 16). Peri- 
pheral circu- 
lation. 
48 hours. 
Gametocytes 
spherical. 
No crescents. 
Male % size of 
female, which 
is 1) times size 
of red-blood 
cell. 
About 14 days. 
Pale, granular, 
slightly en- 
larged red- 
blood cells. 
Finely gran- 
ular pigment 
formed from 
metamor- 
phosed hemo- 
globin. 
Double infec- 
tion may 
cause a par- 
•oxysm every 
day, thus giv- 
ing clinically 
a quotidian 
type of fever. 
H. malaria) 
(parasite of 
quartan 
fever). 
If* to little less 
than size of red- 
blood cell. 
Not very ac- 
tive. 
Within a few 
hours. 
Collected in 
zone on peri- 
phery. 
Coarsely gran- 
ular. 
Slight dancing. 
Regular daisy 
shape. 
6 to 10 (aver- 
age 8). Peri- 
pheral circu- 
lation. 
72 hours. 
Gametocytes 
spherical. 
Fewer than in 
vivax. About 
size of red- 
blood cell. No 
crescents. 
About 3 weeks. 
Red-blood cells 
•may be slight- 
ly shrunken. 
Double infec- 
tion may 
cause a par- 
oxysm every 
day, thus giv- 
ing clinically 
a quotidian 
type of fever. 
H. falcipa- 
rum (para- 
site of esti- 
vo-autum- 
nal (perni- 
cious) fever 
type. 
Smaller than 
others, from 
very small to 
J diameter cor- 
puscle. 
Active, but 
slightly less 
so than ter- 
tian type. 
Within 24 hours. 
Small amount, 
2 to 3 coarse 
granules usu- 
ally central. 
Non-motile or 
sluggish. 
More pr less 
symmetrical 
daisy. 10 to 32 
very small. 
Chiefly in 
bone marrow 
and viscera. 
24 to 48 hours. 
Gametocytes 
crescentic, 
short and 
plump. } size 
of red cell. 
About 10 days. 
Red-blood cells 
unstained, 
greenish, 
shriveled (cre- 
n a t e d ) and 
darkened; 
stained (Giemsa) 
salmon color. 
Table Showing Chief Differences between the Species of Malarial Organisms (Genus ELemameba) Found in Man. 552 
PATHOGENIC MICROORGANISMS 
as the achromatic zone, and of a basic (blue) periphery, the body, 
including a metachromatically stained, rounded, compact (red) 
chromatin mass, the nucleus, which tends to give the parasite the 
form of a signet ring. 
Later stages up to a certain number of hours show simply changes 
in size and outline of the body. The nucleus then divides by simple 
mitosis. Later it breaks up by amitotic division into an increasing 
number of small masses. By the time the chromatin division is 
completed the chromatin masses will have assumed a rounded form, 
and will be seen to exhibit ultimately the same strong affinity for certain 
dyes which is seen in the compact chromatin body of the young ring- 
like form. At this stage the heretofore scattered pigment appears 
in one clump. Good technique will always show a corpuscular remnant 
even at this time. The achromatic zone mentioned will be seen to 
develop with the chromatin, and when the next step, namely, the 
division of the body of the parasite, is seen to be completed, there will 
be as many achromatic bodies as there are chromatin bodies, each 
division having a share of the basic mother-body, each representing 
the young parasite (merozoite). 
A certain number of the full-grown parasites do not segment and 
these are the forms which commence the life cycle in the mosquito. 
These forms grow to produce the sexual forms, the macrogametocyte, 
or female organism, and the microgametocyte, or male organism. 
When mature these forms are generally larger than the mature schizont 
of the same species, the female organism being usually larger than 
the male and containing more food granules and a smaller nucleus. 
In the estivo-autumnal forms they are crescentic in shape, while in 
the other species they are spherical. In the circulating blood of human 
beings they show no further changes except to become freed from the 
corpuscle; but when the blood containing them is withdrawn and 
exposed for a short time to the air, an interesting series of changes 
in the microgametocyte is observed. The crescentic bodies are 
transformed into spherical bodies; the pigment of the microgameto- 
cytes becomes actively motile, due to internal agitation of the chro- 
matin fibrils, which presently emerge as flagella-like appendages. 
Their movements are very rapid, causing corpuscles to be knocked 
about, and finally they become detached as the microgametes, or male 
elements, and go in search of the female element. In birds, one may 
actually observe the process of conjugation in slide preparations even 
without the aid of a moist chamber and heat. This transformation of 
male bodies never occurs in the human blood. It will be seen that 
it belongs to the sexual cycle which occurs in the stomach of the 
mosquito. 
The Sexual Cycle (Sporogony) Developing in the Mosquito.-The 
common mosquito, often day-flying, belongs to the genus Culex; 
it cannot carry human malaria. It is easily distinguished from its 
night-flying or dusk-flying relatives, Anopheles (the malarial carrying 
mosquitoes comprise about eight genera of the subfamily anophelina?), THE SEXUAL CYCLE DEVELOPING IN THE MOSQUITO 
553 
by its assuming a different posture on the perpendicular wall. While 
the Culex holds the body more or less parallel with the surface, the body 
of the Anopheles stands off at a marked angle. Other differential 
points are the following (see Figs. 188-197): 
Wings of Culex are unspotted; those of Anopheles are spotted (except 
in one rare species). 
The proboscis of Anopheles points toward the resting surface, 
while that of Culex does not do so. 
Anopheles species bite usually in the early evening, while those 
of Culex bite almost at any hour of the day. 
The male mosquito is readily told from the female by its plumed 
antennae, those of the female being inconspicuous. 
The eggs and the larvae of the two genera are quite distinct as may 
be readily seen by glancing at Figs. 188 to 197. The anopheles mos- 
quitoes breed in practically any kind of a collection of water, though 
some species prefer slow running water to quiet pools. The best known 
domestic carriers are usually found in barrels and cisterns. 
If an ordinary mosquito {Culex} is allowed to imbibe the blood of 
a malarial patient whose blood shows gametocytes there will be simply 
a digestion of such blood in the mosquito, and no development of the 
malarial organisms results. If, however, certain species of Anopheles 
ingest such blood, immediate changes follow. It should be remem- 
bered that only female mosquitoes are blood sucking; hence, they 
alone can be responsible for the spreading of the disease. It should 
also be remembered that if the blood imbibed by the anopheles does 
not contain gametocytes, though it may contain earlier stages of the 
malarial organisms, no amount of such blood can cause general in- 
fection of the mosquito. The sexual cycle is similar in all species of 
the parasite. 
The flagellation of the male parasite described above will promptly 
take place in the stomach of the anopheles, 4 to 8 microgametes being 
formed; these conjugate with the female element (Plate VII) in a 
manner comparable to the impregnation of the ovum of higher animals 
by spermatozoids. The macrogametocyte becomes a macrogamete 
by the formation of a reduction nucleus which is thrown out of the 
organism. 
The product of conjugation, the ookinet (zygote), remains for a 
number of hours in the juices of the chyme stomach, changing gradually 
from a spherical, immobile body into an elongated motile wormlet 
(Plate VII, Sexual Forms, 1h). This penetrates the epithelial lining 
of the stomach and rests in the tunica elastico-muscularis (Plate 
VII, Sexual Forms, Fig. 2); here it changes into an oval then into a 
round body, which grows in the course of the next few days enor- 
mously, forming a cyst which projects into the body cavity. Meanwhile 
the chromatin will have become very active. It will have divided into 
numerous nuclei, which become arranged around inactive portions, and 
filamentous sporozoites develop from this chromatin and surrounding 
protoplasm (Plate VII, Sexual Forms, Fig. 2). These sporozoites ulti- Fig. 188 
Fig. 189 
Fig. 190 
Fig. 191 
Fig. 192 
Fig. 193 
Fig. 194 
Fig. 195 
Fig. 196 
Fig. 197 
Chief comparative characteristics of Culex and Anopheles. (From Kolle and Hetsch.) 
Egg of Culex, Fig. 188, laid together in "small boat," those of Anopheles, Fig. 189, separate 
and rounded. Larva of C., Fig. 190, hangs nearly at right angles to water surface, those 
of A., Fig. 191, are parallel to surface. Body of C., Fig. 192, when resting is held parallel 
to wall in a curved position, that of A., Fig. 193, stands at an angle of about 45° and is 
straight; wings of C., Fig. 194, are generally not spotted, those of A., Fig. 195, are spotted; 
in C. the palpse, Fig. 196, of the female are very short, of the male are longer than the 
proboscis; in A., Fig. 197, the proboscis of both sexes are about of equal length. TOXIN PRODUCTION 
555 
mately fill the cysts, which rupture, setting them free into the cavity 
of the mosquito's body; they then are carried by the lymph to all 
parts of the body of the mosquito and thus reach a glandular structure 
in the thoracic cavity of the insect, the so-called salivary gland (poison 
gland), in which they accumulate in large numbers. This gland is in 
immediate connection with the biting and sucking apparatus. If, now, 
such an infected mosquito "bites" a human being, the lubricating fluid 
of the puncturing apparatus will carry sporozoites into the latter's 
blood and the human cycle begins. The stages of development in the 
mosquito require from seven to ten days, but only when the temperature 
is favorable. 
Cultivation.-Bass and Johns announced in 1911. that they had 
succeeded, by a rather complicated method, in obtaining a certain 
amount of development of H. Vivax in the test-tube (see p. 106 for 
details of method). Essentially, defibrinated malarial blood is used, to 
which is added | per cent, dextrose. Bass and Johns state that the 
layer of serum about the sedimented red-blood cells must not be too 
deep (| inch) and that the leukocytes must be removed if more than 
one generation of the parasite is wished. The Thompsons (1913) 
state that they have gotten several generations by less attention to 
these details. 
They draw the malarial blood into the sterile test-tube (10 c.c.) in which there 
is a thick wire. One-tenth c.c. of a 50 per cent, aqueous solution of glucose 
is added before adding blood. The blood is defibrinated by gently stirring the 
wire for five minutes. The wire and clot are removed, and the blood is poured 
into smaller tubes, 1 inch in each. Rubber caps are placed over cotton plugs. 
These tubes are kept at 37° to 44° C. The corpuscles settle slowly leaving about 
one-half inch clear serum. They have found it unnecessary to remove the leuko- 
cytes by centrifuging. They state that the malarial organisms are not destroyed 
by the leukocytes in the tube but by the changes in the serum. Their organisms 
grow through the whole depth of the layer of red-blood cells. 
Effect on Man (Pathogenesis).-As the organism grows at the expense of 
the red-blood cells the principal change is in the blood. Melanemia, or the 
formation of pigment granules from the destroyed red-blood cells, is one of 
the most characteristic features of malaria. As the disease progresses the 
red corpuscles show varying changes in form and hemoglobin content, not 
only the infected corpuscles, but others as well, thus showing that the organism 
produces either primarily or secondarily some toxic substances. The pig- 
ment occurs in two forms, melanin and hemosiderin. The second only gives 
the reaction for iron and is found in the internal organs, while the first is found 
everywhere in the circulating blood. The pigment is taken up by the leuko- 
cytes. There is usually a definite reduction of both red- and white-blood cor- 
puscles, which is more marked in tertian and quartan malaria than in estivo- 
autumnal. There is a relative increase in the number of mononuclear leuko- 
cytes. The spleen shows marked hyperplastic inflammation and pigmentation. 
In intense estivo-autumnal cases the capillaries of brain and other organs may 
be filled with the parasites. We have observed parasites also in the large nerve 
cells of the brain. 
Toxin Production.-The relationship between segmentation and 
paroxysm is always noted in tertian cases, and it is reasonable to sup- 
pose that the occurrence of the paroxysm is referable entirely to the Description of Plate VIII. 
1. Typical young tertian form; the corpuscle shows incipient degeneration; cor- 
puscle to left above shows a blood-platelet. 
2. Abnormal young form, showing small accessory chromatin body. 
3. Two parasties; one a normal young form; the second a large form in crenated 
corpuscle is an unusual abnormal form with very large achromatic area. 
4, 5, 6. Estivo-autunmal parasites; single, double, and triple infection; central 
elongated chromatin bodies. These forms are about the largest usually seen in the 
peripheral blood; no degeneration of corpuscle. 
7. Tertian parasite, about ten hours old; marked degeneration of corpuscle. 
8. Double infection of a corpuscle in tertian fever; marked degeneration of 
corpuscle. 
9, 10, 11. Large tertian parasites showing division of chromatin previous to seg- 
mentation. 
12 and 14. Complete segmentation of tertian parasite. 
13. Double infection of corpuscle, one parasite reaching maturity, but showing 
unusually small segments; the second one atrophied. 
15. Tertian parasite, old case; while the parasite is only half-grown, the chroma- 
tin has split into several compact masses. Degeneration of infected corpuscle. 
16. Dwarfed tertian parasite, smaller than a red corpuscle, but showing five 
compact chromatin bodies; resemblance to quartan rosette. 
17. Microgametocyte of tertian malaria; prominence of blackish pigment sur- 
rounding a large achromatic zone in which the microgametes lie coiled up. 
18. Tertian macrogametocyte. 
19 to 23. Crescentic bodies of estivo-autumnal malaria. 
19. Typical gametocyte; pigment surrounding achromatic area; no chromatin 
shown; the "bib" is present. (Male?) 
20. Semiovoid gametocyte. (Female?) 
21. Pigment removed. Elliptical achromatic area in which the microgametes 
are seen. 
22 and 23. Pigment removed; chromatin more compact; possibly female elements. 
24. From a case of perniciqus malaria with rich infection; only hyaline forms in 
peripheral blood. Below, a large blood-platelet. 
Note.-As the amplification is not uniform, a comparison of the parasites with the 
blood-corpuscles shown should be made in order to have a correct conception of 
their size. PLATE VIII 
Photographs of Tertian and Estivo-autumnal Malarial 
Parasites in Different Stages of Development. 
(Goldhorn.) POINTS OF DIAGNOSIS 
557 
liberation of toxic substances resulting from metabolic activity of the 
parasite within the corpuscle. That there should be a toxic product 
seems highly probable, and its amount must be considered in heavy 
infections. Cases showing an infection of 1 to 5 per cent, of all cor- 
puscles are not infrequent; the destruction of from 50,000 to 200,000 
or more corpuscles per cubic millimeter of blood leads to the rapid 
deglobularization of the blood; hence the deficiency in numbers; add 
to this the effects of the metabolic products, and little is left to the 
imagination to explain the pronounced anemia. 
Immunity from malaria appears to exist as natural and acquired 
immunity. 
Prophylaxis.--The fact that, with the extermination of the malarial 
carrying mosquitoes, malarial fevers in man would be made impossible, 
remains established; the parasite must have its chance of rejuven- 
esence in the mosquito's stomach. 
The various methods of extermination are fully described in books 
which go minutely into the subject. The method of giving small 
doses of quinine to human beings exposed to Anopheles, and of thus 
getting rid of the organism itself within man, should be considered. 
In hot climates especially, where it is practically impossible totally to 
destroy the breeding places of the mosquitoes by drainage or oiling, 
this method is especially serviceable. In these countries, too, the use 
of adequate screening is of marked value. 
Points of Diagnosis.-By a study of the circulating parasite the 
examiner should be able to tell not only the species present, but also 
the progress the disease is making. Malarial parasites can always 
readily be found in recent primary infections, and it is usually only in 
old cases that the search becomes difficult; one is, however, generally 
rewarded by finding them if one looks long enough for them. 
A helpful sign is the finding of pigment in mononuclear leuko- 
cytes, which are seen about the time of a chill or of the period symp- 
tomatically corresponding to it. Free pigment cannot be used as a 
means of diagnosis, as it may be impossible to tell it from dirt or dust. 
In a primary infection of long standing, the gametocytes may be found, 
and in relapses and in those cases treated by quinine, many atypical 
forms appear. A small dose of quinine may drive all parasites except 
the sexual forms out of the peripheral circulation; at all events, the 
finding of them becomes, in the absence of gametocytes, a matter of 
time and experience, especially also as they may be much altered in 
appearance. The part most and first affected is the blue staining 
body; later follow eccentricities of the chromatin, such as multiple 
bodies, and dwarfing, just such changes as might have occurred in 
time, if the body had been allowed to combat the parasite without the 
aid of drugs. In both cases the fever curve becomes atypical. It 
should be remembered that there is no quotidian form originating in 
this country. Quotidian paroxysms occurring here are either a double, 
tertian, or a triple quartan infection. The notion that the parasites 
can be found only at the time of the paroxysm is still in the minds of 558 
PATHOGENIC MICROORGANISMS 
many; it is erroneous. The gametocytes are quite resistant to qui- 
nine and other drugs, and it appears as if cases in which these forms 
are seen are much more prone to relapse than promptly treated recent 
primary infections. The macrogametocytes may remain quiescent 
for years in the blood, and then under certain conditions, probably 
through parthenogenesis, may again begin to develop and multiply, 
thus bringing about relapses. 
In the estivo-autumnal forms the crescentic gametocytes are gener- 
ally few, but at times large numbers of them develop. Of course, 
they are absolutely characteristic. The young parasites are more or 
less characteristic in stained preparations (Plate VII). There may be 
as many as seven parasites in one corpuscle. Later the few heavy pig- 
ment granules are characteristic. 
In fatal cases the formation of crescents may not take place; the 
blood infection with young parasites is then enormous, every field of 
the microscope showing numbers of them. 
In the study of estivo-autumnal fever, as well as in that of the other 
forms, it is to be remembered that crescents when found indicate that 
the disease is of some standing, for such sexual forms are not formed 
until the asexual propagation is waning. The recognition of these 
ovoidal and crescentic bodies is easy. But as there are no readily 
discoverable pigmented forms in the peripheral blood in the early 
stages, it is necessary to be thoroughly familiar with the young estivo- 
autumnal forms. Polychrome staining for them cannot be too much 
recommended, as there is little that is characteristic about them when 
they have been stained with methylene blue alone. Many a serious 
error has been made by adhering to the antiquated idea that parasites 
should be looked for in the fresh blood, as these young, non-pigmented, 
so-called hyaline forms cannot be readily recognized by the inex- 
perienced, while it is an easy matter to know and classify them when 
properly stained. 
The recognition of the quartan parasite in its early stages in the 
fresh blood is not as difficult as that of the tertian form, because the 
outline is more distinct; but in stained preparations it is often indis- 
tinguishable from the latter. The living ameboid young form or 
schizont is more refractive than the young living tertian schizont, 
more like the estivo-autumnal form, and it is just as sluggish in its 
movements. Then, too, the corptiscle is often shrunken and looks 
as if it contained more hemoglobin than in the case of infection with 
the tertian parasite. 
The growing parasite rapidly becomes pigmented, but it shows 
fewer, larger, less motile pigment granules than the corresponding 
tertian one; moreover, the pigment is arranged around the periphery 
of the organism, while in the tertian form it is distributed through- 
out the protoplasm. The quartan parasite is apt to form a band across 
the infected corpuscle. Segments are few in number, as a rule, and the 
parasite remains dwarfed while the infected red-blood cells are normal BLACKWATER FEVER 
559 
in size. The segments are generally arranged symmetrically around 
the central pigment, giving the so-called daisy or marguerite appear- 
ance to the parasite at this stage (Plate VII). 
In tertian fever, the granular degeneration which the infected cor- 
puscles early undergo is diagnostic. In the first few hours it resembles 
the ordinary granular stroma degeneration with basic affinity, while 
it is later seen that the affinity of the then more numerous granules 
is more acid, or, at least, the staining is no longer orthochromatic, the 
blue being superimposed by a red; in other words, these granules 
stain later metachromatically. The greater the loss or transformation 
of the hemoglobin the greater the number of granules. This holds 
good only for tertian parasites, the estivo-autumnal variety causing 
practically no appreciable change though the same technique be 
used. 
Malarial-like Parasites in Other Animals.-Two genera of protozoa 
closely related to the malarial organisms have been found in birds: 
(1) the proteosoma or hemoproteus; (2) the halteridium; both found 
in owls {Hemoproteus noctue Celli and Sanfelice). Points in their 
life history have been brought out by various observers, especially 
by Ross and by MacCallum. The complete life cycle of both forms, 
as worked out by Schaudinn, is considered by him and his followers 
to be of fundamental importance to the understanding of the rela- 
tionship of blood parasites. Schaudinn states that these organisms 
pass through a flagellate stage in the intestinal tract of the common 
mosquito {Culex pipiens) which has previously fed on owls infected 
with the intracellular organisms (halteridium and hemoproteus). 
Novy considers that this mosquito flagellate stage of Schaudinn is 
simply a growth of trypanosomes in the mosquito's intestinal tract 
which are normally found there, and that Schaudinn did not sufficiently 
control his work to warrant his conclusions. 
Malarial-like organisms have been found also in monkeys, cattle, 
dogs, and frogs, but they have been little studied. 
An interesting article by Bernberg-Gossler on malarial organisms in 
monkeys was published in 1909. In it the author described a binucleate 
phase of these plasmodia and agrees with Hartmann in his recent 
classification of these organisms. 
Blackwater Fever.-This is a condition which occurs frequently, 
especially in Europeans in tropical countries. Its etiology has been the 
subject of much discussion. The chief symptoms are fever, hemoglo- 
binuria, delirium, and collapse. It frequently ends in coma and death. 
A few consider it a disease entity, but the majority of observers are 
inclined to believe it the terminal stage of a severe malarial infection. 
It is frequently associated with the demonstration of the malarial 
organism, but they are not always found. It may be that the invasion 
of the ganglion cells of the braifi by the malarial organisms are the 
chief cause of the symptoms, aided in certain cases by the lysemia 
noted by Christophers and Bentley. 560 
PATHOGENIC MICROORGANISMS 
GENUS BABESIA (PIROPLASMA). 
It was not until 1888 that there was a hint as to the real nature of 
the actual cause of "Texas fever" (bovine malaria, tick fever, hemo- 
globinuria) and allied diseases which attack field cattle in many parts 
of the world. Then Babes described inclusions in red-blood cells in 
Roumanian cattle sick with the disease, though he did not decide upon 
the nature of the organism. No new studies were reported until 1893, 
when Theobald Smith and Kilborne gave such a complete description 
of this disease and its cause as occurring in Texas cattle that little 
concerning it has since been discovered. 
These authors describe as the cause of Texas fever, pigment-free 
ameboid parasites appearing in various forms within the red-blood cells 
of infected animals. The organisms may be irregularly round and lie 
singly or they may be in pear-shaped twos, united by a fine line of 
protoplasm. 
Because of these double pear-shaped forms Smith and Kilborne 
named the organism Pyrosoma bigeminum1 and they placed it pro- 
visionally among the hemosporidia. These authors also showed 
that the contagion was carried by a tick (see p. 560). Their work 
has been corroborated by many investigators in different parts of the 
world. Hartmann places this genus in his new order Binucleata, 
and he considers it an important form for showing the relationship of 
the endocellular blood-parasites to the flagellates. Schaudinn, in 1904, 
was the first to call attention to the occurrence of nuclear dimorphism 
in B. canis and bovis, and Luhe, Nuttall and Graham-Smith, Breinl and 
Hindle, and others have confirmed this observation. The second 
nuclear mass is generally in the form of a small granule similar to the 
blepharoplast of undoubted flagellates. 
Morphology of the Parasite.-In the examination under 1000 
diameters of fresh blood of sick cattle, according to Smith and Kilborne, 
are seen, in the red blood cells, double pear-shaped forms and single 
rounded or more or less irregular forms. The size varies, though 
generally it is the same among the bodies in the same red-blood cell. 
The average size is 2^ to 4/z long and 1|m to 2^ wide. The pointed 
ends of the double form are in apposition and generally touch, though 
in unstained specimens a connection between them cannot be seen. 
The axis forms either a straight line, or an angle. The protoplasm has 
a pale, non-granular appearance, and is sharply separated from the 
protoplasm of the including red-blood cell. The small forms are generally 
fully homogenous, whereas the larger ones often contain in the rounded 
ends a large rounded body, 0.1/x to 0.2/z in size, which is very glistening 
and takes a darker stain. Within the largest forms in the centre of the 
1 The generic name Pyrosma, already in use for a well-known Ascidian genus, was 
altered to Piroplasma by Patton in 1895. In the meantime Starcovici (1893) had given 
the name Babesia bovis to the form described by Babes; and as this form seems to be iden- 
tical with that described by Smith and Kilborne the correct name of the genus should be 
Babesia, while the species parasitic in cattle should be called Babesia bigemina. GENUS BABESIA 
561 
thick end is a large round or oval body, 0.5/z to 1/z, which sometimes 
shows ameboid motions. Piana and Galli-Valerio (1895 and 1896) and 
other observers have since described definite ameboid motion of the 
whole parasite. The motion of the whole parasite on the warm stage 
is not produced by the formation of distinct pseudopodia, but by a 
constant change of the boundary. The changes can succeed each other 
so quickly that it is scarcely possible to follow them with the eye. The 
motion may persist for hours. The single ones show motion, while the 
double ones remain unchanged. The parasites take most basic aniline 
stains well. The Romanowsky method or its modifications gives the 
best results (Plate IV, Fig. in, b). 
Stained by this method, the smallest forms appear as tiny rings, 
about one-sixth the diameter of the red-blood cell. A part of the 
rim takes the red nuclear stain, the rest is blue. In the large mature 
pear-shaped organisms a loose mass of chromatin is at the rounded end 
and a dense, compact mass is situated nearer the pointed end. These 
mature, pear-shaped forms, Nuttall states, are the mark of distinction 
between Piroplasma (Babesia) and other intracorpuscular blood-para- 
sites. These pyriform bodies are generally present in pairs, and 
occasionally, in the acute form of the disease, sixteen pairs may be 
seen in a single blood cell. 
The number of red 'pells infected is about 1 per cent, of the whole. 
If the number increases to 5 per cent, or 10 per cent., it generally 
means the death of the animal. The parasites quickly disappear 
from the blood after the disappearance of the fever. In fatal cases 
many parasites are found in the red-blood cells of the internal organs. 
They vary in number according to the stage at which death occurs, 
are most abundant in the kidneys (50 to 80 per cent, of all red corpuscles 
infected), and are found in fewer numbers in the liver, spleen, and other 
internal organs. 
R. Koch has described a bacillar form which he found in large 
numbers in red-blood cells of acute fatal cases in East Africa. Be- 
tween these and the pear-shaped forms he found all grades. This 
variety is probably a distinct species. 
Flagella-like appendages in Babesia have been described by several 
observers as occurring in the blood in mammals. More frequently 
they have been seen in the tick and in attempted cultures. Some of 
them have been interpreted as possible microgametes (Hartmann, 
Calkins), others as true flagella (Breinl and Hindle), still others as 
fine pseudopodia (most observers). 
Smith and Kilborne showed that the infection is caused by a species 
of tick, Margaropus annulatus Say (Bodphilus boms) (Fig. 198), and 
Kossel gives Ixodes redivius as the tick causing transmission of the 
germ in the hemoglobinuria of Finland cattle. 
The ticks feeding upon the blood of cattle and other mammals 
become sexually mature at their last moult. They then pair, and 
the fertilized females, after gorging themselves with the blood of their 
host, drop to the ground. Each female then lays about 2000 eggs, 
36 562 
PATHOGENIC MICROORGANISMS 
and within the shell of each egg a large quantity of blood is deposited 
to serve as food for the developing embryo. The female then shrivels 
up, becoming a lifeless skin. The newly hatched larvae containing 
in their abdomens some of the mother-blood, crawl about until they 
either die from starvation or have the opportunity of passing to the 
skin of a fresh host. If the mother-tick has drawn its supply of blood 
from cattle infected with piroplasma, her larvae are born infected with 
the parasite and become the means of disseminating the disease further. 
This mode of dissemination explains the long incubation period of 
the disease (forty-five to sixty days-thirty days for the development 
of the larvae and the remainder for the development of the parasite 
within the host). It is possible that the tick embryo acquires the 
infection secondarily from the blood it absorbs in the egg, and that the 
parasites do not pass through the ovum itself as in Nosema bombycis. 
This species of tick M. annulatus has been found also on sheep and 
ponies. 
Fig. 198 
No. 1. Texas fever tick, Margaropus annulalus (Boophilus bovis). X 15. 
No. 2. Natural size. (Mohler.) 
So far, it has not been possible experimentally to inoculate animals 
other than cattle with these parasites. Calves withstand the infec- 
tion better than older animals and a certain degree of immunity is 
reached in some of the older cattle in infected districts. The piro- 
plasmata taken in by such animals may remain as harmless parasites 
for some time. If, however, such cattle are weakened from any cause, 
their resistance to the organism may be lowered and they may there- 
fore pass through a more or less severe attack of the disease. 
Symptoms of the Disease.-Fever (40° to 42° C.), anorexia, weakness, 
increased pulse and respiration, decreased secretion of milk, hemoglobinuria 
at the height of the fever, causing the urine to appear dark red like port wine 
or darker. The urine may contain albumin even if the hemoglobinuria .is 
absent, but there are no red-blood cells present, the color being due to the 
coloring matter of the blood only. There is icterus of the mucous membrane 
if much blood is destroyed. GENUS BABESIA 
563 
The prognosis varies in different epidemics from 20 to 60 per cent. Death 
may occur in three to five days after first symptoms appear. Recovery is 
indicated by a gradual fall of the fever. 
Treatment.-Quinine in large doses seems to have helped in some epidemics. 
Nuttall, Graham-Smith, and Hadwen have reported curative effects from 
trypanblau in both canine and bovine babesiosis (Piroplasmosis). 
Prophylaxis.-Stalled cattle are not infected, but it is impracticable 
to keep large herds of cattle stalled. If the cattle are kept from infected 
fields for one or two years and other animals (horses and mules) 
are allowed to feed there the ticks may disappear. The burning of the 
field for one season may have a good effect. If animals cannot be taken 
from infected fields such fields should be enclosed. 
Ticks on animals may be killed by allowing the cattle to pass through 
an oil bath (paraffin, cottonseed oil, etc.), whereupon the ticks die 
from suffocation. The bath should be repeated after a week in order 
to kill any larvae which may have developed. All animals sent from 
infected regions should receive this treatment. Animals apparently 
healthy before the treatment, after the disturbing influence of the 
bath often develop the disease in an acute form and die. 
Certain birds in Australia seem to feed on the ticks, therefore such 
birds might be propagated. 
Various attempts have been made to give protection by the inocu- 
lation of fresh (not older than two or three days) blood from slightly 
infected animals. Some partial results have been reported, especially 
when the inoculations were made during the cold months. In Aus- 
tralia, the inoculation of defibrinated blood from animals which have 
just recovered from the infection, but whose blood still contains some 
parasites, has been tried. So far no absolute protection has been pro- 
duced, neither does the parasite-free serum of animals which have 
entirely recovered from the disease seem to contain protective qualities. 
Blood-organisms.-Blood-organisms similar to those described in 
the hemoglobinuria of cattle have been found in cases of red water 
fever of cattle in England. They also occur in monkeys, dogs, sheep, 
horses, and pigeons. Nocard and Motas, who have made an extensive 
study of these parasites in the malignant jaundice (hemoglobinuria, 
malaria, or biliary fever) of dogs, state that though the parasites are 
morphologically similar to those infecting cattle, yet it is impossible 
to infect cattle or any other animal tried with them. They must 
therefore be considered a physiologic variety. 
Cultivation.-Thompson and Fantham have reported successful 
development in the test-tube after the method of Bass and Johns for 
malaria. 
Nuttall and Graham-Smith report a study of canine piroplasmosis, and have 
drawn a cycle showing the usual mode of multiplication in the circulating blood. 
They consider B. canis a species distinct from B. bovis and B. pithed (found by 
Ross, in 1905, in blood of a species of cercopithecus) though no morphologic 
differences are given. 
Christophers has described probable sexual stages of development in the tick 
R. sanguineus, so that he has drawn a complete life cycle of the organism, 564 
PATHOGENIC MICROORGANISMS 
References. 
Bass and Johns. Jour. Exp. Med., 1912, xvi, 567. 
Berenbreg-Gossler. Beitrage zur Naturgeschichte der Malariaplasmodien, Archiv. fiir 
Protistenkunde, 1909, xvi, 245. 
Christophers. Jour. Trop. Med., 1907, x, 323. 
Craig. The Malarial Fevers, in Osler's Modern Medicine. Philadelphia, 1907, vol. i; 
also The Malarial Fevers, etc., 1909, Wm. Wood & Co., New York, first edition. 
Howard. Mosquitoes, in Osler's Modern Medicine, Philadelphia, 1907, vol. i. 
Kinoshita. Arch. f. Protistenk., 1907, viii, 294. 
Koch. Zeitschr. f. Hygiene, 1901, xlv, 1. 
Marchiafava and Bignami. Malaria, in Twentieth Century Practice, New York, 1900. 
Miyajami. Philip. Jour. Science, 1907, ii, 83. 
Nuttall and Graham-Smith. Jour. Hygiene, 1905-1906-1907. Also in Parasitology, 
1909, ii, 215, 229, 236. 
Rowley. Jour. Exp. Med., 1914, xix, 450. 
Ruge. Kolle and Wassermann's Handbuch der Pathogenen Mikroorganismen, 1913, 
2d ed., Jena. 
T. H. Smith and Kilborne. United States Department of Agriculture, 1893, Bulletin 
No. 1. 
Thayer and Hewetson. The Malarial Fevers of Baltimore, Johns Hopkins Hospital 
Rep., 1895, vol. v. 
Thompson and Thompson. Ann. Par. and Trop. Med., 1913, vii, 509. 
Thompson and Fantham. Ann. Par. and Trop. Med., 1913, vii, 621. CHAPTER XL1V. 
SMALLPOX (VARIOLA) AND ALLIED DISEASES. 
Introduction.-The diseases smallpox, cow-pox, vaccinia, horse-pox, 
sheep-pox, if not identical, are closely allied. Indeed, the following 
facts seem to prove that at least cow-pox and variola are very closely 
related, if not essentially the same disease: First, smallpox virus in- 
oculated into calves produces, after passage through several animals, 
an affection exactly similar to cow-pox. The successful inoculation 
of the first series of cattle from smallpox is a matter of great difficulty, 
but so many experimenters have asserted that this has been done 
that there seems to be no doubt as to its truth. In our laboratory 
not one of many attempts to accomplish it has been successful. Second, 
both when occurring in nature and when produced by experiment the 
lesions of the two diseases are similar. Third, monkeys have been 
successfully protected against either disease by previous inoculation 
of the other; also, observations go to show that human beings inoculated 
with cow-pox vaccine are not susceptible to inoculation with smallpox 
virus, and that those who have within a varied time passed through 
an attack of smallpox cannot be inoculated successfully with cow-pox 
vaccine. These facts seem positively to prove that the two diseases 
are produced by organisms originally identical, one being modified 
by its transmission through cattle, the other through human beings. 
Variola is perhaps the most regularly characteristic of the diseases 
of man. It is highly infectious and is controlled only by vaccination. 
Notwithstanding the fact that we know definitely the exact site of the 
infective agent in this disease and that certain experimental animals 
are susceptible to inoculation of the material containing the infective 
agent, most investigators are still undecided in regard to the nature 
of the chief exciting factor. A few, however, claim that certain bodies 
found chiefly in the epithelial cells of the skin and mucous membranes 
in the specific lesions are protozoa causing the disease. 
Definition.-Smallpox (Synonyms: Variola, la variola, Blattern, 
Pocken, Vajuola) is an acute infectious disease characterized by an 
epidermic eruption of vesicles and pustules which, upon healing, produce 
cicatrices of varying extent and depth. 
Historical Note.-The first undoubted description of the disease was given 
by Rhazes in the tenth century, but it is evident that he did not consider it a 
new disease. To trace its original home seems to be impossible. It may have 
developed first in certain regions in Asia and Central Africa where it is at present 
endemic and is said to be uncontrolled by vaccination. Many outbreaks of the 
disease in the United States can be traced directly to the importation of African 
negroes. 
The disease, carried by the intercommunication, principally of war and com- 566 
PATHOGENIC MICROORGANISMS 
merce, was widespread when Edward Jenner showed conclusively in 1798 that 
vaccination with cow-pox afforded protection. Now the few cases of variola 
that occur are seen in those who, through neglect or ignorance (sometimes 
willful), have not been vaccinated. 
Etiology of Variola and Cow-pox.-It has been repeatedly shown 
that no bacteria similar to any of the known forms have a causal 
relation to these diseases. In our own laboratory we are able, by the 
inoculating of rabbits' skins, to produce extremely active vaccine 
virus in large quantities, absolutely free from microorganisms which 
grow under the conditions of our present methods of bacterial culti- 
vation. Such pure active vaccine, when emulsified in equal parts of 
glycerin and water and filtered through two or three thicknesses of 
the finest filter paper, gives a slightly opalescent filtrate, which in 
the hanging drop under high magnification shows many very tiny 
granules with an occasional larger one, and in smears shows no formed 
elements giving characteristic stains. This filtrate, from which no 
growth was obtained on artificial culture media, when rubbed over 
a freshly shaved rabbit's skin after the method of Calmette and Guerin, 
or when used to vaccinate human beings, gives an abundant typical 
reaction. 
These facts show that some, at least, of the infective forms cannot 
as yet be made to grow outside of the body, that such forms are very 
minute, and that they do not stain characteristically with our usual 
methods of staining. In a few experiments we were unable to filter the 
virus through a Berkefeld filter under forty pounds' pressure, but this 
may have been due to the fact that we did not dilute our virus sufficiently. 
Since then Bertarelli and a few others have reported that the virus is 
slightly filtrable under pressure. Park and Wilson, however, on later 
experiments (1913) still obtain only negative results. 
Steinhardt, Israeli, and Lambert (1913) report that evidence of 
multiplication of germs may be obtained by growing the virus on living 
tissue in vitro. 
Since Guarnieri in 1892 claimed that certain inclusions present 
in the epithelial cells of the lesions of smallpox in a rabbit's cornea 
(Fig. 199) were parasites, much attention has been given to the study 
of these bodies, commonly known as "vaccine bodies," yet opinions 
still differ as to their nature. Among the more important studies of 
these bodies are those, on the one hand, by Councilman and his asso- 
ciates, who believe them to be protozoa, and, on the other, by Ewing, 
who believes that all of the forms so far described are degeneration 
products, some specific, others not. 
Calkins, working with Councilman, thinks that his original tentative 
cycle is too elaborate. He still firmly believes that the bodies are 
protozoa, but that they belong among the rhizopoda and not among 
the microsporidia where he first placed them. 
Prowazek and others believe that the organisms of this group of diseases, as 
well as of rabies, scarlet fever, trachoma, and a few others, are all minute coccus- 
like forms which have the power of producing an envelope from the host cell SMALLPOX AND ALLIED DISEASES 
567 
substance, such envelope with its contained organism constituting the specific 
body which others have called a protozoon. Prowazek calls the group Chla- 
mydozoa and says they probably stand between the bacteria and the protozoa 
in systematic classification. From our studies on this whole group of diseases 
we have come to the conclusion that there is no close relationship between the 
trachoma bodies and the intracellular bodies of rabies, smallpox and scarlet 
fever (see pp. 439 and 571). 
Fig. 199 
Epithelial cells of a rabbit's cornea, containing many "vaccine bodies." Tissue fixed 
three days after inoculation with smallpox virus, a and d, vaccine bodies; b and c, nuclei. 
X 1500 diameters. 
In our own work on sections, which has extended irregularly over a period 
of several years, we have gotten results which are somewhat confusing, princi- 
pally so because of the non-uniformity of the appearances of these bodies, both 
by different methods and by the same methods at different times. There is 
no doubt that, whatever the nature of the bodies, they are easily affected by 
methods used for fixing, hardening, and staining them. This accounts in part 
for the varied results reported. However, in the most perfectly prepared 
specimens, judged according to the appearance of the red-blood cells, leukocytes, 
and tissue cells at a distance from the lesions, we have found that the vaccine 
bodies, especially in corneal infection, show a more or less constant series of 
changes, somewhat similar to those described by Calkins in his "gemmule 
formation" and by Tyzzer in his development of the vaccine bodies. 
Our best results on corneas have been obtained with the following tech- 
nique: Fix in Zenker's fluid for from four to eight hours; wash in running water 
overnight; place in 95 per cent, alcohol '(changing in two hours to fresh) for 
twenty-four hours, then in absolute alcohol for twenty-four hours. Imbed in 
paraffin. The cuts should be from 3m to 5m thick. Stain with (1) eosin and 
methylene blue (Mallory)-eosin half an hour, methylene blue two minutes; 
(2) Heidenhain's iron hematoxylin; (3) Borrel modified by Calkins. 
The vaccine bodies may be studied for a short time in the living 
cornea by rapidly excising an inoculated cornea, spreading it on a 
shallow agar plate and dropping a thin cover-glass over it. The 
structured bodies are very clearly differentiated from the rest of the 
cell contents, and interesting changes have been observed in them. 
Too little work has been done, however, by this method, to draw 
any further conclusions in regard to their nature. Councilman and 568 
PATHOGENIC MICROORGANISMS 
Tyzzer photographed these living cornea bodies with the ultraviolet 
light, and the structure came out as the chromatin structures of known 
living cells. 
Pathogenesis.-For Lower Animals.-Various animals seem to contract the 
disease, or a modification of it, in nature. Horse-pox, sheep-pox, and cow-pox, 
all show similar pathologic changes. Experimentally, probably all mam- 
mals are susceptible though in varying degrees. Most of them are more sen- 
sitive to vaccinia than to variola. The epidermis of rabbits, for instance, 
shows a beautifully typical eruption after inoculation with vaccine virus, while 
material from smallpox eruptions produces only diffused redness. The corneal 
"take," however, in both instances, is similar in intensity. Monkeys are equally 
susceptible to both forms of the disease. 
For Man.-Without vaccination human beings seem to be equally suscep- 
tible to infection with variola, whatever their race or their condition in life or 
in whatever part of the world they live. 
The immunity caused by successful vaccination is not permanent, 
and varies in its duration in different individuals. Although it usually 
gives protection for several years and may give it for ten or fifteen 
years, it is not well to count on immunity for more than one year, 
and whenever one is liable to exposure it is well to be vaccinated. 
If this vaccination were unnecessary it will not be successful, while 
if it is successful we have reason to believe the individual was open 
at least to a mild smallpox infection. 
Protective Substances Present in the Serum of Animals after Suc- 
cessful Vaccination.-It has been frequently shown that the blood- 
serum of a calf some days after an extensive vaccination possesses 
feeble protective properties, so that the injection of one or two liters 
of it into a susceptible calf would prevent a successful vaccination. 
A further and more convincing fact has been demonstrated by Huddle- 
ston and others, namely, that when active vaccine is mixed in certain 
proportions with serum from an animal which had just recovered from 
a successful vaccination, and the mixture is inoculated into a susceptible 
animal, there is no reaction. 
The Preparation of Vaccine.-For the following points we are indebted 
to Dr. Fielder who is now in charge of this work in New York Health 
Department. 
Seed Virus.-This may be prepared by one of the following methods: 
1. Glycerinized bovine virus which has been ripened for two or three 
months. Such virus becomes attenuated after a varying number of 
calf-passages. 
2. Rabbit Virus.-Rabbits vaccinated with stock bovine virus. This 
may also result in deterioration. 
3. Human Virus.-This consists of the serum collected on sterilized 
bone slips from the vaccine vesicles of previously unvaccinated children. 
This produces good vaccine on calves; it is manifestly difficult to secure. 
4. Glycerinized vaccine from calves which have been vaccinated with 
an emulsion of human vaccine crust. This also gives good virus, but 
it necessitates the constant collection of human crusts. SMALLPOX AND ALLIED DISEASES 
569 
5. Human-Calf-Rabbit Seed.-This has been found to be the most 
economic, efficient, and reliable seed yet found by us. It is produced 
as follows: Crusts are collected from healthy children about nineteen 
days after successful vaccination. These crusts are cut up and emulsified 
with boiled water to a mucilaginous paste. This humanized seed 
is inoculated into an area about 6 inches square upon the abdomen 
of a calf, the remainder of the calf being vaccinated in the ordinary 
way. The pulp from this special area is separately collected and glycerin- 
ized in the usual way. It is then tested bacteriologically and clinically. 
This bovine virus from human seed is now used in a dilution of 1 part 
to 12| parts of normal salt solution to vaccinate rabbits. The seed 
is rubbed sufficiently upon the freshly shaven skin of the back. Five 
days after vaccination the pulp is removed with a curette, weighed 
and emulsified in a mortar with the following solution: glycerin 50 
per cent., sterile water 49.5 per cent., and carbolic acid 0.5 per cent., 
in the proportion of 1 part of pulp to 8 parts of the solution. Four 
rabbits should yield from 15 to 20 c.c. of this emulsion, an amount 
sufficient to vaccinate one calf. The regular supply of vaccine is pro- 
duced by vaccinating calves with this rabbit seed in the manner to 
be described. 
Animals.-The preferable animals are female calves, from two to 
four months of age, in good condition and free from any skin disease. 
These can easily be vaccinated on the posterior abdomen and insides 
of the thighs by placing them on an appropriate table. It is possible 
that, on account of the character of the available supply, older animals 
may be desirable, but the calves take more typically and are more 
easily handled. When an animal is too old to be thrown and held with- 
out difficulty it may be vaccinated on the rump each side of the spine; 
but the skin there is tougher than on the posterior abdomen and insides 
of the thighs, and the resulting virus, though efficient, is not so easily 
emulsified. 
Vaccination.-The hair should be clipped from the entire body when, 
the animal is first brought into the stable and the calf should be cleaned 
thoroughly, including the feet and the tail. Just before vaccination 
the posterior abdomen and insides of the thighs are shaved and the skin 
beneath washed in succession with soap and water, sterilized water 
and alcohol, and then dried with a sterile towel. Oh this area there are 
now made superficial linear incisions with a sharp knife, about a fourth 
of an inch apart. After they have been made they should be dried with 
a sterile towel or with sterile cotton and rubbed with the charged slips. 
One to two slips, depending on the amount of virus each slip holds, 
should be sufficient for vaccinating each vesicle. 
Collection.-On the fifth or sixth day, depending upon the rate of 
development of the vaccine vesicles, they should be ready for collection. 
The entire shaved area is washed with sterile water and sterile cotton, 
and the crusts are picked off. The soft, pulpy mass remaining is then 
curetted off with an ordinary steel curette and the pulp placed in a 
sterilized vessel. The pulp should be mixed with four times its weight 570 
PATHOGENIC MICROORGANISMS 
of glycerin and water (50 per cent, glycerin, 49 per cent, water, 1 per 
cent, carbolic acid). This may be done by passing the mixture through 
a special vaccine mill. The more watery the pulp, especially if it is 
not to be used immediately, the smaller should be the proportion of 
glycerin. The emulsion so produced can then be put up for issue in 
vials. Capillary tubes require especial means of filling, and small 
vials filled and corked answer the purpose admirably. 
Care of the Calves.-The calves are inspected by a Department 
Veterinarian at the time of purchase and during the period of detention 
previous to vaccination. After the vaccine has been collected the calves 
are immediately killed and their organs examined by the Veterinarian. 
If, at autopsy, an animal be found tuberculous or otherwise diseased, 
the vaccine is discarded. 
The vaccine stable at Otisville, N. Y., is a new building with screened 
windows and concrete floors and stalls, which are kept flushed with 
water to wash away the dejecta of the animals. 
The calves stand upon raised racks of galvanized iron. They are 
fed on milk, no hay or straw being used for any purpose. 
The calves are vaccinated and the vaccine pulp collected under 
careful aseptic precautions in a separate operating room, which has 
tiled walls and a concrete floor. 
The vaccine pulp when collected is placed in sterile glass containers, 
sealed, packed in ice and shipped at once by express to the vaccine 
laboratory. 
Preparation and Testing of Finished Product.-As soon as received, 
the vaccine pulp is ground up and emulsified in a solution of glycerin, 
50 per cent.; sterile water, 49 per cent.; carbolic acid, 1 per cent.; in 
the proportion of 1 gram of pulp to 4 c.c. of diluent. 
Laboratory Tests for Purity.-The following tests for purity are now 
made: 
1. Plating upon agar and counting colonies of organism. This is done 
weekly for five weeks. After one to three weeks the plates usually 
show no growth, the carbolic acid and glycerin having killed off the 
extraneous organisms. 
2. Inoculation of glucose broth in fermentation tubes for evidence 
of gas-forming organisms. 
3. Tests for Tetanus.-Two tests are carried out at the same time, 
using two different anaerobic methods. After the cultures have incu- 
bated six days, they are filtered and guinea-pigs are inoculated with the 
filtrate, and are watched for ten days for evidences of tetanus. 
4. Test for Streptococcus.-A guinea-pig is inoculated subcutaneously 
with the freshly prepared vaccine and watched for ten days for evidences 
of streptococcus infection. 
Clinical Tests for Efficiency.-After all the laboratory tests for purity 
have been made and found satisfactory, and not until then, the vaccine 
is ready for use upon human beings, provided it be found to be efficient. 
To determine this point, fifteen inoculations of the vaccine are made 
upon previously unvaccinated children. These must all show a perfect SMALLPOX AND ALLIED DISEASES 
571 
take in order to pass the vaccine as up to standard. A clinical test 
of such vaccine is made every two weeks thereafter so long as the 
vaccine is on the market. If one of these tests fails before the end of 
the period of guarantee, the vaccine is called in. 
Keeping of Vaccine.-Bulk vaccine is. kept in cold storage at a 
temperature of 4° to 8° F. below zero. 
Vaccine which has been put up in packages ready for issue is kept in 
an ice-box at a temperature of 33° to 40° F. 
References. 
Councilman and Co-workers. Jour. Med. Research, 1904, xii, 1. Osler's Modern 
Medicine, Philadelphia, 1907, vol. ii. 
Ewing. Jour. Med. Research, 1904, xii, 509. 
Prowazek and Halberstaedter. Zur Aetiologie des Trachoms, Deutsche med. Woch., 
1907, vol. xxxiii. 
Williams and Flournoy. Studies from the Rockefeller Institute for Medical Research, 
1905, vol. iii. 
Steinhardt, Israeli, and Lambert. Jour. Inf. Dis., 1913, xiii, 294, and 1914, xiv, 87. 
Williams. Rabies, in Forscheimer's System of Med., 1914, vol. v. CHAPTER XLV. 
RABIES. YELLOW FEVER. 
RABIES. 
Introduction.-Rabies (synonyms: Hydrophobia, Lyssa, Hundswuth, 
Rage) is an acute infectious disease of mammals, dependent upon 
a specific virus, and communicated to susceptible animals by the 
saliva of an infected animal coming in contact with a broken surface, 
usually through a bite. The name rabies is given to the disease 
because of its most frequent and characteristic symptom-furor or 
madness. Hydrophobia (Greek, fear of water) is another name com- 
monly used, which is also given because of a frequent symptom of the 
disease, the apparent fear of water. Lyssa is a Greek word meaning 
hydrophobia. Within the gray nervous tissue of rabid animals are 
peculiar protozoon-like structures known as "Negri bodies" which 
are diagnostic of rabies. The nature of these bodies is still a question 
of dispute (see below). 
Historical Note.-Rabies is probably one of the oldest diseases in exist- 
ence, but because of the occurrence of so few human cases, and because the 
disease develops so long after the bite, its source was for a long time not known 
nor was it recognized as a separate disease. Hippocrates does not mention 
it in his writing, but Aristotle about 50 years later (about 300 b. c.) speaks 
of its being purely an animal disease and being carried by the bite of one animal 
to another. Celsus in the first century was the first to give in writing a detailed 
description of human rabies. He speaks of it being produced by the bite of 
rabid animals and states that the wound must be thoroughly bathed and then 
burned with a hot iron in order to prevent the development of the disease, for 
after symptoms appear death always follows. As Celsus was not a physician 
he must have gotten his knowledge from writings which have since been lost. 
Other writers soon after gave very true descriptions of the symptoms and 
handling of the 'disease. 
Many hundred years passed after this without adding anything to our knowl- 
edge of the disease, though authors on the subject were numerous. Van Sweiten 
in 1770 observed the paralytic form of rabies in human beings. At this time 
several authors, among them. Morgagni and Zwinger, believed that the bite 
of a dog which was not suffering from rabies might produce the disease in 
man. In 1802 Bosquillon brought forth the original idea that belief in tbe 
existence of infectious material in rabies was a chimera and that hydrophobia 
was simply due to fright. This false idea had adherants for a long time; even 
now, by a few people, it is thought to be a true one. 
Among the host of good observers who studied the disease during the latter 
part of the nineteenth century, Pasteur stands out as the discoverer, in 1880, 
of the fact that the disease may be prevented by inoculating gradually increas- 
ing doses of the virus into the person or animal bitten. This treatment with 
some modifications, the details of which will be given later, is still used, though 
many efforts have been made to develop an efficient serum treatment. Pasteur, PLATE IX 
Nerve Cells in Spreads from Ammon's Horn. 
Magnification 1200 diameters. 
Figs. 1 and 2 from dog "street rabies" show "Negri bodies" (NB); Fig. 8 
from non-rabie eat, and Fig. 4 from dog distemper show indefinite 
inclusion (I) that might be mistaken for Negri bodies by the inexperienced. 
Negri bodies are structured, more intensely staining, and more refractive. 
Figs. 1, 3, and 4 are stained by fuchsin-methylene blue mixture, which 
stains Negri bodies (NB) red with blue granules, nucleus (NUC) of nerve 
cells blue, and red-blood cells yellow (RB). Fig. 2 is stained by Giemsa's 
mixture, which stains Negri bodies a robin's egg blue with red granules, 
nucleus of nerve cells red, and red-blood cells salmon pink. RABIES 
573 
as well as numerous other investigators, tried to discover the specific cause of 
rabies, but all of the results were negative. The importance of making a quick 
diagnosis had become so evident that the efforts of many workers were directed 
toward this end alone. 
Pasteur and his immediate followers relied for their diagnosis entirely upon 
rabbit inoculations, and this meant a fifteen to twenty days' wait before the 
patient knew whether or not the treatment he was receiving was necessary. 
In 1898 this time was shortened to about nine days in our laboratory by Wilson, 
who found that guinea-pigs came down with the disease much more quickly 
than rabbits. From time to time it has been thought that certain histologic 
findings were diagnostic; for instance, the "rabic tubercles" of Babes, and 
the areas of ".round and oval-celled accumulation in the cerebrospinal and 
sympathetic ganglia" of Van Gehuchten and Nelis, were said to be specific, 
but further study has shown that they are not absolutely specific for rabies. 
In many cases the whole picture of the grosser histologic changes is sufficiently 
characteristic to warrant the diagnosis of rabies, but often it is not so. 
It was not until Negri, in 1903, described certain bodies (Negri bodies) seen by 
him in large nerve cells in sections of the central nervous system, that anything 
was found which seemed absolutely specific for hydrophobia. Negri claims 
that these bodies are not only specific for rabies, but that they are probably 
animal parasites and the cause of the disease. 
Negri's later studies confirm his previous work and add some new facts in 
regard to the structure of the larger bodies. 
His work, especially as far as the diagnostic value of these bodies is con- 
cerned, has been corroborated by investigators in almost all parts of the scientific 
world, among them workers in our own laboratory who not only determined their 
worth in diagnosis, but investigated their nature. 
In our work emphasis was placed upon the fact that the demonstration of 
the "Negri bodies" by our "smear method" (see p. 572) wonderfully sim- 
plified the process of diagnosis. As a result of our studies we concluded that 
the Negri bodies are not only specific for rabies, but that they are living organ- 
isms, belonging to the protozoa, and are the cause of the disease; giving as our 
reasons the following facts: (a) They have a definite, characteristic mor- 
phology; (b) This morphology is constantly cyclic, that is, a definite series of 
forms indicating growth and multiplication can be demonstrated; (c) The 
structure and staining qualities, as shown especially by the smear method of 
examination, resemble those of protozoa. 
Since this report was published many more cases of rabies have been 
more or less studied by us and our former conclusions have been more 
firmly established. Indeed, the evidence as to animal nature of these 
cell inclusions seemed so convincing that Williams in 1906 gave them 
the name Neuroryctes hydrophobia J Calkins has since studied these 
bodies and agrees with Williams as to their nature. He called attention 
to the similarity between their structure and that of the rhizopod a. 
A number of observers, however, still believe that the Negri body 
as a whole is principally the result of cell degeneration and that the 
specific organism may be contained within it. Prowazek includes 
rabies with his " chlamydozoan diseases" (see p. 565). To anyone 
who has made a long and minute study of the two diseases, however, 
there can be no question in regard to the essential difference between 
the "trachoma bodies" and the "Negri bodies." 
1 Proceedings of the N. Y. Pathological Society, 1906, vi, 77. 574 
PATHOGENIC MICROORGANISMS 
Material and Methods for Study.-In New York one may still frequently 
obtain fresh brains of rabid animals, from veterinary hospitals or from the 
laboratories handling this material. Two methods have been used in helping 
to study the principal site of infection. 
(1) Animal inoculations. (2) Sections and smears. 
The first method is used as a decisive test in diagnosis when results from 
the second method are doubtful. 
The technique of the smear method used at present in the Research Labora- 
tory of the New York City Health Department is as follows: 
1. Glass slides and cover-glasses are washed thoroughly with soap and 
water, then heated in the flame to get rid of oily substances. 
2. A small bit of the gray substance of brain chosen for examination is 
cut out with a small sharp pair of scissors and placed about one inch from 
the end of the slide, so as to leave enough room for a.label. The cut in the 
brain should be made at right angles to its surface and a thin slice taken, avoid- 
ing the white matter as much as possible. 
3. A cover-slip placed over the piece of tissue is pressed upon it until it 
is spread out in a moderately thin layer; then the cover-slip is moved slowly 
and evenly over the slide to the end opposite the label. Only slight pressure 
should be used in making the smear, but slightly more should be exerted on the 
cover-glass toward the label side of the slide, thus allowing more of the nerve 
tissue to be carried farther down the smear and producing more well-spread nerve 
cells. If any thick places are left at the edge of the smear, one or two of them 
may be spread out toward the side of the slide with the edge of the cover-glass. 
4. For diagnosis work such a smear should be made from at least three 
different parts of gray matter of the central nervous system; first, from the 
cortex in the region of the fissure of Rolando or in the region corresponding 
to it in lower animals (in the dog, the convolution around the crucial sulcus); 
second, from Ammon's horn, and, third, from the gray matter of the cerebellum. 
5. The smears are partially dried in air and fixed for about ten seconds in 
acid-free methyl alcohol to which 0.1 per cent, picric acid is'added. 
6. The excess of alcohol is removed by pressing fine filter paper gently over 
the smear. 
7. The methylene-blue-fuchsin staining mixture recommended by Van 
Gieson is poured over the slide, warmed until it steams, poured off, and the 
smear is washed in running tap-water, and allowed to dry, the excess of water 
being removed with fine filter paper. 
The staining mixture recommended by Van Gieson is made by us at present 
in the following proportions: 30 c.c. distilled H2O; 10 c.c. sat. ale. sol. meth, 
blue; 0.5 c.c. sat. ale. sol. basic fuchsin. 
The mixture in room temperature in diffuse daylight will keep for a day, and 
possibly two. In the dark at room temperature it retains its staining powers 
a little longer. At ice-box temperature it lasts a much longer time, probably 
indefinitely. 
With this method the Negri bodies stain magenta, their contained granules 
blue, the nerve cells blue, and the red-blood cells yellow (Plate IX, Fig. 1). 
Other methods we have found useful for staining smears are: (1) Giemsa's 
(p. 81), by which generally the "bodies" are a blue and the contained granules 
are azure. The cytoplasm of the nerve cells stains blue also, but with a success- 
fully made smear the cytoplasm is so spread out that the outline and struc- 
ture of most of the "bodies" are seen distinctly within it. The nuclei of the 
nerve cells are stained red with the azur, the nucleoli a dull blue, the red-blood 
cells a pink-yellow, more pink if the decolorization is used (Plate IX, Fig. 2). The 
"bodies" have an appearance of depth, due to their refractive qualities. (2) The 
eosin-methylene-blue method of Mallory (p. 83). With this method of staining, 
the cytoplasm is a magenta, light in the small bodies, darker in the larger; 
the central bodies and chromatoid granules are a very dark blue, the nerve- 
cell cytoplasm a light blue, the nucleus a darker blue, and the blood cells a RABIES 
575 
brilliant eosin-pink. With more decolorization in the alcohol the "bodies" arc 
not such a deep magenta, and the difference in color between them and the red- 
blood cells is not so marked. 
In the technique of the section work (p. 84) the most important point is the 
time the material is allowed to remain in Zenker. According to our experience 
two hours' fixation is not enough, three to eight hours is very good, and with 
every hour after eight hours the results become less satisfactory. Left in 
Zenker overnight the tissue is granular and takes the eosin stain more or less 
deeply, both of which results interfere with the appearance of the tiniest 
"bodies," especially of the very delicate, minute forms found by us in sections 
from fixed virus. 
In regard to the rest of the technique, it is sufficient to say that the changes 
to the different fluids are made with great regularity, and the final differentia- 
tion in alcohol of the stained sections is done most carefully. 
The sections may be stained by the eosin-methylene-blue method of Mallory 
(p. 83). In the sections made in this way we have been able to demonstrate 
clearly very minute .forms, as well as good structures in the larger forms. 
Giemsa's method for sections may also be used. Mann's method, recom- 
mended by others, has not given us such good results. 
Harris has published a staining method for both sections and smears (see 
References), which brings the larger bodies out clearly, but which does not 
seem to give enough differentiation between the smaller bodies and the nucleoli 
of the nerve cells. 
Fig. 200 
Negri body showing central chromatin with ring of small granules. X 2000. 
Morphology of the Negri Bodies.-The largest forms measured are 
about 18m and the smallest about 0.5/1. They are round, oval, oblong, 
triangular, or ameboid. The latter are more numerous in the fixed 
virus of rabbits and guinea-pigs. Their structure is shown especially 
well in smears. Whatever the variety or species of animal infected, 
the bodies present the same general characteristic structure; i. e., a 
hyaline-like cytoplasm with an entire margin, containing one or more 
chromatin bodies having a more or less complicated and regular arrange- 
ment. 
Their structure varies to a certain extent with their size. In fixed 
virus, with an occasional exception, only tiny forms are found. These 576 
PATHOGENIC MICROORGANISMS 
are rounded or sometimes wavy in outline, as if possessing slight 
ameboid motion, sometimes elongated, extending along the rim of the 
host-cell nucleus, or along^one of the nerve fibrils, as if moving there; 
with eosin and methylene blue they take a delicate light magenta 
stain, very similar to that taken by the small serum globules in the 
bloodvessels. Many of the organisms, however, show a small chromatin 
granule, situated more or less eccentrically, sometimes on the very rim 
of the body. In the larger forms the granule is large, in the smaller 
it cannot always be seen; some of the larger forms show from two 
to several granules and occasionally there is a body with the definite 
central body and the small granules about it. 
Detailed Characteristics of Structure in the Large Forms (Fig. 200). 
-In smears, as well as in sections, the cytoplasm appears quite homo- 
geneous; there is no evidence of a reticulum or of a granular structure 
outside of the definite chromatoid granules. The smears, however, 
have brought out one important point in regard to the cytoplasm more 
clearly than the sections, and that is that it is more basophilic than 
acidophilic in staining qualities. With the Giemsa stain, as we have 
already seen, it takes the methylene-blue stain more than the eosin- 
red, and even with the simple eosin-methylene-blue stain the protoplasm 
appears as a deep magenta unless much decolorized. 
In studying the central bodies of these organisms, as they appear 
in the smears, one of the first things noticeable is that they are not 
surrounded by a clear space-that there is no sign of a vacuolar ap- 
pearance in the body as there usually is in the sections. We notice 
next that in the great majority of the organisms the central body stands 
out clearly, as decidedly different in structure, and slightly so in staining 
qualities, from the chromatoid granules which surround it. The general 
type of the structure of the central body is that of ma^y well-known 
protozoan nuclei; that is, the chromatin is arranged in a more or less 
granular ring around the periphery of the central body or nucleus, 
leaving an achromatic or more acid-staining centre in which is situated, 
generally eccentrically a varying-sized karyosome. There are a number 
of variations from this principal type, according to stage of development 
(Plate IX, Figs. 1 and 2). 
Fragmented particles seem to be leaving the nucleus in certain 
forms, and in this way presumably the chromatoid granules are pro- 
duced, thus forming chromidia. 
The chromatoid granules are most frequently arranged in a more 
or less complete circle about the nucleus. They are somewhat ir- 
regular in outline and size, being occasionally ring-shaped, some- 
times elongated, often in twos, due probably to active changes of 
growth and division. They take generally a more mixed chromatin 
stain than the chromatin of the nucleus. 
Evidences of Division.-All stages in transverse division are seen. 
Many evidences of budding are also seen. The chromatoid granules 
seem to divide and pass out with part of the cytoplasm as a bud. This 
budding or unequal division appears to take place very early in the RABIES 
577 
growth of the organism and to continue throughout growth until the 
parent1 body forms a mass of small organisms which may then break 
apart at the same time. The budding accounts for the number of small 
and large forms in a single cell (Plate IX, Fig. 1). 
Number.-They vary in number according to the stage of the 
disease and to the infectivity of the part. 
Site.-They are situated chiefly in the cytoplasm and along the 
fibers in the branches of the large nerve cells of the central ner- 
vous system. In parts of smears which are more broken up the bodies 
may appear as if lying free, and it is these bodies, if the pressure be 
not too great in smearing, that show the structure best. In some 
cases the bodies are distinctly localized in small scattered areas of the 
central nervous system. We have always found bodies in the spinal 
cord in abundance, but here they are especially prone to be localized 
in discrete groups of cells. 
Manouelian has found them in the ganglion cells of the salivary 
gland. 
That the organisms are present in various glands of the body (salivary 
thyroid, suprarenal capsule, etc.) is shown by the virulence of emul- 
sions from these organs. Cows' milk (Westbrook, McDaniel) and 
blood (Marie) have also been shown to be slightly virulent. 
Cultivation of the Rabies Organism.-Many attempts have been 
made to produce artificial cultures, but no reports have been so far 
corroborated. Noguchi, the latest claimant, states that he has obtained 
virulent cultures on the twenty-first transplant. Williams, Krauss, 
Barbara, and Volpino have not been able to get the same results. 
Diagnosis of Rabies.-In our laboratory, for the past ten years, 
or since we have used the smear method in routine diagnosis, there have 
been about 4500 cases in all examined, including suspected rabies and 
controls. 
These are divided into two groups, the first comprising the cases 
sent in from outside, for diagnosis only, and the second, the experi- 
mental cases. 
Since the publication of our work in May, 1906, in our routine 
work we have considered the presence of the Negri bodies in smears 
as diagnostic of rabies and have made no further tests except in those 
cases which we have used in our experimental work. Through this 
experimental work, however, we have added many hundred cases 
to the list of those which had the comparative tests, and our former 
conclusions have been more firmly established. 
In all of our work controlled by careful animal inoculations we have 
never yet failed to have typical rabies develop in animals inoculated 
with material showing definitely structured Negri bodies. Negative 
results after inoculation with such material must be interpreted at 
present as due to some error in technique, such as regurgitation, or 
hemorrhage at the time of inoculation, emulsion improperly made, 
not enough of the virulent material taken because of localization of 
the organisms, etc. 578 
PATHOGENIC MICROORGANISMS 
Possibly individual resistance of the animal inoculated might play a part. 
We have used principally guinea-pigs, and some of them have shown enough 
irregularity in regard to the time in which they have come down with the 
disease to suggest a varied individual susceptibility, if other .factors can be 
ruled out. 
On the other hand, material in which we have failed to demon- 
strate typically structured bodies has produced rabies. All of this 
material, however, since we have improved our technique, has shown 
suspicious small forms similar to those found in rabbit-fixed virus. 
But any decomposing brain may also show in smears bodies very 
similar to these tiny forms, therefore it is difficult to rule out rabies 
in such cases. Of course the animal test will probably always have 
to be used with brains that are too decomposed to show any formed 
elements except bacteria, unless a reliable chemical test can be dis- 
covered. Brains from animals dying of distemper may show small 
non-structured forms, somewhat like "fixed virus" forms (Plate IX, 
Figs. 3 and 4). 
So far we have not had rabies produced by fresh brains showing no 
Negri bodies and no suspicious forms, but a few observers have claimed 
that such material has produced the disease. Therefore, until we can 
standardize our technique, we must in all such cases use animal inocu- 
lations. We may, however, be reasonably certain that a case showing 
such negative material was not a case of rabies. We may summarize 
our knowledge in regard to the worth of the smear method in diagnosis 
as follows: 
1. Negri bodies demonstrated, diagnosis rabies. 
2. Negri bodies not demonstrated in fresh brains, probably not rabies 
(except 4). 
3. Negri bodies not demonstrated in decomposing brains, uncertain. 
4. Suspicious bodies in fresh brains, probably rabies. 
The localization of the Negri bodies is an important point in mak- 
ing diagnoses. We have found well-developed bodies distinctly local- 
ized in different parts of the brain in several instances. In horses 
there may be small, widely scattered areas of well-structured forms 
throughout the cerebellum, while tiny, indefinite forms are scattered 
through the rest of the brain examined. In human brains well-developed 
forms are found in the corpus striatum and not in the rest of the brain. 
In several dogs the localization has also been marked. 
The Complement Binding Test in Rabies.-This test has been 
tried by Heller (1907), Friedberger (1907), and Baroni (1908), with 
negative results. Berry (1910), in our research laboratory went over 
this work thoroughly and obtained similar negative results. 
Effect of Chemical and Physical Agents on Rabic Virus. Rabic 
virus appears to become attenuated under certain conditions of tempera- 
ture; indeed, if it be subjected for about an hour to 50° C. or for 
half an hour to 60° C., its activity is completely destroyed. A 5 per 
cent, solution of carbolic acid, acting for the same period, exerts a RABIES 
579 
similar effect, as do likewise 1 :1000 solutions of bichloride of mercury, 
acetic acid, or potassium permanganate. Cumming has shown that 
formalin is especially deleterious to the virus. The virus also rapidly 
loses its strength by exposure to air, especially in sunlight; when, 
however, protected from heat, light, and air it retains its virulence 
for a long period. 
The virus is readily filtered through all grades of Berkefeld filters, 
and from the glands through the coarser Chamberland. Poor and Stein- 
hardt have shown that the filtered gland and brain virus seem similar. 
Pathogenesis.-Natural Infection.-The disease occurs in nature 
among the following animals given in order of their frequency: dogs, 
cats, wolves, horses, cows, pigs, skunks, deer, and man; in fact, as all 
warm-blooded animals are more or less susceptible to inoculations, all 
may presumably contract the disease when an open wound is brought in 
contact with infectious material of a rabid animal. 
Rabies occurs in almost all parts of the world. It is most common in 
Russia, France, Belgium, and Italy; it is not infrequent in Austria and 
in those parts of Germany bordering on Russia. In this hemisphere 
it is infrequent in Canada, but in the United States the cases are in- 
creasing in numbers. In Mexico, and South America it occurs occa- 
sionally; while in England, North Germany, Switzerland, Holland, 
and Denmark, because of the enforced quarantine laws, and of the wise 
provision that all dogs shall be muzzled, it is extremely rare. In 
Australia it is unknown, probably because the law that every dog 
imported into the island must first undergo a six months' quarantine 
has always been enforced. In the vicinity of New York the disease 
seems to be on the increase. 
In this connection the question as to how long the sputum of a rabid dog 
may remain virulent after it drops from the animal is an interesting one. A 
case came under our observation in 1906 which illustrates this point. A child 
of six years came down with typical rabies in a neighborhood where there 
had recently been several cases of canine rabies, but no history of a bite could 
be obtained. The parents were sure she had not been bitten. Six weeks before, 
however, the child had fallen in the street and cut her cheek severly on a jagged 
stone. The wound was cauterized and healed without further trouble. A mad 
dog had been on that street just before this occurred. It is reasonable to sup- 
pose that the stone had on it some of the sputum from that dog, and so the child 
was infected. Such a case would not occur very often, but the possibility should 
be considered. 
In regard to the question as to whether the bite of apparently healthy 
animals may give the disease, it may be said that, judging from labora- 
tory experiments, some animals may have a light attack of the disease 
and recover spontaneously; though such cases, if they occur, are prob- 
ably extremely rare. That the bite of an infected animal may give the 
disease before that animal shows symptoms has been proved. Fifteen 
days is the longest time reported between a bite and the appearance of 
symptoms in the dog. Therefore, if an animal is kept under observation 
three weeks after biting another, without developing symptoms, he may 
be pronounced free from suspicion. 580 
PA THOGENIC MICROORGA NISMS 
Occupation seems to have an effect upon the number of cases among 
humans in one way. Those people who are much in the country or on 
the streets-in other words, those who might come most frequently 
in contact with rabid animals-most frequently contract the disease; 
otherwise neither age, sex, nor occupation has any effect. 
The time of the year seems to have little effect, though most cases 
are said to occur during the summer months. The numbers vary 
with different years. In 1907 for instance we had as many cases in 
January as in August and in September and more in June than in any 
other month. In another year February gave the largest number of cases. 
The certainty with which the disease may be produced after a bite 
and the rapidity of its development have been found to be governed 
by three factors: (1) the quantity of the rabic virus introduced; 
(2) the point of inoculation; (3) the strength of the virus as deter- 
mined by the kind of animal which affords the cultivation ground 
for the growth of the organism. It is a matter of common observation 
in man that slight wounds of the skin of the limbs and of the back or 
wherever the skin is thick and the nerves few either produce no results, 
especially when bites are made through clothes, or are followed by the 
disease after an extremely long period of incubation; while in lacerated 
wounds of the tip of the fingers where small nerves are numerous or 
where the muscles and nerve trunks are reached, or in lacerated 
wounds of the face where there is also an abundance of nerves the 
period of incubation is usually much shorter and the disease generally 
more rabid. 
These facts explain why only about 16 per cent, of human beings 
bitten by rabid animals and untreated appear to contract hydrophobia. 
Since the establishment of the Pasteur treatment for the disease, 
the percentage of developed cases after bites is very much less, a 
fraction of 1 per cent. 
Symptoms.-There is always a decided incubation period after the bite 
which varies within quite wide limits, but in the great majority of cases it is 
from twenty to sixty days. Any period after six months is an exception; the 
shortest we have on record is fourteen days and the longest authentic period 
is seven months. A very few apparently authentic cases have been reported 
as developing in about one year, but reports of any time beyond this must be 
received with doubt. After treatment, however, a few cases have been reported 
as occurring later than this, but even here the question of reinfection is not 
absolutely ruled out. 
The wound heals as other wounds and sometimes shows no further symp- 
toms. Occasionally, however, redness and swelling of the scar have been 
reported; oftener there are pains extending from the scar along the nerve paths 
of the brain. 
The symptoms may be divided into three stages. First, the prodromal or 
melancholic stage; second, the excited or convulsive stage; and, third, the 
paralytic stage. 
When the second stage is the most pronounced the disease is called furious 
or convulsive rabies; when this stage is very short or practically lacking and 
paralysis begins early, the disease is called dumb or paralytic rabies. 
In the dog rabies appears in the two typical forms, the furious and the 
paralytic. The principal symptoms of each form may be summarized as fol- RABIES 
581 
lows: (a) Furious rabies: change of behavior, biting (especially at those to 
whom the animal has been affectionate before), increased aggressiveness, 
characteristic restlessness, loss of appetite for ordinary food, with desire to eat 
unusual things, intermittent disturbance of consciousness, paroxysms of fury, 
peculiar howling bark, rapid emaciation, paralysis, beginning in the hind limbs, 
death in great majority of cases in three to six days (exceptionally slightly 
longer) after the beginning of symptoms. (6) Paralytic rabies: short period 
of excitation, paralysis of the lower jaw, hoarse bark, appetite and consciousness 
disturbed, weakness, with paralysis spreading in great majority of cases, and 
death four to five days after first symptoms. There may be a number of cases 
showing transition types between these two forms. 
In Human Beings.-Furious Rabies.-The first definite symptoms are 
difficult and gasping breath with a feeling of oppression and difficulty in swallow- 
ing, the latter the most characteristic symptom. It is caused by convulsive 
contraction of the throat muscles. The attacks are brought out when attempt- 
ing to drink or swallow. The very thought of drinking may bring one on; and 
though there is no fear of water itself, there is fear of taking it because of the 
effect it produces. The convulsive attacks finally become more or less general 
over the whole body; in certain cases some parts are more affected by reflex 
excitation than others; for instance, there may be slight or no photophobia, 
while in exceptional cases, more frequently in dogs, the hydrophobia is also 
absent. 
Most of the special reflexes are increased. Pupils become irregularly con- 
tracted and widened until they finally remain fixed. 
Human beings are seldom dangerous to the people about them; they do 
not make aggressive bites. In their convulsions they may bite things placed 
between their teeth, but not otherwise. At this time there is an increased flow 
of saliva, and one should avoid the contact of this with opened wounds. It may 
be so increased that the patient may try to get rid of it by taking it from the 
mouth with the hand and throwing it about. As a general thing, however, 
the patient has full possession of his senses between the convulsive attacks until 
very late in the disease. 
The temperature is increased from 38° to 40° C., at first with morning remis- 
sions. Just before death it may rise as high as 42.8° C. (In lower animals the 
temperature sinks below normal just before death.) The pulse is generally 
over 100 and is irregular. This stage lasts from one to four days. Death may 
occur during a convulsion, but more often there is a paralytic stage, which lasts 
from two to eighteen hours. The convulsions become less frequent and the 
patient becomes weaker until finally there is a complete paralysis. At the 
beginning of this stage the patient may be able to drink water better than 
formerly. Death may occur at any time through paralysis of the heart or 
respiratory centre. 
Paralytic Rabies.-This form occurs quite seldom in human beings, more fre- 
quently in dogs, but not so often as a convulsive form. It is supposed to occur 
in humans and dogs after a more severe infection. Instead of periods of con- 
vulsions, the various muscles simply tremble and become gradually weaker 
until complete general paralysis supervenes. Sometimes paralysis develops 
very quickly and may be general before death from syncope or asphyxia occurs. 
This form generally lasts longer than ordinary rabies. Between these two typical 
forms of rabies there are many different types, giving quite different pictures of 
the disease. 
Length of the Disease.-The majority of the cases of furious rabies die on 
the third or fourth day after the symptoms show themselves. The limits of 
the reported cases are one to fifteen days, though there are reports of only 
one or two cases dying on any day over the ninth to the fifteenth. As the 
time when the symptoms really begin is difficult to notice, these statistics are 
probably only approximately correct. In paralytic rabies the average time 
in which death occurs is five days. 582 
PATHOGENIC MICROORGANISMS 
Treatment.-The old treatment of rabies consisted simply in 
encouraging bleeding from the wound, or in first excising the wound 
and then encouraging bleeding by means of ligatures, warm bathing, 
cupping-glasses, etc.; the raw surface was then freely cauterized with 
caustic potash, nitric acid, or the actual cautery. It is doubtful whether 
the disease ever manifested itself after such heroic treatment if the wound 
were small and the treatment was begun soon after the bite; but when 
the wounds were numerous or extensive, the mortality was still high. 
As it was often impossible to apply cauterization to the wound rapidly 
or deeply enough to insure complete destruction of the virus, Pasteur 
and others were led to study the disease experimentally in animals 
with the hope of finding some means of immunization or even cure; 
these investigations finally resulted in the discovery of methods of 
preventive inoculation applicable to man. 
Pasteur's Method of Preventive Inoculation.-Pasteur's treatment 
is based upon the fact that rabic virus may be attenuated or intensified 
under certain conditions. He first observed that the tissues and 
fluids taken from rabid animals varied considerably in their virulence. 
Then he showed that the virus may be intensified by successive 
passage through certain animals (rabbits, guinea-pigs, cats) and 
weakened in passing through others (monkeys). If successive inocu- 
lations be made into rabbits with virus, either from the dog or the 
monkey, the virulence may be so exalted beyond that of the virus 
taken from a street dog, in which the incubation period is from twelve 
to fourteen days, that at the end of the fiftieth passage the incubation 
period may be reduced to about six or seven days when it remains 
fixed. This "fixed virus" was used by Pasteur and those after him in his 
preventive treatment because the dose could be more definitely regu- 
lated by subsequent attenuation or dilution. 
Original Method.-A series of spinal cords from rabbits dead from 
"fixed virus" infection are cut into segments and suspended in sterile 
glass flasks plugged with cotton stoppers and containing a quantity 
of some hygroscopic material, such as caustic potash; these are kept 
at a temperature of about 22° C. The cord when taken out at the 
end of the first twenty-four hours is found to be almost as active as 
the fresh untreated cord; that removed at the end of forty-eight 
hours is slightly less active than that removed twenty-four hours pre- 
viously; and the diminution in virulence, though gradual, progresses 
regularly and surely until, at the end of the eighth day the virus is in- 
active. Pasteur began his treatment with an emulsion of the cord 
kept until the fourteenth day. A certain quantity of this was injected 
into the animal that had been bitten; this was followed by an injection 
of an emulsion of a twelve-day cord; and so on until the animal had 
been injected with a perfectly fresh and, therefore, extremely active 
cord, corresponding to the fixed virus. Animals treated in this way 
were found by Pasteur to be absolutely protected, even against subdural 
inoculation with consideable quantities of the most virulent virus, 
and thus Pasteur's protective inoculation against rabies became an RABIES 
583 
accomplished fact. As it would be undesirable to inject any but persons 
who had actually been bitten by a rabid, or presumably rabid, animal 
Pasteur continued his experiments in order to see whether it would not 
be possible to cure a patient already bitten. He carried on, therefore, 
a series of experiments which led to the discovery that if the process 
of inoculation be begun within fix^e days of the bite in animals in which 
the incubation period was at least fourteen days, almost every animal 
bitten can be saved; and that even if the treatment be commenced 
at a longer interval after the bite a certain proportion of recoveries 
can be obtained. Thus the application of this method of treatment 
to the human subject was not tried until it had been proved in animals 
that such protection could be obtained and that such protection would 
last for at least one year and probably longer. 
The chance of success in the human subject appears to be even 
greater than in the dog or rabbit. Man's period of incubation is 
comparatively prolonged. Thus there is an opportunity of obtaining 
immunity by beginning the process of vaccination soon after the bite 
has been inflicted, the protection being complete before the incubation 
period has passed. 
Present Administration of Pasteur's Treatment in Human Beings.- 
The original method of Pasteur in its entirety was soon adopted in 
many lands, and his results were corroborated. Before long, how- 
ever, a number of modifications were suggested by different observers, 
some slight, others more fundamental. Some have been widely used, 
such as Hogyes' dilution method; others have had a limited application 
in lower animals and are probably only of theoretic interest as regards 
man. Such are the intravenous inoculation of brain emulsions from 
street rabies into herbivora (Nocard and Roux, Protopopoff), and the 
intraperitoneal inoculations of large doses of fully virulent fixed virus 
into dogs, cats, or rabbits (Hellman, Heim, Remlinger). Immunity 
has been produced also in rats by allowing them to feed on rabid brains 
(Fermi, Repetto, Remlinger). 
Hogyes in Budapest was one of the first to use a different procedure. 
He claimed that the virus by Pasteur's method was attenuated only 
through the death of some of the specific organisms, that is, that there 
were simply fewer living organisms in the early doses given than in the 
later, and that therefore the same result might be obtained perhaps with 
even more accurate dosage by giving gradually decreasing dilutions of a 
fresh virulent cord. By diluting sufficiently he obtained a mixture which 
when inoculated did not produce rabies in the test animals, a result simi- 
lar to that following an 8- to 10-day dried cord. This dilution he used 
for the first inoculation and gradually stronger dilutions for the succeed- 
ing ones. 
Other methods of attenuating or diluting fixed virus have been used, 
such as exposure to the action of heat, cold, gastric juice, glycerin, or 
carbolic acid. 
The mixed treatment with specific serum and vaccine has also been 
employed, chiefly by Marie, by Remlinger, and by Babes. 584 
PATHOGENIC MICROORGANISMS 
Methods of Attenuation by Gradual Drying.-Pasteur's classic method 
has undergone modifications in three general directions: (1) lengthening 
or shortening the period of treatment; (2) starting the inoculations with 
a less attenuated cord; (3) increasing or decreasing the amount given at 
each injection. The method of drying the cords, however, has remained 
essentially the same as that used by Pasteur. 
Fig. 201 
One corner of constant temperature room showing drying bottle containing fixed 
virus cords being prepared for vaccine. 
The cord is removed by a modification of the method of Oshida in the 
following manner: Strict asepsis is preserved. The rabbit when com- 
pletely paralyzed (seventh day) is killed by gas or chloroform and is 
dropped into a 5 per cent, solution of carbolic acid for 5 minutes. It is 
then removed, the excess of carbolic solution is drained off, and an 
incision through the skin at the upper and inner part of the thigh is 
made. The skin is loosened by cutting around the lower portion of 
the trunk. It is then pulled by the hands toward the upper extremity 
of the animal and over the head to the ears, leaving the back exposed 
and sterile throughout the entire length of the spine. The spine 
is then divided transversely near each extremity by bone-cutting 
forceps. The muscles are cut through about these areas so the spine 
may be more easily reached. With a long wire probe swabbed with 
cotton at one end the cord is pushed upward from its canal, freed from 
its nerves and membranes. The spine is steadied by lion-jawed forceps. 
The cord curls in a spiral as it emerges and rests on the sterile muscles 
of the neck. It is lifted with forceps, placed in a Petri dish and 
cut in two. A small piece is cut from one end and is dropped into a tube RABIES 
585 
of broth to test its purity. A ligature with one long end is placed about 
each piece, both of which are then hung in a drying bottle (Fig. 201). 
Drying the Cord.-The drying bottles are sterile aspiration bottles 
with both openings plugged with cotton. A layer one inch high of sticks 
of caustic potash covers the bottom, and the pieces of cord are suspended 
from the top cotton plug by their attached ligatures. The bottles are 
then labeled and placed in the constant temperature room (Fig. 201) 
or incubator, which is kept at a temperature of about 21° C. (70° F.). 
After 24 hours' drying the cord is known as 1-day cord; after two days, 
2-day cord, etc. Pieces of cord cut off at any time and put into glycerin 
will retain about the same strength for several weeks. This procedure 
is followed in regions where there are few cases of rabies, and the daily 
killing of rabbits to keep up the vaccine would be a large expense. It 
may also be followed where treatment is sent by mail. 
The New York City Health Department used Pasteur's first schemata, 
with modifications, up to January, 1906, when they began treatment 
with a 10- and 9-day cord and finished with a 1-day. They continued 
with this until August, 1913. Since then they have been using the 
more intensive method of the Hygienic Laboratory at Washington. 
From 1906 to 1913 inclusive they treated 4282 cases infected by rabid 
animals, with a total mortality of 0.54 per cent, and a corrected mor- 
tality of 0.19 per cent. They have had 7 cases of definite paralysis 
with two deaths; 6850 cases in all, including those not bitten by rabid 
animals, were treated. 
Since it had been found that fresh rabbit-fixed virus inoculated sub- 
cutaneously into man is apparently harmless, the Berlin Institute, with 
the hope of obtaining an earlier immunization and a shorter treatment, 
began to give still earlier cords. In 1901 it began with the 8-day cord 
on the first inoculation, and was inoculating a 2-day cord on the eighth 
day of treatment. Its treatment lasted 21 days. This method was 
adopted at the Hygienic Laboratory in Washington in 1908, with slight 
variations for the different degrees of bites. Now only the intensive 
schema is used for all cases as follows: 
Days. 
1 2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
Age of 
cord 
8 
1 
6 
4 
3 
5 
4 
3 
3 
2 
2 
1 
5 
4 
4 
3 
3 
2 
2 
4 
3 
2 
3 
2 
1 
Amount 
injected 
in c.c. 
Adults 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.52.5 
2.5 2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.52.5 
2.5 
2.5 2.5 
2.5 
5 to 10 years 
2.5 
2.5 
2.5 
2.5 
2.5 
2 
2.5 1.5 
2.52.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 2.5 
2.5 
2.5 2.5 2.5 
1 to 5 years 
2.5 
2 
2.5 
2 
2 
1.5 
2 
1 
2.5 2.5 
2.5 
2 
2 
2 
2 
2.5 2.5 
2 
2 
2.5 
2 
Each dose contains | cm. of the indicated cord. 
The New York City Health Department has been using this schema 
since August, 1913, for severe head and face bites, but it reduces all 
doses by one-fifth, i. e., | cm. of the indicated cord is emulsified in 3 c.c. 
of normal salt solution, and 2| c.c. of this emulsion is inoculated. It 
also substitutes a 2-day cord for the 1-day cord of the eighth and twenty- 586 
PATHOGENIC MICROORGANISMS 
first day in other than severe cases. In cases with very slight wounds 
which have begun treatment immediately the inoculations are carried 
only as far as the fifteenth day. 
Treatment by Mail.-The New York City Health Department was 
the first to send out treatment by mail to physicians for their own 
patients. Full directions are sent in the mailing case. One-fourth 
per cent, of carbolic acid is added to the emulsions prepared as above 
for the first three days' treatment; 20 per cent, glycerin is added to all 
other emulsions. The carbolic acid and the glycerin are added as pre- 
servatives, and are therefore omitted when the vaccine is administered 
to patients at the laboratory. 
The results from the treatment sent in this way seem to be equally as 
good as those from the treatment administered at the laboratory. 
More Intensive Treatment.-In Berlin, where intensive treatment 
has been longest used, they began to employ even fresher cords for begin- 
ning doses because they continued to have late deaths, though not quite 
so often, after the more intensive methods they were using. Since 
1910 Joseph Koch, the present chief of the Institute, has been using the 
following schema: 
Days 
1 
2 
3 
4 
5 
6 
7 
8 9 
10 
11 
12 13 
14 
15 
16 
17 
18 
19 
20 
21 
Age of cords .... 
3 
2 
1 
1 
3 
2 
1 
1 3 
2 
1 
1 3 
2 
1 
1 
3 
2 
1 
1 
1 
The dose is 2 c.c. of cord emulsion (1 part of cord in 5 parts of sterile 
physiologic salt solution) inoculated once a day into the subcutaneous 
tissue of the abdomen. Children and adults receive the same dose. 
Simon gives the following statistics of the results of Berlin's increas- 
ingly intensive methods: 
Berlin Statistics. 
Period. 
Age of cord used 
for beginning 
inoculation. 
Cases. 
Paralysis. 
Mortality. 
Per cent. 
I. 1898-1906 
Chiefly 8-day cord 
2,896 
0 
21 
0.7 
II. 1906-1909 
4-day cord. Sometimes 
3-day cord . 
1,490 
2 
7 
0.47 
III. 1909-1910 
3-day cord for all cases. 
819 
3 
5 
0.6 
Several other institutes are employing very intensive treatments, but 
their cases are still too few for consideration. Other directors still use 
the older methods. 
Rapid Drying of Rabies Virus.-Recently Harris, of St. Louis, has 
published a new method of drying rabies virus and of regulating the 
dosage. 
Technique.-The brain and cord are removed aseptically and ground 
up in a sterile mortar with a sufficient quantity of CO2 snow thoroughly RABIES 
587 
to freeze the tissue. The frozen nerve tissue and snow are then placed 
in a Scheibler jar over H2SO4, the jar being kept in a Frigo apparatus. 
A vacuum of from 5 to 2 mm. is produced in the jar, which is then kept 
at the temperature of 18° C. by an ice and salt mixture for a sufficient 
length of time to dry thoroughly the nerve substance, which then appears 
as a dry powder. About two days are required for one brain and cord, 
which lose about one-half of their virulence in the process. The powder 
is then sealed in tubes in vacuo and kept at a temperature below 0° C. 
until required for use. It has been found that by keeping the powder 
thoroughly dry and cold practically no further loss of virulence occurs 
for at least six months. Before storing the virus for use its strength in 
units is computed, the unit being the minimal infecting dose (M. I. D.) 
for a rabbit when injected intracerebrally. 
The advantages claimed for this method are: (1) the ease and econ- 
omy with which a large amount of virus can be prepared, it being neces- 
sary to prepare the virus for use even in large laboratories only at inter- 
vals of several months; (2) the possibility of more accurate dosage for 
the patients; (3) a shortened period of treatment; and (4) the inoculation 
of more virus units. The required amount of powdered virus is weighed 
out each morning, and the necessary dilutions in salt solution for the 
various patients are made from this. 
Up to October 13, 1913, Harris had treated 240 cases exposed to 
infection from dogs, in which the diagnosis of rabies was either proved 
by laboratory methods or strongly probable from veterinary diagnosis. 
Of this number one patient, who had started treatment six days after 
the bite, died of rabies during the period of observation. No cases of 
paralysis have been reported. 
We must wait for further statistics before being able to judge of the 
efficiency of this method. 
Fixed Virus Modified by Dialysis.-Cumming, of Ann Harbor, 
has devised a method of antirabic vaccination, by which he uses fixed 
virus which has been rendered avirulent by dialysis. The emulsion of 
fixed virus is placed in collodion sacs (prepared by the Novy method and 
sterilized in the autoclave at 105° C. for 20 minutes) and dialyzed in 
distilled water for from 12 to 24 hours. The resulting vaccine does not 
produce rabies on intracranial inoculations, but does produce immunity 
on subcutaneous inoculations. Experiments by Cumming on rabbits 
show that whereas the original Pasteur method protects against only 
twice the minimum lethal dose (minute directions for obtaining the 
M. L. D. are given) injected intracerebrally, and the Hogyes method 
against one and one-half times the fatal dose, the dialysis method 
protects against at least three times the fatal dose. He also claims 
that immunity is produced at an earlier date than by the other methods. 
Treatment (2 c.c. of the vaccine) is given daily for from 15 to 25 days. 
Cumming reports over 800 cases (62 per cent, bitten by animals proved 
to have been rabid) treated without a death and without complications. 
We must wait for further practical testing of this interesting method 
before deciding as to its comparative worth. 588 
PATHOGENIC MICROORGANISMS 
Marie's Method.-For several years past the use of virus serum 
mixture has been in vogue at the Pasteur Institute in Paris, the tech- 
nique of which is as follows: 1 gm. of the medulla of a rabbit dead of 
fixed virus is finely emulsified with 9 c.c. of 0.8 per cent, salt solution 
and filtered through linen. Two c.c. of this emulsion and 4 c.c. of 
antirabic serunr (obtained from sheep, and inactivated at 56° C. for 
30 minutes) are carefully mixed after standing for a time. Six c.c. of 
this mixture, which contains an excess of virus, is injected into the 
patient. These injections are repeated on the next three days, after 
which the treatment proceeds according to the regular Pasteur schema, 
beginning with the use of a 6-day cord on the fifth day. The antirabic 
serum is obtained from sheep which have been subjected to a long and 
strong course of treatment with fixed virus. It is claimed that a quicker 
immunity is produced by the serum-virus mixture than by the original 
Pasteur scheme, an advantage of especial value in the treatment of 
cases liable to become infected with a short incubation, such as bites 
on the head. 
Antirabic Serum.-The possibility that the serum of animals im- 
munized against rabies contains protective substances was suggested by 
Pasteur as early as 1889. The following year Babes recommended the 
use of the serum of vaccinated animals in combination with the Pasteur 
treatment. Since then the study of the amount and character of the anti- 
body content of animals immunized against rabies has been carried on 
more or less extensively both from the theoretic and the practical sides. 
It was hoped that a serum could be obtained that would effect a cure for 
developed rabies just as diphtheria antitoxin does for developed diph- 
theria. But such a definite applicability of the serum has not developed. 
It was soon found that, while serum of certain vaccinated animals pos- 
sessed the property of neutralizing rabies virus in vitro, it had only 
a slight inhibiting power when inoculated into the living animal, and 
apparently no action at all by any method of inoculation after the 
disease had become manifest. Babes still claims, however, that the 
serum has enough effect in vivo to be used in treatment, and his serum 
treatment is based upon this claim. He gives as his reason for em- 
ploying serum at the end of treatment that he wishes to introduce into 
the patient at the time he most needs it the largest amount of anti- 
bodies. He also claims that the serum so given will prevent or cure 
the occasional paralyses which occur during treatment. 
Those who did not agree with Babes were led to test the practical 
use of the serum combined with the beginning vaccine inoculations. 
Remlinger, Marie, and others showed that a serum-virus mixture 
with a slight excess of virus will protect an animal against infection into 
the anterior chamber of the eye when inoculated during the three days 
following the vaccination. Thus Marie showed that immunity is pro- 
duced more quickly by these unsaturated mixtures of virus and serum 
than by the virus alone. If a surplus of serum is present the animals 
are not protected from a later infection. 
Marie, who has used the serum in his treatment of humans since 1904, RABIES 
589 
prepares it as follows: The brains of two rabbits dying from fixed virus 
infection are finely rubbed up with physiologic salt solution in the pro- 
portion of 20 gm. in 180 c.c. This emulsion is filtered through fine 
cloth and heated for one-half hour at 37° C. Sheep are used for the 
inoculation. Each sheep receives intravenously 30 c.c. (3 gm. fixed 
virus) a week for six to eight weeks. Thirteen days after the last inocu- 
lation the first blood is drawn. Then in a period of two weeks, at 4 
bleedings, 200 c.c. of blood are drawn. After a 14-day pause another 
series of inoculations are given and the animal is ready for another 
series of bleedings. From each animal yearly about 3 liters of antirabic 
serum are obtained. A strong serum is one that neutralizes 40 virus 
units in 1 c.c. 
A virus unit is 1 c.c. of five times the dilution of fixed virus that will 
surely kill a rabbit inoculated intracerebrally, e. g., the unit of a fixed 
virus that will surely kill a rabbit in 1 : 500 dilution is 1 c.c. of a 1 : 100 
dilution. 
The nature of the antibodies in rabies serum has been the subject of 
many studies. Fermi and a few others claim that the antibodies are 
not specific. They say that they can obtain a similar serum after the 
inoculation of normal brain emulsions. Some even use normal brain 
emulsions in the treatment of their lighter cases. 
Certain investigators (Kraus, Marie, and others), while not able to 
corroborate all of these claims, have found that the sera of certain 
animals which are more or less refractory to rabies possess a small 
amount of rabicidal strength; e.g., 0.5 c.c. of normal chicken serum 
mixed with one unit of fixed virus (1 c.c. of 1 :100 dilution) causes the 
latter to become neutral in eighteen hours. 
The neutralizing property is not due to a neurotoxic substance since 
animals stand very large doses of the serum without harm. 
All species of animals tried produce the specific antibodies, but not 
to an equal degree. Human beings and monkeys are said to have more 
antibodies after vaccination than rabbits. 
Centanni showed that immediately after vaccination the animal is 
not fully protected, though its serum may contain antirabic qualities, 
while later the animal is immune, though its serum may not be able 
to neutralize the rabies virus. These facts point to a cellular immunity. 
Results of Antirabic Treatment.-On the whole the results of pro- 
tective inoculations against rabies are marked. One has only to compare 
the statistics of mortality after bites from animals suffering from hydro- 
phobia with those given after any of the methods of treatment employed 
to see the benefit. As regards the best method to use, the case is dif- 
ferent. With many methods tried in many lands on a large number of 
cases, it would seem that we should be able by this time to determine 
their comparative worth. But the trouble is that the improvement 
on the whole is not great and the statistics are not kept uniformly 
or minutely enough to draw trustworthy comparisons. 
A slight decrease in mortality has been shown in the statistics from 
most of the antirabic institutes of the world. 590 
PATHOGENIC MICROORGANISMS 
But these figures tell us little about the actual value of the different 
methods. In order to be able better to judge, the statistics should uni- 
formly give many more details. Some institutes give such details, 
others do not. Until some such scheme as the following is carried out 
by all, we must change cautiously a treatment that has already given 
good results. 
1. Diagnosis of biting animal: 
(a) Rabies, (6) probably rabies, (c) questionable, (d) not 
rabies, (e) nothing known. 
2. Manner of making diagnosis: 
(a) By animal inoculation, (6) by microscopic examination, 
(c) by clinical diagnosis. 
3. Site and character of bites (e.g., number, depth, laceration, pro- 
tected by clothing, etc.): 
(a) Head, (6) hands, (c) other parts of body. 
4. Time elapsing between bite and beginning of treatment. 
5. Method of treatment used. 
6. Complications during or after treatment, particularly paralysis. 
7. Character and time of death. 
That the time after the bite makes a great difference is shown by the 
following table: 
Time intervening between bite and 
beginning treatment. 
Number of 
cases treated. 
Death. 
Percentages. 
Babes . . 1 to 2 days . 
3,406 
3 
0.088 
3 to 5 days . 
2,541 
2 
0.077 
5 to 6 days . 
809 
1 
0.124 
Diatroptoff . 1 week 
4,602 
26 
0.560 
2 weeks . 
961 
16 
1.660 
3 weeks . 
313 
10 
3.190 
Immunity.-The immunity in human beings produced by the anti- 
rabic treatment apparently lasts a variable time. That it may not last 
more than fourteen months is shown by the history of one of our cases. 
The patient was an assistant in a hospital for dogs. He was given eighteen 
days' treatment after a light wound in the hand from a rabid dog. Four- 
teen months later he came down with typical hydrophobia. Since his 
treatment he had become very careless with cases of rabies, exposing 
wounded hands to saliva because he considered himself immune. He 
was warned that there might be danger. Six weeks before his death he 
put a wounded hand into the mouth of a rabid animal. There is little 
doubt but that this is a case of reinfection after loss of protection from 
the treatment rather than one of delayed hydrophobia. 
Marie has found complete immunity in dogs eighteen months after 
treatment. 
Hl Effects of Treatment.-Local.-There is only slight local dis- 
comfort, increased a little if the emulsion contains glycerin. During 
the second week an erythema often appears about the point of inocula- 
tion, which Stimson regards as a manifestation of hypersusceptibility to 
foreign nerve tissue. It disappears in a few days. RABIES 
591 
Constitutional.-Ever since the beginning of treatment occasional 
non-fatal affections of the nervous system have been reported, which 
occurred during or shortly after the course of treatment. These have 
varied in degree all the way from a slight neuritis, through paraplegias 
to paralyses of various parts of the body. Very occasionally the paralyses 
are marked and the patient dies. Cases of true paralytic rabies which 
may occur within the period required for the establishment of im- 
munity by the treatment must be differentiated from cases occurring 
as a result of treatment. 
Simon published an extensive report of 84 cases occurring during the 
years 1888 to 1911 inclusive, and mentions a few others about which he 
could not get sufficient data. Simon classifies the cases collected by 
him according to the diagnosis of the biting animal, with the mortality 
in each group as follows: 
Positive 
group. 
Probable 
group. 
Questionable 
group. 
Negative 
group. 
Not known 
group. 
Number Per 
Number Per 
Number Per 
Number Per 
Number Per 
cases. cent. 
cases. cent. 
cases. cent. 
cases. cent. 
cases. cent. 
25 29.76 
(2) 
11 13.0 
(4) 
21 25 
(5) 
17 20.23 
(3) 
10 11.9 
(5) 
Nineteen deaths occurred, as seen from the figures in parentheses, 
or 22 per cent, of the 84 cases. 
In analyzing the effect of different methods of treatment on paralyses, 
Simon gives the following summary: 
Number of 
cases treated. 
Cases of 
paralysis. 
Proportion. 
Classic Pasteur method . 
32,676 
6 
1: 5446 
Modified Pasteur method 
8,657 
16 
1: 541 
Hogyes method 
51,417 
3 
1: 17139 
It is seen that the number of paralyses following the Hogyes method 
are markedly less than those following the other methods. 
From the studies so far made of these paralyses the possibility of 
there being different causes for different cases cannot yet be ruled out. 
The chief theories advanced as to factors in producing the condition 
are six: 
1. Due to "laboratory rabies" from the fixed virus vaccine inocu- 
lated. 
2. Due to "modified rabies" resulting from the treatment on the 
street virus infection. 
3. Due to a toxin produced by the rabies organisms. 
4. Due to infection with extraneous organisms introduced with 
the virus during treatment. 
5. Due to psychologic disorders. 
6. Due to the inoculation of a foreign proteid with a subsequent 
anaphylactic reaction. 
The Cauterization of Infected Wounds.-We believe that in cases 
in which the Pasteur treatment cannot be applied great benefit may be 
derived from the correct use of cauterization with fuming nitric acid, 592 
PATHOGENIC MICROORGANISMS 
even twenty-four hours after infection, and that even in cases in which 
the Pasteur treatment can be given, an early cauterization will be of 
great assistance as a routine practice and should be very valuable, as 
the Pasteur treatment is frequently delayed several days for obvious 
reasons, and then does not always protect. In the case of small wounds 
all the treatment probably indicated will be thorough cauterization 
with nitric acid within twelve hours from the time of infection. Our 
experience in dealing with those bitten by rabid animals goes to show 
that physicians do not appreciate the value of thorough cauterization 
of the infected wounds. 
Preventive Measures in Animals.-Far more important than any 
treatment, curative or preventive, for hydrophobia in man is the preven- 
tion of rabies in dogs, through which this disease is usually conveyed. 
Were all dogs under legislative control and the compulsory wearing 
of muzzles rigidly enforced for two years where rabies prevails, hydro- 
phobia would practically be stamped out. This fact has been amply 
demonstrated by the statistics of rabies in countries (e. g., England) 
where such laws are now in force. New York City is now trying to 
enforce such laws. 
YELLOW FEVER. 
Yellow fever is an acute infectious disease of tropical countries with 
no characteristic lesions except jaundice and hemorrhage. Other 
lesions that exist are those common to toxemia. One attack usually 
produces complete immunity. 
Historical Note.-There have been, many extensive studies on the etiology 
of this disease with numerous announcements of the discovery of its specific 
cause. Not one of the latter, however, has been corroborated. The Bacillus 
icteroides of Sanarelli (1897), found in the circulating blood and in the tissues 
of most yellow fever patients, was thought by many to be the real organism, 
and for some time it was the subject of most minute studies with the result 
that it, too, has been placed with the rejected organisms. 
The epoch-making investigations of the United States Army Commission 
composed of Walter Reed, James Carroll, Aristides Agramonte, and Jesse 
W. Lazear (1901), established the truth, that this disease, like malaria, is 
carried from one infected person to another through the agency of a mosquito. 
Finley in 1881 was the first positively to assert that the mosquito was the 
transmitter of the disease. He was, however, unable to prove his theory, and 
it remained for the commission conclusively to show that a distinct species 
of mosquito carried the infection. 
The work of the American commission was fully corroborated by the French 
commission and by other workers. 
The principal facts established by the commission have been summed 
up by Goldberger as follows: 
1. Yellow fever is transmitted, under natural conditions, only by 
the bite of a mosquito (Aedes calopus) that at least twelve days before 
had fed on the blood of a person sick with this disease during the first 
three days of his illness. YELLOW FEVER 
593 
2. Yellow fever can be produced under artificial conditions by the 
subcutaneous injection of blood taken from the general circulation 
of a person sick with this disease during the first three days of his 
illness. 
3. Yellow fever is not conveyed by fomites. 
4. Bacillus icteroides Sanarelli stands in no causative relation to 
yellow fever. 
Though the specific parasite remains yet undiscovered, facts have 
been brought out by these studies which give some idea of its character. 
1. It seems to require two hosts (a mammal and an arthropod) 
for the completion of its life cycle (analogies, Plasmodium malaria, 
Babesia bigeminum). 
2. There is a definite time after the bite of the mosquito before the 
blood of the person bitten becomes infective (average five days), and 
a definite time that the blood remains infective (three days). 
3. The blood during these three days is still infective after passing 
through the finest-grained porcelain filters (Chamberland B and F). 
4. The blood loses its virulence quickly (forty-eight hours) when 
exposed to the air at a temperature of 24° to 30° C. When protected 
from the air by oil and kept at the same temperature it remained 
virulent longer (five to eight days). Heated for five minutes at 55° C. 
it becomes non-virulent. 
5. The bite of an infected mosquito does not become infectious 
until twelve days (at a temperature of 31° C.) after it has bitten the 
first patient. 
The cause of the disease still remains undiscovered, notwithstanding 
much study of human blood and other tissues and of infected mos- 
quitoes. The infective blood-filtrates show nothing with the dark- 
field illumination except small motile granules similar to those found 
in healthy persons. 
Certain facts relating to the disease seem to point to protozoa as 
the cause; for instance, the necessity for a second host and the long 
incubation time required before that host becomes infective after 
biting a yellow-fever patient. 
Seidelm's paraplasma has been shown by Agramonte to be tissue 
changes. 
The higher monkeys seem to be susceptible, though no complete 
experiments have been made with them. 
The Yellow-fever Mosquito (Figs. 202-207).-The name Stegomyia for 
this small tropical mosquito was suggested by the English entomologist 
Theobald, who separated this genus from the genus Culex, with which 
it was formerly classed. It was first given the specific name Fasciata, 
but Blanchard proved that this had already been used and the name 
Calopus (Meigen, 1818) was found to be the proper one. Later the 
genus Stegomyia was shown to be invalid and the organism now goes 
by the name Aedes calopus Meigen. Salient characteristics of Aedes 
are: (1) The palpi in the male are as long or nearly as long, as the 
proboscis; in the female the palpi are uniformly less than one-half 594 
PATHOGENIC MICROORGANISMS 
as long; (2) the legs are destitute of erect scales; and are alternately 
banded white and black; (3) the thorax is marked with lines of silvery 
scales. Aedes calopus is spread over a wide range of territory, embrac- 
ing many varieties of climate and natural conditions. It has been 
found as far north as Charleston, S. C., and as far south as Rio de la 
Plata. There is no reason to believe that it may not be present at some 
time or other in any of the intermediate countries. In the United 
States specimens of Aedes calopus have been captured in Georgia, 
Fig. 202 
The yellow-fever mosquito (Aedes calopus). Adult female. Much enlarged. 
(Howard.) 
Louisiana, South Carolina, and eastern Texas. The island of Cuba is 
overrun with this insect. The fact that Aedes calopus has been known 
to exist at various times in Spain and other European countries may 
account for the spread of yellow fever which has occurred there once 
or twice in former times; the same may be said of the country farther 
north in the United States, where Aedes calopus has not yet been 
reported, but which have suffered from invasions of yellow fever. 
The adult mosquito is so small that mosquito netting of 19 or YELLOW FEVER 
595 
Fig. 203 
The yellow-fever mosquito. Adult male. Much enlarged. (Howard.) 
Fig. 204 
The yellow-fever mosquito. Adult female, side view. Much enlarged. (Howard.) 
Fig. 205 
The yellow-fever mosquito. Egg. Greatly enlarged. (Howard.) 596 
PATHOGENIC MICROORGANISMS 
20 strands or meshes to the inch is required to keep the insect from 
entering a place. 
Brackish water is unsuited for the development of Aedes larvae. 
The species Aedes calopus seems to select any deposit of water which 
is comparatively clean. The defective drains along the eaves of tile 
roofs are a favorite breeding place in Havana and its suburbs; indoors 
they find an excellent medium in the water of cups of tin or china 
into which the legs of tables are usually thrust to protect the contents 
from the invasion of ants, a veritable pest in tropical countries. The 
same may be said of shallow traps, where the water is not frequently 
disturbed. 
Fig. 206 
Fig. 207 
The yellow-fever mosquito. Larva. 
Much enlarged. (Howard.) 
The yellow-fever mosquito. Pupa. 
Much enlarged. (Howard.) 
Like other Culicid(E, it prefers to lay at night. It is eminently a 
town insect, seldom breeding far outside of the city limits. Agramonte 
never found Aedes calopus resting under bushes, in open fields, or in 
the woods; this fact explains the well-founded opinion that yellow 
fever is a domiciliary infection. 
The question of hibernation in the larval stage is important. Agra- 
monte failed to get larvee that could resist freezing temperature, and YELLOW FEVER 
597 
found that in the case of Aedes calopus this degree of cold was invariably 
fatal. 
The possibility of their being capable of life outside their natural 
element must also be considered from an epidemiologic point of 
view. The dry season in the countries where this species seems to 
abound is never so prolonged as completely to dry up the usual breed- 
ing places. Experimentally, adult larvae removed from the water and 
placed overnight upon moist filter paper could not be revived the 
following morning. 
The question of the life period of the female insect is of the greatest 
importance when we come to consider the apparently long interval 
which at times has occurred between the stamping out of an epidemic 
of yellow fever and its new outbreak without introduction of new cases. 
The fact is that Aedes calopus is a long-lived insect; one individual 
was kept by Agramonte in a jar through March and April into May, 
in all for seventy-six days after hatching in the laboratory. 
These mosquitoes bite principally in the late afternoon, though 
they may be incited to take blood at any hour of the day. They are 
abundant from March to September, and even in November Agra- 
monte was able to capture them at will in his office and laboratory. 
The mosquito is generally believed to be incapable of long flight 
unless very materially assisted by the wind. At any rate, the close 
study of the spread of infection of yellow fever shows that the tendency 
is for it to remain restricted within very limited areas, and that when- 
ever it has travelled far beyond this, the means afforded (railway 
cars, vessels, etc.) have been other than the natural flight of the insect. 
Experiments have demonstrated that not all mosquitoes which 
bite a yellow-fever patient become infected, but that of several which 
bite at the same time some may fail either to get the parasite or to 
allow its later development in their body. This condition is similar 
to that seen in Anopheles, with regard to malaria. 
The question as to the length of time infected mosquitoes remain 
dangerous to the non-immune community cannot be definitely answered 
at present; there is good presumptive evidence that the mosquito may 
harbor the parasite through the winter and be enabled to transmit in 
the spring an infection acquired in the fall. There is reason to believe 
that the mosquito, once infected, can transmit the disease at any 
time during the balance of its life. Freezing temperature, however, 
quickly kills the insect. 
Carrying out preventive measures based on the knowledge gained by 
the splendid work of the American Army Commission, yellow fever 
has been practically wiped out of Cuba, the Isthmian Canal Zone, and 
other infected areas. 
References. 
Berry. The Complement Binding Test in Rabies. Jour. Exp., Med., 1910, vol. xii. 
Harris. A Method for the Staining of Negri Bodies. Jour, of Infect. Diseases, 1908, 
v, 566; also Treatment of Rabies. 
Hogyes, Lyssa. Nothnagel's Specielle Pathologie u. Therapie, Wien, 1897. 598 
PATHOGENIC MICROORGANISMS 
Howard. The Yellow Fever Mosquito, Farmers' Bulletin, 547, United States Depart- 
ment Agriculture, Washington, D. C. 
Kerr and Stimson. The Prevalence of Rabies in the United States. The Jour, of the 
Amer. Med. Assoc., 1909, liii, 989. 
Kraus and Barbara. Deut. med. Woch., 1914, xl, 1507. 
Marie. L'Etude experimentale de la Rage, Paris, 1909. 
Manouelian. Ann. Inst. Past., 1914, xxviii, 233. 
Noguchi. Jour. Exp. Med., 1913, xviii, 314. 
Otto. Gelbfieber. In Kolle and Wassermann's Handbuch d. path. Mikrobrg, 1913, 
2d ed., Jena. 
Poor and Steinhardt. Jour. Inf. Dis., 1913, xiii, 203. 
Reed and Carroll. Jour. Exp. Med., 1900, v, 215. 
Reed and Carroll and Agramonte. Jour. Amer. Med. Assoc., 1901, xxxvi, 413. 
The Yellow Fever Institute Bulletin, No. 16, Yellow Fever, Etiology, Symptoms, and 
Diagnosis, by Goldberger, gives a good resume with full literature to 1907. 
Volpino. Presse Med., 1914, p. 79. 
Williams. Rabeis, in Forscheimer's Therapeusis, 1914, vol. v, Sec. Ed., New York. 
With references. 
Williams and Lowden. Jour, of Infect. Dis., 1906, iii, 460, with full list of references 
to date on Negri bodies. PART III. 
APPLIED MICROBIOLOGY. 
CHAPTER XLVT. 
THE BACTERIOLOGICAL EXAMINATION OF WATER, AIR, 
AND SOIL. THE CONTAMINATION AND PURIFICA- 
TION OF WATER. THE DISPOSAL OF SEWAGE. 
The bacteriological examination of water is undertaken for the 
purpose of discovering whether any pathogenic bacteria are liable 
to be present. The determination of the number of bacteria in water 
was for a tune considered of great importance, then it fell into disrepute, 
and the attempt was made to isolate the spepific germs of diseases 
which were thought to be water-borne. At first these attempts seemed 
very successful in that supposed typhoid bacilli and cholera spirilla 
were found. Further study revealed the fact that there were common 
water and intestinal bacteria which were so closely allied to the above 
forms that the tests applied did not separate them. Even the use of a 
serum from an animal immunized to injections of the typhoid bacillus 
was found to agglutinate some other bacteria in high dilutions; so that 
the test as usually carried out was insufficient. With the latest tech- 
nique it is probable, but not certain, that absorption tests with the 
serum from an immunized animal will be sufficient to decide whether a 
suspected bacillus is the typhoid bacillus or not. The improbability 
of getting typhoid bacilli from suspected water except under unusually 
favorable conditions caused a return to the estimation of the number 
of bacteria in water and above all to the estimation of the number of 
intestinal bacteria. It is known that the group of colon bacilli have 
a somewhat longer existence than the typhoid bacilli, and as the colon 
bacilli come chiefly or wholly from the intestinal passages of men and 
animals, it was fair to assume that typhoid bacilli could not occur 
without the presence of the colon bacillus except in rare cases, as, for 
example, pollution with urine alone. The latter could, of course, 
occur abundantly without the typhoid bacillus. 
During the past few years the attention of sanitarians has been 
seriously devoted to the interpretation of the presence of smaller or 
larger numbers of colon bacilli in water, until at present, upon the 
quantitative analysis (measuring, within certain limits, decomposing 600 
APPLIED MICROBIOLOGY 
organic matter) and the colon test (indicating more specifically pollution 
derived from intestinal discharges of man or animals), the bacteriological 
analysis of water is based. The determination of the number of bacteria 
is also of value. 
Technique for Quantitative Analysis.-The utmost care is necessary 
to get reliable results. A speck of dust, a contaminated dish, a delay 
of a few hours, an improperly sterilized agar or gelatin, a too high or 
too low temperature, may introduce an error or variation in results 
which would make a reliable test impossible. 
Collection of Samples.-The small sample taken must represent the 
whole from which it was drawn. If a brook-water, it must be taken 
some distance from the bank; if from a tap, the water in the pipes 
must first be run off, for otherwise the effect of metallic substances 
will invalidate the results; if from lake or pond, the surface scum 
or bottom mud must be avoided, but may be examined separately. 
The utensils by which the water is taken should be of a good quality 
of glass, clean and sterile. From a brook the water can be taken 
directly into a bottle, the stopper being removed while it fills, avoiding 
the surface film and its attending excessive numbers of bacteria; from 
a river or pond it can be taken from the bow of a small boat, or from 
a bottle properly fastened on the end of a pole so as to avoid con- 
tamination; from a well a special apparatus has been devised by Abbott, 
where a bottle with a leaded bottom is so held that when lowered to 
the proper depth a jerk will remove the cork and allow the bottle to 
fill. The same device or another accomplishing the same purpose 
can be rigged up readily by anyone. The sample of water should 
be tested as soon as possible, for the bacteria immediately begin to 
increase or decrease. In small bottles removed from the light preda- 
tory microorganisms and many bacteria begin to increase, and among 
these are the members of the colon group. Thus, the Franklands 
record a case in which in a sample of well-water kept during three days 
at a moderate temperature the bacteria increased from 7 to 495,000; 
while Jordan found that in a sample the bacteria in forty-eight hours 
fell from 535,000 to 54,500. In a sample we kept at room temperature 
the colon bacilli during twenty-four hours increased from 10 to 100 
per c.c. The only safe way to prevent this increase is to plate and 
plant the water in fermentation tubes within a space of one or two 
hours or to keep it at a temperature under 5° C. (41° F.). If it cannot 
be kept cold, it is far better to make the cultures in the open field or in 
a house rather than to wait six to twelve hours for the conveniences and 
advantages of the laboratory. 
The third matter of great importance is the adding of proper amounts 
of water to the broth in the fermentation tubes and the media for plant- 
ing. Usually 1 c.c., 0.1 c.c., and 0.01 c.c. are added to the fermenta- 
tion tubes and to 10 c.c. of the melted nutrient agar or gelatin. If 
possible, duplicate tests should always be made. When it is desired to 
know whether colon bacilli are present in larger amounts than 1 c.c., 
quantities as great as 10 or 100 c.c. can be added to bouillon, and then TECHNIQUE FOR QUANTITATIVE ANALYSIS 
601 
after a few hours 1 c.c. added to fermentation tubes. Less than twenty 
colonies and more than two hundred on a plate give inaccurate counts, 
the smaller number being too few to judge an average and the larger 
number interfering with each other. When as many as 10,000 colonies 
develop in the agar contained in one plate, it will be found that there 
will develop in a second plate containing but one-tenth the amount of 
waiter from 20 to 50 per cent, as many colonies. This shows that the 
crowding of the colonies had prevented the growth or caused a fusion 
of all but one-fifth to one-half of them. 
The chemical composition of the medium on which the bacteria are 
grown affects the result of the analysis. Nutrient 1.5 per cent, agar 
gives slightly lower counts than gelatin, but on account of its con- 
venience in summer and its greater uniformity it is being more and 
more generally used for routine quantitative work. There is an 
optimum reaction for every variety of bacteria, and to ensure uniformity 
the committee of the American Public Health Association adopted a 
standard reaction of +1 per cent.,1 which was as near as possible 
to the average optimum for water bacteria. Such a uniform standard 
is a necessity to secure comparability of results. At best only a certain 
proportion of bacteria develop, and it is only important that our counts 
represent a section through the true bacterial flora which fairly represents 
the quick-growing sewage forms. Comparability is the vitally essential 
factor. 
The temperature at which the bacteria develop is of great importance, 
and they should be protected from light. The access of oxygen which 
prevents the growths of anaerobes must also not be forgotten. As a 
rule, the plate cultures are developed for four days at 20° to 21° C., 
and for twenty-four or forty-eight hours at incubator temperature. 
Some bacteria do not develop colonies in four days, but these are 
neglected. The number of bacteria growing at room temperature is 
usually much greater than those growing at 37°. As all the intestinal 
groups of bacteria grow at body temperature, while many of the water 
types do not, some investigators believe it important to develop the 
bacteria at both temperatures so as to compare the results. We have 
not found this to be of any advantage when tests are also made for the 
colon group of bacilli. 
The lactose broth is placed at 37° C. for the development of the 
colon bacilli. The fermentation tubes not showing gas are recorded 
as negative and usually discarded. Those showing gas are suspected 
to contain colon bacilli. To a number of tubes containing melted 
litmus-lactose agar at about 44° C. are added 1, 0.1, and 0.01 loop 
of the culture fluid. Plates are poured and the whole placed in the 
incubator. The Bacillus coli ferments lactose and thus produces 
acid, so that if colon bacilli are present we have a number of red colonies 
on a blue field. Later, if many colon bacilli were present, the whole 
medium becomes acid. At forty-eight hours, on account of alkali 
being produced by the formation of NH3, the blue may return. If 
1 To phenolphthalein. 602 
APPLIED MICROBIOLOGY 
after inspection red colonies are seen, four or five are picked and planted 
into lactose bouillon and other media. Litmus lactose agar is frequently 
used for the original plating of water samples, the absence or presence 
of acid-producing colonies being thus immediately noted. The results 
of these tests are all more or less presumptive evidence of the bacilli 
belonging or not belonging to the intestinal colon bacillus group. Un- 
fortunately there are some types of bacilli growing in the soil which 
resemble them, if it is necessary to be more accurate. The colon-like 
cultures should be subjected to the Vosges reaction (page 120), and 
should be kept for one month at 20° C. in gelatin before a decision 
is made. Colon bacilli do not liquefy gelatin nor give the Vosges re- 
action. There are a few colon-like bacilli in the intestinal tract that give 
the Vosges reaction. For a more complete understanding of the technique 
and the interpretation of results of the bacteriological examination of 
water see Elements of Water Bacteriology-Prescott and Winslow. 
For the characteristics of the colon bacilli the Massachusetts State 
Board of Health uses six media-gelatin, lactose agar, dextrose broth, 
milk, nitrate solution, and peptone solution, determining, respectively, 
absence of liquefaction, production of gas, turbidity, coagulation with- 
out liquefaction of the coagulum, production of nitrite, and of indol. 
Lactose-bile-peptone solution has been much used. In badly con- 
taminated waters this has a distinct advantage in that the bile inhibits 
many varieties of bacteria more than those of the colon-typhoid group. 
In good waters the results are very similar from the lactose-peptone 
and lactose-bile-peptone solutions. 
Significance of the Colon Bacillus.-The colon test has been received 
by the majority of engineers and practical sanitarians with great satis- 
faction, and has been applied with confidence to the examination 
not only of water, but of shell-fish and other articles of food as well. 
On the other hand, some have denied its value. Bacteriologists have 
found bacilli like certain members of the colon group in apparently 
unpolluted well-water. The discovery that animals have colon bacilli 
identical in the usual characteristics studied with those of man has 
complicated matters. Thus a fresh hillside stream may be loaded with 
colon bacilli from the washings of horse or cow manure put on the 
fields through which it runs or polluted by a stray cow or horse. Swine, 
hens, birds, etc., may contaminate in unsuspected ways. The number 
of colon bacilli rather than their presence in any body of surface water 
is therefore of importance. In well- and spring-water the presence 
of colon bacilli indicates contamination. The absence of colon bacilli 
in water proves its harmlessness so far as bacteriology can prove it. 
When the colon bacillus is present so as to be isolated from 1 c.c. of 
water in a series of tests, it is reasonable proof of animal or human 
pollution and the conditions should be investigated. Ten colon bacilli 
in 1 c.c. indicates serious pollution. Surface waters from inhabited 
regions will always contain numerous colon bacilli after a heavy rain, 
storm, or shower. The washings from roads and cultivated fields con- 
tain necessarily large numbers. Winslow reports that in only two INTERPRETATION OF THE QUANTITATIVE ANALYSIS 
603 
out of fifty-eight samples of presumably non-polluted waters did he 
get colon bacilli in the 1 c.c. samples. Even in twenty-one stagnant 
pools he only found colon bacilli in five of the 1 c.c. samples. 
The experience of all who have studied the subject practically is 
that in delicacy the colon test surpasses chemical analysis: in con- 
stancy and definiteness it also excels the quantitative bacterial count. 
All these tests must, however, be supplemented by inspection. 
Interpretation of the Quantitative Analysis.-The older experi- 
menters attempted to establish arbitrary standards by which the 
sanitary quality of water could be fixed automatically by the number 
of germs alone. This has been largely given up. Dr. Sternberg con- 
siders that a water containing less than 100 bacteria is presumably 
from a deep source and uncontaminated by surface drainage; that 
one with 500 bacteria is open to suspicion; and that one with over 
1000 bacteria is presumably contaminated by sewage or surface drain- 
age. Even this conservative opinion must be applied with caution. 
The source of the sample is of vital importance in the interpretation; 
thus, a bacterial count which would condemn a spring or well might 
be normal for a river. In woodland springs and lakes several hundred 
bacteria per c.c. are frequently found. In lakes the point at which 
the sample is taken is of great importance, as the bacterial count varies 
with the distance from the shore and with the depth. The weather 
also is an influence, since the wind causes currents and waves which 
stir up the bottom mud, bringing up organisms which have been 
sedimented. Rains greatly influence streams by flooding them with 
surface water bringing a huge number of bacteria at times. The season 
of the year is an important factor. The counts are highest in the 
winter and spring months, and lower from April to September. 
The following figures illustrate this point: 
Water. 
Observers. 
Year. 
Jan. 
Feb. 
Mar. 
April. 
May. 
June. 
New York City tap-water 
Houghton 
1904 
890 
1100 
650 
240 
350 
370 
Boston tap-water . 
Whipple 
1892 
135 
211 
102 
52 
53 
86 
Merrimac River tap-water 
Clark 
1899 
4900 
5900 
6300 
2900 
1900 
3500 
The winter and spring increases are not exceptions to the rule that 
high numbers indicate danger, but an indication of its truth; for it 
means a melting of the snow and a flow of surface water into the streams 
without the usual filtering soil filtration. A number of severe epidemics 
of typhoid fever have been produced in this way. It is only the fact 
that typhoid fever is at a minimum in winter that prevents more frequent 
pollution. Although, as a rule, a series of tests are necessary to pass 
judgment on a water, a single test may be very important. A large 
increase in the number in tap-water a day after a storm points unerringly 
to surface pollution, and if towns exist in the water-shed, to street and 
sewer pollution. The Croton water frequently jumps from hundreds 
to thousands after such a storm. 
In a typhoid epidemic at Newport, Winslow reports that a test 
of the water supply showed but 334 bacteria per cubic centimeter, 604 
APPLIED MICROBIOLOGY 
but one from a well showed 6100. The suspicion aroused was justified 
by finding all the typhoid cases had gotten water from this well. 
The study of the bacterial effluent from municipal water filters 
is the only way in which the efficiency of the filter and the accidents 
which occur can be determined. In Germany these regular tests are 
obligatory. The filter should remove about 99 per cent, of the bacteria. 
Elaborate studies have recently been made of the exact distribution 
of streams of sewage in bodies of water into which they flow, their 
disappearance by dilution and sedimentation, and their removal by 
death. Under peculiar conditions bacteria in water may increase for 
a time, but here the prevailing bacteria belong almost exclusively to 
one type. 
Streptococci in Sewage.-The varieties of streptococci found most 
often in polluted water correspond to the streptococci described by 
Houston. In some water in which these are found no B. coli have 
been found and there is considerable doubt in such cases as to whether 
the streptococci imply serious pollution. The streptococci remain 
alive longer than the colon bacilli. In England the examination for 
streptococci in water is much more regularly done than in America. 
Other Bacteria.-Most of the bacteria which develop in the intes- 
tines of man and animals necessarily occur in polluted water, and an 
examination for some of these has been advocated by many, such as 
the B. enteritidis sporogenes, other anaerobic spore formers, the various 
members of the typhoid-colon group, and the proteus group. 
Isolation of the Typhoid Bacillus from Water.-If it were possible 
to readily obtain the typhoid bacilli from water, when they were present 
in small numbers, its examination for that purpose would be of much 
greater value than it is now; but we have to remember that we can 
only examine at one time a few cubic centimeters of water by bacterio- 
logical methods, and that although the typhoid bacilli may be sufficiently 
abundant in the water to give, in the quantity that we ordinarily 
drink, a few bacilli, yet it must be a very lucky chance if they happen 
to be in the small amount which we examine. Still, further, although 
it is very easy to isolate typhoid bacilli from water when they are in 
considerable numbers, yet when they are a very minute proportion 
of all the bacteria present it is almost impossible not to overlook them. 
Many attempts have been made to devise some method by which the 
relative number of the typhoid and other parasitic bacteria present in 
water could be increased at the expense of the saprophytic bacteria. 
Thus, to 100 c.c. of water 25 c.c. of a 4 per cent, peptone nutrient 
bouillon is added, and the whole put in the incubator at 37° C. for 
twenty-four hours. From this, plate cultures are made. As a matter 
of fact, the typhoid bacillus is rarely found, even in specimens of water 
where we actually know that it is or has been present because of cases 
of typhoid fever which have developed from drinking the water. From 
these facts we must consider our lack of finding the bacillus in any 
given cases as absolutely no reason for considering the water to be 
free from danger. Another serious drawback to the value of the ex- CONTAMINATION AND PURIFICATION OF DRINKING WATERS 
605 
aminations for the typhoid bacillus is that they are frequently made at 
a time when the water is really free from contamination, though both 
earlier and later the bacillus was present. It is hardly worth while, 
therefore, except in careful experimental researches, to examine the 
water for the typhoid bacillus, but rather study the location of the 
surrounding privies and sources of contamination. A number of ob- 
servers, resting on the agglutination test, have thought they have 
isolated typhoid bacilli from the soil and water, but these investigators 
had not considered sufficiently the matter of group agglutinins, and 
their results are not trustworthy. 
CONTAMINATION AND PURIFICATION OF DRINKING WATERS. 
Brook-water and river-water are contaminated in two ways: through 
chemicals, the waste products of manufacturing establishments, and 
through harmful bacteria by the contents of drains, sewers, etc., the 
latter method being far the more dangerous. 
When water, which has been soiled by waste products of manu- 
factories only, becomes so diluted or purified that the contamination 
is not noticeable to the senses and shows no dangerous products on 
chemical analysis, it is probably safe to drink. When sewage is the 
contamination, this rule no longer holds, and there may be no chemical 
impurities and no pathogenic bacteria found, and yet disease be pro- 
duced. That river-water which has been fouled by sewage will, by 
oxidization, dilution, sedimentation, action of sunlight, and predatory 
microorganisms, become greatly purified is an indisputable fact. The 
increase in bacteria which occurs from contamination is also largely 
or entirely lost after ten to twenty miles of river flow. Nevertheless, 
the history of many epidemics seems to show that a badly contaminated 
river is never an absolutely safe water to drink, although with the lapse 
of each day it becomes less and less dangerous, nor will sand filter-beds 
absolutely remove all danger. These statements are founded upon the 
results of numerous investigations; thus the marked disappearance of 
bacteria is illustrated by the following: Kummel found below the town 
of Rosbock 48,000 bacteria to the cubic centimeter; twenty-five kilo- 
meters farther down the stream only 200 were present-about the same 
number as before the sewage of Rosbock entered. On the other hand, 
the doubtful security of depending on a river purification is proved 
by such experiences as the following: In the city of Lowell, Massa- 
chusetts, an alarming epidemic followed the pollution of the Merrimac 
River three miles above by typhoid feces, and six weeks later an alarming 
epidemic attacked Lawrence, nine miles below Lowell. It is estimated 
that the water took ten days to pass from Lowell to Lawrence and 
through the reserviors. Typhoid bacilli usually die in river-water in 
from three to ten days, but they may live for twenty-five days in other 
water; the Lawrence epidemic is easily explained. Newark-on-Trent, 
England, averaged seventy-five cases a year from moderately well- 
filtered water and only ten when it was changed to deep-well supply. 606 
APPLIED MICROBIOLOGY 
Purification of Water on a Large Scale.-For detailed information 
on this subject the reader is referred to works on hygiene. Surface 
waters, if collected and held in sufficiently large lakes or reservoirs, 
usually become so clarified by sedimentation, except shortly after 
heavy rains, as to require no further treatment so far as its appearance 
goes. The collection of water in large reservoirs allows not only the 
living and dead matter to subside, but allows time also for the patho- 
genic germs to perish through light and antagonistic bacteria and other 
deleterious influences, as sand, or mechanical coagulant. Filtration of 
water exerts a very marked purification, taking out 99.8 per cent, of 
the organisms in those best constructed and at least 95 per cent, in 
those commonly used in cities. The construction of filters is too large 
a subject to enter on minutely here; sand filters consist, as a rule, of 
several layers, beginning with fine sand, and then smaller and larger 
gravel, and finally rough stones. A certain time elapses before the 
best results are obtained; this seems to wait for the formation of a 
film of organic material on the sand, which is full of nitrifying bacteria. 
Even the best filters only greatly diminish the dangers of polluted 
water. Spring-water and well-water are, in fact, filtered waters. 
Water which is subject to serious pollution must be submitted to 
a preliminary purification before it can be considered a suitable source 
for a drinking-water supply. The means employed for its purification 
depend to a large extent upon the character of the water and the nature 
of the pollution. Filtration through slow sand filters, three to five 
feet in depth, removes 98 to 99.5 per cent, of the bacteria and organic 
matter; so that effluents from the best constructed sand filtration 
beds constitute safe and reliable drinking waters. Five hundred 
thousand to one or two million gallons, depending somewhat upon the 
extent of pollution and the fineness of the sand, can be filtered daily 
per acre. Only the surface of the sand filter becomes in any way clogged 
and as thin a layer as can be scraped off is removed one or more times a 
month. This surface sand is washed with clean water and several scrap- 
ings replaced at one time. Sand filtration beds are very widely used 
abroad and are coming into extensive use in this country. The filter- 
beds at Lawrence, Mass., have been used over ten years with marked 
success; when properly managed, they render the highly polluted 
Merrimac River a fairly safe drinking water; the filter-beds there 
are scraped about thirteen times a year. 
Mechanical filtration plants find considerable favor where clarifica- 
tion as well as bacterial purification is desired. A coagulant such as 
sulphate of aluminum is employed and forms in the water a flocculent 
precipitate which carries down with it all suspended matter; 125,000,000 
or more gallons of water can be filtered on an acre daily, but the filters 
must be washed daily by reversing the flow and cleansing the clogged 
filter with a stream of the purified water. Chlorinated lime when 
added to drinking water to the extent of one-eighth to one-twelfth of 
a grain per gallon will destroy all intestinal bacteria of the typhoid- 
colon group within a few hours. This is a very useful means of puri- 
fication for emergencies. It does not injure the water. THE DISPOSAL OF SEWAGE 
607 
Under special conditions other methods, such as the passage of 
ozone, have proved successful. 
Domestic Purification.-Water which requires private filtering should 
not be supplied for drinking purposes. Unhappily, however, it often is. 
Domestic filters may be divided, roughly, into those for high and low 
pressure. The former are directly connected with the water main, 
while the others simply have the slight pressure of the column of water 
standing in the filter. Many household filters contain animal charcoal, 
silicated carbon, etc., either in a pressed condition or in one porous 
mass. These filters remove much of the deleterious matter from the 
suspected waters, but the majority cannot be depended upon to remove 
all bacteria. Even those which are equipped for self-cleansing become 
in a little while foul, and, if not cleaned, unfit for use. The best of the 
filters are of porous stone, such as the Berkefeld and Pasteur filters. 
These yield a water, if too great a pressure is not used, almost absolutely 
free from bacteria, and if they are frequently cleansed they are reliable. 
A large Berkefeld filter will allow sixty gallons of water to pass per 
hour. The Pasteur filter is more compact and slower. From the best 
Pasteur filters sterile water may be passed for two to three weeks; 
from the Berkefeld usually only a few days. A single typical low- 
pressure filter is that of Bailey Denton. The upper compartment 
contains the filtering material, which may be sapd or charcoal, and is 
fed from a cistern or hydrant. After a certain quantity of water has 
passed in, the supply is automatically cut off until the whole amount 
is filtered. A fairly efficient filter is the following: Take a large-sized 
earthenware pot and plug the hole in the bottom with a cork, through 
which pass a short glass tube. Upon the bottom place an inch of small 
pieces of broken flower-pot; upon this a couple of inches of well-washed 
small gravel, and upon this six to twelve inches of well-washed, fine, 
sharp sand. Cover the sand with a piece of filter-paper and hold this 
down with a few small stones. Mount the pot on a tripod, and it is 
ready for use. The paper prevents the sand being disturbed when water 
is added, and as it also holds most of the sediment, this can be readily 
removed. Every few months the sand can be washed and replaced. 
Animal charcoal is not a good substance for permanent filters, as 
bacteria grow wrell in it. Whenever water is suspected, and there 
is any doubt as to the filters, it should be boiled for ten minutes; this 
will destroy all bacteria. This precaution should always be taken 
in the presence of typhoid fever and cholera epidemics. 
THE DISPOSAL OF SEWAGE. 
The disposal of sewage is becoming a vital question with all towns 
and cities which are not situated near salt-water outlets, since the 
present tendency in legislation is to compel such towns to dispose of 
their waste so that it shall not be a menace to drinking-water streams, 
destructive to fisheries, or a nuisance to harbors. 608 
APPLIED MICROBIOLOGY 
Methods of sewage purification depend upon the character of the 
sewage and the kind of effluent desired. 
Two hundred thousand gallons of crude sewage may be filtered 
upon an acre of land daily and an effluent obtained which will com- 
pare favorably in every way known to the chemist and bacteriologist 
with the best mountain springs. This is, however, a slow process, 
and it is rare that such a pure effluent is required. Similar results 
may be obtained by utilizing the septic-tank method, running the 
sewage from the septic tank to contact beds and thence to sand filter- 
beds; where, because of the partial " self-purification of the sewage'" 
in the septic tank and contact beds, 2,500,000 gallons of sewage can 
be filtered daily on an acre of surface. In this process less land is 
required and both these effluents can be safely turned into drinking- 
water streams. 
If, however, a merely non-putrescible effluent is required, one which, 
though high bacterially, will not be offensive in any way, or subject 
to further decomposition, it may be obtained by passing crude sewage 
to septic tanks, thence to double contact beds, the resulting effluent 
having merely, an earthy, humus-like odor and being non-putrescible. 
Where acid wastes, tannery wastes, dyestuffs, etc., from various 
factories enter into sewage, its disposal becomes a more complicated 
problem and chemical .precipitation by the use of lime or other chemicals 
is generally employed for such sewage purification, which at best is 
only partial and is sometimes supplemented by sand filtration. 
Sea-water.-This is only feebly bactericidal. The salty tidal waters 
of rivers allow tyhpoid bacilli and other members of the typhoid-colon 
group to live for a number of days. 
Saprophytic bacteria are always present in considerable numbers 
in the air except far out at sea or on high mountains. They reach the 
air from the earth's surface and are most plentiful nearest to it. They 
are more abundant where organic matter abounds and in dry and windy 
weather. 
The air is kept constantly in motion by winds so that fine particles 
are constantly being carried into it from the ground, especially in an 
inhabited area with its dusty streets. The rays of sunlight visibly 
reveal these particles to us. The bacteria in the dust of the fields and 
streets are carried along with these dust particles. They are usually 
the harmless soil bacteria or the almost equally harmless intestinal 
bacteria of animals. Pathogenic human bacteria happen rarely to be 
carried in harmful numbers except under exceptional circumstances and 
usually as spores, such as those of anthrax bacilli from the dust from 
the wool and hides of infected animals or of tetanus bacilli from the 
infected manure. After a storm few bacteria are in the air, while on 
a dry windy day many thousands exist in a cubic meter. In warm 
weather rain carries down the bacteria of the air. The bacteria in the 
BACTERIA IN AIR-EXAMINATION OF AIR. BACTERIOLOGICAL EXAMINATION OF THE SOIL 
609 
air of the country are much less than in the city air. Forests decrease 
the number of bacteria. 
On high mountains and on the sea far from land bacteria are very 
scarce. The bacteria that multiply in the soil of street and country 
are almost entirely saprophytic types. Sunlight and drying rapidly 
destroy bacteria. In dwellings the bacterial content depends on many 
factors, of which the chief are the opening of windows to the outside 
dust-laden air, the cleanliness of the dwelling, and the amount of 
stirring up of the dust by sweeping. It is almost impossible to separate 
the effect of the bacteria which we inhale from that of the dust particles 
which they accompany. Both probably act as slight irritants and so 
predispose to definite infections. 
Pathogenic bacteria are only occasionally present in the air. The 
practical results obtained from the examination of air for pathogenic 
bacteria have been slight. We know that at times they must be in the 
air, but unless we purposely increase their numbers they are so few in 
the comparatively small amount of air which it is practicable to examine 
that we rarely find them. Examination of dust, however, in hospital 
wards and sick-rooms, in places where only air infection was possible, 
have occasionally revealed tubercle bacilli and other pathogenic bacteria. 
Although very light they generally settle to the ground. The importance 
of air infection in the spread of infectious diseases is considered as much 
less than it was formerly thought. 
The simplest method of searching for the varieties of bacteria in 
the air and their number in any place is to expose to the air for longer 
or shorter periods nutrient agar spread upon the surface of the Petri 
dish. After exposure the plates are either put in the incubator at 37° 
C. or kept at room temperature. The more careful quantitative 
examination is made by drawing a given quantity of air through tubes 
containing sterile sand, which is kept in by pieces of metal gauze. 
When the operation is completed the sand is poured into a tube con- 
taining melted nutrient gelatin or nutrient agar, and after thoroughly 
shaking, the mixture is poured into a Petri dish and the bacteria allowed 
to develop, either at 37° or 23° C., according as the growth of the 
parasitic or saprophytic varieties is desired. Instead of agar or gelatin, 
ascitic broth or animals may be inoculated. Such examinations are 
occasionally made of the air of theatres, crowded streets in cities, etc. 
They give interesting, but hardly valuable, results. 
BACTERIOLOGICAL EXAMINATION OF THE SOIL. 
The subject from its agricultural side is considered on p. 627. Speci- 
mens of deep soil can be gathered in sterile, sharp-pointed, sheet-iron 
tubes. Through the examination, we wish to learn either the number 
of bacteria or the important varieties of bacteria present. To estimate 
the number, small fractions of a gram are taken and planted in nutrient 
agar or in special media contained in Petri dishes. Anaerobic as well 
as aerobic cultures should be made. 610 
APPLIED MICROBIOLOGY 
According to Houston, uncultivated sand soil averages 100,000 
bacteria per gram, garden soil 1,500,000, and sewage-polluted 115,- 
000,000. The most important bacteria to be sought for are bacilli 
of the colon group and streptococci. Both of these suggest fairly 
recent excremental pollution. 
The period during which typhoid bacilli remain alive in soil is variable, 
since it depends on so many unknown factors and differs so in different 
places. The typhoid bacilli probably rarely increase in the soil and 
probably rarely survive a month in it. The main danger of soil bacteria 
is their being washed into water supplies by rains or carried to them 
by the wind. 
Reaction of Vosges and Proskauer.-Grow the culture in 1 per cent, 
glucose-peptone water in a fermentation tube for four days at 37° C. 
Add 1 c.c. of 50 per cent. KOH solution to the open end and allow the 
mixture to stand for two days at room temperature. With certain 
varieties of bacteria a red color like that of eosin develops after twenty- 
four to forty-eight hours. With true B. coli this color does not develop. CHAPTER XLVII. 
THE BACTERIOLOGY OF MILK IN ITS RELATION TO 
DISEASE. 
From the stand-point of the dairy many of the different varieties 
of bacteria found in milk are of importance, which have little or no 
medical interest. We have space here only to consider the bacteri- 
ology of milk so far as it is related to health and disease. The 
saprophytic bacteria taken collectively have importance because one 
can determine from their number something as to the care taken in 
handling the milk and also because, when numerous, they produce 
chemical changes in the milk which are harmful for infants. 
Numerical Estimation of Bacteria-The number of bacteria in a cubic 
centimeter of milk is usually estimated from the colonies developing in 
nutrient agar plate cultures during a period of forty-eight hours when 
kept at a temperature of 37° C. This allows in market milk in which 
bacteria have developed at low temperature only a certain proportion 
of the varieties of bacteria to develop colonies. Sometimes fully twice 
as many colonies develop at 20° to 27° C. during three to five days 
as at 37° for two days. The advantages of the shorter time and of 
uniformity have led to the adoption of the technique given in Part I. 
Any culture method necessarily underestimates their number, as many 
of the bacteria remain after vigorous shaking in pairs or small groups. 
In order to overcome this and also to note the morphological types, 
the direct microscopical examination of smears of the sediment has 
been urged. A great practical objection to this is that, if a heated 
milk is examined, the dead as well as the living bacteria are counted. 
This method has, however, great advantages at the creamery or farm 
in that one can immediately tell whether a sample has few or many 
bacteria and also note the presence of streptococci and leukocytes. The 
microscopic count gives from two to ten times as many bacteria as 
the routine culture method. 
Smear Method for Direct Examination of Milk.-The Prescott- 
Breed method is the most accurate. That of Slack is also used. 
In the Slack method one cubic centimeter is withdrawn and put into 
a tube of small caliber having two rubber corks and is centrifugalized 
for ten minutes. After centrifugalization the upper cork is removed 
and the supernatant cream and milk are gently poured off; the lower 
cork which holds the sediment is then removed and the sediment is 
spread as evenly as possible on slides in areas of two square centimeters 
upon which a drop of sterile water has been previously placed. 612 
APPLIED MICROBIOLOGY 
Prescott and Breed1 explain their smear method briefly as follows: 
The sample of milk to be examined is shaken thoroughly and 0.01 c.c. 
is withdrawn by means of a specially constructed capillary pipette. 
The milk so obtained is spread evenly over an area of one square centi- 
meter on an ordinary glass slide. These areas may be easily determined 
by placing the glass slide over paper or glass on which areas of this 
size have been accurately ruled out. More satisfactory results can be 
obtained by using circular instead of square areas. Duplicate smears 
should be made on the same slide. The milk is then dried with gentle 
heat, the fat dissolved out with xylol or other fat solvent, the smear 
again dried, immersed in alcohol for a few minutes to fix the film, 
again dried and stained with methylene blue or other stain. In the 
Slack a more simple method for fixing and staining is used in their 
laboratory. The smears are allowed to dry in the air, fixed in methyl 
alcohol and then stained with a watery solution of methylene blue. 
Alkaline stains or others which attack the casein and loosen the smear 
must be avoided. The glassware used must be cleansed but need not 
be sterilized as the bacteria have no chance to increase in number. 
The counting of the bacteria is done by a microscope and an oil 
immersion lens. If the diameter of the field is so adjusted, by means 
of the draw tube, that it equals 0.16 mm., then each field of the micro- 
scope covers approximately one-five-thousandth (0.0002) of a square 
centimeter. On this basis, each bacterium seen in a field taken at ran- 
dom represents 500,000 per c.c. if they are evenly distributed. However, 
it is impossible to distribute them evenly so that at least 100 fields of 
the microscope should be counted if accurate results are required. 
The total number of bacteria seen in ten fields multiplied by 50,000 
or the total number seen in 100 fields multiplied by 5000 gives approxi- 
mately the total number of bacteria per cubic centimeter. 
Identification of Bacteria.-The milk is plated in a 2 per cent, lactose- 
litmus nutrient gelatin or agar, and the bacteria, after development 
of colonies, isolated and grown upon the usual identification media. 
The pathogenic properties of the different bacteria can be tested by 
intraperitoneal and subcutaneous inoculation in guinea-pigs with 2 c.c. 
of a forty-eight-hour broth culture, and by feeding young kittens for 
several days with 3 to 6 c.c. daily of a twenty-four-hour broth culture 
by means of a medicine dropper. 
Varieties.-Bacteria in milk can be divided into two great groups- 
those which get into the milk after it leaves the udder and those which 
come from the cow. The first group comprises bacteria from dust, 
hands, milking pails, strainers, etc. 
The extraneous bacteria are of importance because they produce 
changes in the chemical composition of the milk when they have 
developed in great numbers. The number of bacteria in any sample 
of milk depends on three factors: the number deposited in the milk 
from the cow's udder, from the air, and utensils; the time during 
1 Central, f. Bakt., Parasiten. u. Infektionskrankheiten, 1911, Band xxx, Heft 16/18. VARIETIES 
613 
which they have developed, and the temperature at which the milk 
has stood. The last is perhaps the most important factor. The attempt 
was made during a period of one year to connect illness in infants 
and children with special varieties of saprophytic bacteria in milk. 
As a matter of fact, no such connection was made out. 
From the milks altogether 239 varieties of bacteria were isolated 
and studied. These 239 varieties, having some cultural or other 
differences, were divided into the 31 classes, each class containing 
from 1 to 39 more or less related organisms. 
As to the sources of bacteria found in milk, we made sufficient experi- 
ments to satisfy us that they came chiefly from outside the udder 
and milk-ducts. 
Bacteria were isolated from various materials which under certain 
conditions might be sources of contamination for the milk, and the 
cultures compared with those taken from milk. Thus there were 
obtained from 20 specimens of hay and grass, 31 varieties of bacteria; 
from 15 specimens of feces, manure, and intestinal contents, 28 varieties; 
from 10 specimens of feed, 17 varieties. Of these 76 varieties there 
were 42 which resembled closely those from milk-viz., 11 from grass 
or hay; 26 from manure; 5 from feed. 
During the investigation a number of the varieties isolated from 
milk were shown to be identical with types commonly found in water. 
From the few facts quoted above and from many other observa- 
tions made during the course of the work, it would seem that the term 
"milk bacteria" assumes a condition which does not exist in fact. 
The expression would seem to indicate that a few varieties, especially 
those derived in some way from the cow, are commonly found in milk, 
which forms having entered the milk while still in the udder or after 
its withdrawal, are so well fitted to develop in milk that they outgrow 
all other varieties. 
As a matter of fact, it was found that milk taken from a number 
of cows, in which almost no outside contamination had occurred, 
and plated immediately, contained, as a rule, very few bacteria, and 
these were streptococci, staphylococci, and other varieties of bacteria 
not often found in milk sold in New York City; the temperature at 
which milk is kept being less suitable for them than for the bacteria 
which fall into the milk from dust, manure, etc. A number of speci- 
mens of fairly fresh marked milk averaging 200,000 bacteria per cubic 
centimeter were examined immediately, and again after twelve to 
twenty-four hours. In almost every test the three or four predominant 
varieties of the fresher milk remained as the predominant varieties 
after the period mentioned. 
The above experiments seem to show that organisms which have 
gained a good percentage in the ordinary commercial milk at time of 
sale will be likely to hold the same relative place for as long a period 
as milk is usually kept. After the bacteria pass the ten or twenty 
million a change occurs, since the increasing acidity inhibits the growth 
of some forms before it does that of others. Thus some varieties of 614 
APPLIED MICROBIOLOGY 
the lactic acid bacteria can increase until the acidity is twice as great 
as that which inhibits the growth of many bacteria. Before milk 
reaches the curdling point, the bacteria may have reached over a billion 
to each cubic centimeter. For the most part specimens of milk from 
different localities showed a difference in the character of the bacteria 
present, in the same way that the bacteria from hay, feed, etc., varied. 
Even the intestinal contents of cows, the bacteriology of which might 
be expected to show common characteristics, contained, besides the 
predominating colon types, other organisms, which differed widely 
in different species and in different localities. Cleanliness in handling 
the milk and the temperature at which it had been kept were also 
found to have a marked influence on the predominant varieties of 
bacteria present. 
Pathogenic Properties of the Bacteria Isolated.-Intraperitoneal 
injection of 2 c.c. of broth or milk cultures of about 40 per cent, of 
the varieties tested caused death. Cultures of most of the remainder 
produced no apparent deleterious effects even when injected in larger 
amounts. The filtrates of both cultures of a number of varieties were 
tested, but only one was obtained in which poisonous products were 
abundantly present. Death in guinea-pigs weighing 300 grams followed 
within fifteen minutes after an injection of 2 c.c.; 1 c.c. had little effect. 
As bacteria in milk are swallowed and not injected under the skin, 
it seemed wise to test the effect of feeding them to very young animals. 
We therefore fed forty-eight cultures of 139 varieties of bacteria to 
kittens of two to ten days of age by means of a glass tube. The kittens 
received 5 to 10 c.c. daily for from three to seven days. Only one 
culture produced illness or death. Very young guinea-pigs were fed in 
the same manner with similar results. A full report on the identification 
of bacteria met with in this investigation can be found in an article by 
Dr. Letchworth Smith in the 1902 Annual Report of the Department 
of Health of New York City. 
After five years of effort to discover some relation between special 
varieties of bacteria found in milk and the health of children the con- 
clusion has been reached that neither through animal tests nor the 
isolation from milk given sick infants have we been able to establish 
such a relation. Pasteurized or "sterilized" milk is rarely kept in 
New York longer than thirty-six hours, so that varieties of bacteria 
which after long standing develop in such milk did not enter into 
our problem. The harmlessness of cultures given to healthy young 
kittens does not of course prove that they would be equally harmless 
in infants. Even if harmless in robust infants, they might be injurious 
when summer heat and previous disease had lowered the resistance 
and the digestive power of the subjects. 
Streptococci in Relation to Disease.-In an investigation by Dr. 
D. H. Bergey connection between diarrhea and pus and streptococci 
was sometimes found. 
The results of this investigation appear to warrant the following 
conclusions: DELETERIOUS EFFECT OF BACTERIA IN MILK ON INFANTS 
615 
1. The occurrence of an excessive number of leukocytes in cows' 
milk is probably always associated with the piesence in the udder of 
some inflammatory reaction brought about by the presence of some 
of the ordinary pyogenic bacteria, especially of streptococci. 
2. When a cow's udder has once become infected with the pyogenic 
bacteria, the disease tends to persist for a long time, probably extending 
over several periods of lactation. 
3. Lactation has no causative influence per se upon the cellular 
and bacterial content of cows' milk, though it probably tends toward 
the aggravation of the disease when the udder is once infected. 
It is impossible to differentiate in routine milk examinations the 
pathogenic streptococci of diseased cows from saprophytic varieties. 
Thus it happens that a milk which contains great numbers of strep- 
tococci may or may not be more dangerous than one which contains 
an equal number of other apparently less harmful bacteria. 
The Deleterious Effect of Bacteria in Milk on Infants-We have 
tested this ourselves in the following way: During each of the summers 
of 1902, 1903, and 1904 a special lot of milk was modified for a group 
of fifty infants, all of whom were under nine months of age, and dis- 
tributed daily. To one half the milk was given raw; to the other half 
a similar milk heated at 60° for twenty minutes. 
The modified milk was made from a fairly pure milk mixed with 
ordinary cream. The bacteria contained in the milk numbered on the 
average 45,000 per cubic centimeter, in the cream 30,000,000. The 
modified raw milk taken from the bottles in the morning averaged 
1,200,000 bacteria per cubic centimeter, or considerably less than the 
ordinary grocery milk; the Pasteurized, about 1000; taken in the 
late afternoon of the same day they had, respectively, about 20,000,000 
and 50,000. 
Twenty-one predominant varieties of bacteria were isolated from 
six specimens of this milk collected on different days. The varieties 
represented the types of bacteria frequently found in milk. The infants 
were selected during the first week in June, and at first all were placed 
on Pasteurized milk. The fifty infants which had been selected were 
now separated into two groups as nearly alike as possible. On the 
fifteenth of June the milk was distributed without heating to one half 
the infants, the other half receiving as before the heated milk. In 
this way the infants in the two groups received milk of identically the 
same quality, except for the changes produced by heating to 165° F. 
for thirty minutes. The infants were observed carefully for three 
months and medical advice was given when necessary. When severe 
diarrhea occurred barley-water was substituted for milk. 
The first season's trial gave the following results: Within one week 
20 out of 27 infants put on raw milk suffered from moderate or severe 
diarrhea; while during the same time only 5 cases of moderate and 
none of severe diarrhea occurred in those taking Pasteurized milk. 
Within a month 8 of the 27 had to be changed from raw back to heated 
milk, because of their continued illness; 7, or 25 per cent., did well 616 
APPLIED MICROBIOLOGY 
all summer on raw milk. On the other hand, of those receiving the 
Pasteurized milk, 75 per cent, remained well, or nearly so, all summer, 
while 25 per cent, had one or more attacks of severe diarrhea. There 
were no deaths in either group of cases. 
During the second summer a similar test was made with 45 infants. 
Twenty-four were put on raw modified milk; 13 of these had serious 
diarrhea, in 5 of whom it was so severe that they were put back upon 
heated milk; 10 took raw milk all summer without bad effects; 2 
died, 1 from gross neglect on the part of the mother, the other from 
diarrhea. Of the 21 on Pasteurized milk, 5 had severe attacks of 
diarrhea, but all were kept on this milk except for short periods, when 
all food was omitted; 16 did well throughout the summer. One infant, 
markedly rachitic, died. The third summer's results have not been 
tabulated, but were similar to those of the first two tests. 
The outcome of these observations during the first two summers 
are summarized in the following table: 
Num- 
Kinds of milk. ber of 
infants. 
Re- 
mained 
well for 
entire 
sum- 
mer. 
Number 
having 
severe or 
moderate 
diarrhea. 
Average 
number 
days off 
milk dur- 
ing sum- 
mer. 
Average 
weekly 
gain in 
weight 
Aver- 
age 
num- 
ber of 
days 
diar- 
rhea. 
Deaths. 
Pasteurized milk, 1,000 to 50,- 
000 bacteria per c.c. . . 41 
Raw milk, 1,200,000 to 20,000- 
000 bacteria per c.c. . . 511 
31 
17 
10 
33 
3.0 
5.5 
4.0 oz. 
3.5 oz. 
3.9 
11.5 
1 
2 
Although the number of cases was not large, the results, almost 
identical during the three summers, indicate that even a fairly pure 
milk, when given raw in hot weather, causes illness in a much larger 
percentage of cases than the same milk given after Pasteurization. 
A considerable percentage of infants, however, do apparently quite 
as well on raw as on Pasteurized milk. 
Bacteria in Milk. Effect on Older Children.-The children over 
three years of age who received unheated milk, containing at different 
times from 145,000 to 350,000,000 bacteria per cubic centimeter, 
showed almost no gastro-intestinal disturbance. The conditions at 
three institutions will serve as examples. 
In the first of these an average grade of raw milk was used which, 
during the summer, contained from 2,000,000 to 30,000,000 bacteria 
per cubic centimeter. This milk was stored in an ice-box until 
required. It was taken by children unheated and yet no case of diar- 
rhea of sufficient gravity to send for a physician occurred during the 
entire summer. This institution was an orphan asylum containing 
650 children from three to fourteen years of age-viz., three to five 
years, 98; five to eight years, 162; eight to fourteen years, 390. 
1 Thirteen of the fifty-one infants on raw milk were transferred before the end of the 
trial to Pasteurized milk because of serious illness. If these infants had been left on raw 
milk it is believed by the writers that the comparative results would have been even more 
unfavorable to raw milk. RESULTS WITH HEATED IMPURE AND PURE MILK 
617 
A second institution used an unheated but very pure milk which 
was obtained from its own farm. This milk averaged 50,000 bac- 
teria per cubic centimeter. The inmates were 70 children of ages 
ranging from three to fourteen years. In this institution not a single 
case of diarrheal disease of any importance occurred during the 
summer. 
In a third institution an average grade of milk was used which 
was heated. This milk before heating contained 2,000,000 to 
20,000,000 bacteria per cubic centimeter. The institution was an 
infant asylum in which there were 126 children between the ages 
of two and five years. There were no cases of diarrhea during the 
summer. 
These clinical observations taken in connection with the bacterio- 
logical examination at the laboratory show that although the milk 
may come from healthy cattle and clean farms and be kept at a tem- 
perature not exceeding 60° F., a very great increase in the number 
of bacteria may occur. Furthermore, this may occur without the 
accumulation in the milk of sufficient poisonous products or living 
bacteria to cause appreciable injury in children over three years of 
age, even when such milk is consumed in considerable amount and 
for a period extending over several months. Milk kept at tempera- 
tures somewhat above 60° F. was not met with in our investigations, 
but the histories of epidemics of ptomain poisoning teach that such 
milk may be very poisonous. It is also to be remembered that milk 
abounding in bacteria on account of its being carelessly handled is 
also always liable to contain pathogenic organisms derived from 
human or animal sources. 
Results with Very Impure Milk Heated vs. Those with Pure or 
Average Milk Heated.-During the summer of 1901 we were able to 
observe a number of babies fed on milk grossly contaminated by bac- 
teria. In 1902 systematic supervision of all stores selling milk was 
instituted by the Health Department, so that the very worst milk was 
not offered for sale that summer. 
The observations upon the impure milk of 1901 are of sufficient 
importance to be given in detail, although already mentioned in the 
report of the observations upon infants of both summers which were 
fed on "store milk." A group of over 150 infants was so divided 
that 20 per cent, were-allowed to remain on the cheapest store milk 
which they were taking at the time. To about the same number was 
given a pure bottled milk. A third group was fed on the same quality 
of milk as the second, but sterilized and modified at the Good Samar- 
itan Dispensary. A fourth group received milk from an ordinary 
dairy farm. This milk was sent to a store in cans and called for by 
the people. A few infants fed on breast and condensed milk were 
observed for control. 
In estimating the significance of the observations recorded in the 
tables, one should bear in mind that not only do different infants 
possess different degrees of resistance to disease, but that, try as hard 618 
APPLIED MICROBIOLOGY 
as the physicians could, it was impossible to divide the infants into 
groups which secured equal care and were subjected to exactly the 
same conditions. It was necessary to have the different groups in 
somewhat different parts of the city. It thus happened that the 
infants on the cheap store milk received less home care than the 
average, and that those on the pure bottled milk lived in the coolest 
portion of the city. Certain results were, however, so striking that 
their interpretation is fairly clear. It is to be noted that the number 
of infants included in each group is small. 
There is nothing in the observations to show that fairly fresh milk 
from healthy cows, living under good hygienic conditions and con- 
taining, on some days, when delivered, as many as 200,000 bacteria 
per cubic centimeter, had any bacteria or any products due to bac- 
teria that remained deleterious after the milk was heated to near the 
boiling point. 
Table Showing the Results of Feeding during July and August, 1901, in Tenement 
Houses, of 112 Bottle-fed Infants under one year of Age, and of 47 Bottle- 
fed Infants between One and Two Years of Age with Milk from Different 
Sources, and the Number of Bacteria Present in the Milk. 
Infants under one year. 
Infants over one year. 
Character of the milk. 
'o . 
co 
,ge 
;am. 
Diarrhea. 
o 
tn 
Average 
weekly gain. 
Diarrhea. 
X « 
g-S 
s.S 
Avera 
weekly 
Mild. 
' Severe. 
Deaths 
s 
£ 
Mild. 
Severe. 
1 Deaths 
1. 
Pure milk boiled and modified 
at dispensary or stations; given 
out in small bottles. Milk be- 
fore boiling averaged 20,000 
bacteria per c.c.; after boiling 2 
per c.c. 
41 
3 oz. 
10 
8 
I1 
2. 
Pure milk, 24 hours old, sent in 
in quart bottles to tenements, 
heated and modified at home, 
20,000 to, 200,000 bacteria per 
c.c. when delivered 
23 
4J^ oz. 
8 
5 - 
0 
24 
4j oz. 
8 
2 
0 
3. 
Ordinary milk, 36 hours old, 
from a selected group of farms, 
kept cool in cans during trans- 
port; 1,000,000 to 25,000,000 
bacteria per c.c., heated and 
modified at home before using. 
18 
4 oz. 
6 
6 
I2 
12 
4 oz. 
1 
2 
0 
4. 
Cheap milk, 36 to 60 hours old, 
from various small stores, de- 
rived from various farms, some 
fairly clean, some very dirty; 
400,000 to 175,000,000 bacteria 
per c.c. 
21 
1 QZ. 
4 
13 
43 
_ 7 
H oz. 
1 
3 
0 
5. 
Condensed milk of different 
brands. 'Made up with hot 
water. As given, contained bac- 
teria from 5,000 to 200,000 per 
c.c. 
9 
i oz. 
5 
2 
3 
4 
3% oz. 
1 
3 ' 
0 
6. 
Breast milk 
16 
2| oz. 
5 
2 
0 
1 This infant died from enteritis and toxemia. 
2 This infant died of pneumonia. There had been no severe intestinal disorder noted. 
3 One of the four had pertussis, the remaining three died from uncomplicated enteritis. BACTERIAL CONTAMINATION OF MILK 
619 
On the other hand, it is possible that certain varieties of bacteria 
may, under conditions that are unsanitary, find entrance to milk and 
survive moderate heat or may develop poisonous products resistant 
to heat in sufficient amount to be harmful, even when they have ac- 
cumulated to less than 200,000 per c.c. 
Turning now to the results of feeding with milk which has been 
heated and which before sterilization contained from 1,000,000 to 
25,000,000 bacteria per cubic centimeter, averaging about 15,000,- 
000, though obtained from healthy cows living under fairly decent 
conditions and although the milk was kept moderately cool in transit, 
we find a distinct increase in the amount of diarrheal diseases. Though 
it is probable that the excessive amount of diarrhea in this group 
of children was due to bacterial changes which were not neutralized 
by heat or to living bacteria which were not killed, yet it is only fair 
to consider that the difference was not very great and that the infants 
of this group were under surroundings not quite as good as those on 
the pure milk. 
Finally, we come in this comparison to the infants who received 
the cheap store milk after heating. This milk had frequently to be 
returned because it curdled when boiled, and contained, according 
to the weather, from 4,000,000 to 200,000,000 bacteria per cubic 
centimeter.. In these infants the worst results were seen. This is 
shown not only by the death rate, but by the amount and by the 
severity of the diarrheal diseases, and the general appearance of the 
children as noted by the physicians. Although the average number 
of bacteria in the milk received by this group is higher than that re- 
ceived by the previous group, the difference in results between this 
group and the previous one can hardly be explained by the difference 
in the number of bacteria. The varieties of bacteria met with in 
this milk were more numerous than in the better milk, but we were 
unable to prove that they were more dangerous. Probably the higher 
temperature at which the milk was kept in transit, and the longer 
interval between milking and its use, allowed more toxic bacterial 
products to accumulate. 
Bacterial Contamination of Milk-General Conclusions1 as to Rela- 
tive Importance.-1. During cool weather neither the mortality nor 
the health of the infants observed in the investigation was appre- 
ciably affected by quality of the market milk or by the number of bac- 
teria which it contained. The different grades of milk varied much 
less in the amount of bacterial contamination in winter than in sum- 
mer, the store milk averaging only about 750,000 bacteria per cubic 
centimeter. 
2. During hot weather, when the resistance of the children was 
lowered, the kind of milk taken influenced both the amount of illness 
and the mortality; those who took condensed milk and cheap store 
milk did the worst, and those who received breast milk, pure bottled 
1 These conclusions were drawn up by the writer in association with Dr. L. E. Holt, 
after a joint study of the results obtained in the studies above recorded. 620 
APPLIED MICROBIOLOGY 
milk, and modified milk did the best. The effect of bacterial con- 
tamination was very marked when the milk was taken without pre- 
vious heating; but, unless the contamination was very excessive, only 
slight when heating was employed shortly before feeding. 
3. The number of bacteria which may accumulate before milk 
becomes noticeably harmful to the average infant in summer differs 
with the nature of the bacteria present, the age of the milk, and the 
temperature at which it has been kept. When the milk is taken 
raw, the fewer the bacteria present the better are the results. Of the 
usual varieties, over 1,000,000 bacteria per cubic centimeter are 
certainly deleterious to the average infant. However, many infants 
take such milk without apparently harmful results. Heat of 145° F. 
for thirty minutes or of 170° F. for a shorter period not only destroys 
most of the bacteria present, but, apparently, some of their posionous 
products. No harm from the bacteria previously existing in recently 
heated milk was noticed in these observations unless they had amounted 
to many millions, but in such numbers they were decidedly dele- 
terious. 
4. When milk of average quality was fed, sterilized and raw, those 
infants who received milk previously heated did, on the average, 
much better in warm weather than those who received it raw. The 
difference was so quickly manifest and so marked that there could 
be no mistaking the meaning of the results. 
5. No special varieties of bacteria were found in unheated milk 
which seemed to have any special importance in relation to the sum- 
mer diarrheas of children. A few cases of acute indigestion were seen 
immediately following the use of Pasteurized milk more than thirty- 
six hours old. Samples of such milk were found to contain more 
than 100,000,000 bacteria per cubic centimeter, mostly spore-bear- 
ing varieties. The deleterious effects, though striking, were neither 
serious nor lasting. 
6. After the first twelve months of life, infants are less and less 
affected by the bacteria in milk derived from healthy cattle and the 
air. According to these observations, when the milk had been kept 
cool, the bacteria did not appear to injure the children over three years 
of age at any season of the year, unless in very great excess. 
7. While it is true that even in tenements the results with the 
best bottle feeding are nearly as good as average breast feeding, it is 
also true that most of the bottle feeding is at present very badly done; 
so that, as a rule, the immense superiority of breast feeding obtains. 
This should, therefore, be encouraged by every means, and not dis- 
continued without good and sufficient reasons. The time and money 
required for artificial feeding, if expended by the tenement mother 
to secure better food and more rest for herself, would often enable her 
to continue nursing with advantage to her child. 
Influence of Temperature upon the Multiplication of Bacteria in 
Milk.-Few, even of the well informed, appreciate how great a difference 
a few degrees of temperature will make in the rate of bacterial mul- MULTIPLICATION OF BACTERIA IN MILK 
621 
tiplication. Milk rapidly and sufficiently cooled keeps almost unaltered 
for thirty-six hours, while milk insufficiently cooled deteriorates rapidly. 
The majority of the bacteria met with in milk grow best at tem- 
peratures above 70° F., but they also multiply slowly even at 40° 
F.; thus, of 60 species isolated by us, 42 developed good growth at 
the end of seven days at 39° F. Our observations have shown that 
the bacteria slowly increase in numbers after the germicidal prop- 
erties of the milk have disappeared, and the germs have become ac- 
customed to the low temperature. In fact, milk cannot be perma- 
nently preserved unaltered unless kept at 32° F. or less. The degree 
of cooling to which ordinary supplies of milk are subjected differs 
greatly in various localities. Some farmers chill their milk rapidly, 
by means of pipe coils over which the milk flows; others use deep 
wooden tanks filled with water into which the cans of milk are placed 
soon after milking. In winter these methods are very satisfactory 
for the water runs into the pipes or tanks at about 38° F. In warmer 
weather they are unsatisfactory, unless ice is used, as the natural 
temperature of the water may be as high as 55° F. A considerable 
quantity of milk is not cooled at all at the farms. It is sent to the 
creamery or railroad after two to six hours, and is then more or less 
cooled. These few hours in summer, when the milk is left almost at 
blood heat, allow an enormous development of bacteria to take place, 
as is shown in the table below'. 
Table 1.-Showing the Development of Bacteria in Two Samples of Milk Main- 
tained at Different Temperatures for Twenty-four, Forty-eight, and 
Ninety-six Hours, Respectively. The first Sample of Milk was Obtained 
UNDER THE BEST CONDITIONS POSSIBLE, THE SECOND IN THE USUAL WAY. WHEN 
Received, Specimen, No. 1 Contained 3000 Bacteria per c.c., Specimen No. 2, 
30,000 per c.c. 
Temperature. 
Fahrenheit. 
32° 
39° 
42° 
40° 
50° 
55 0 
60° 
68° 
Time which elapsed before making test. 
24 hours. 
2400 
30,000' 
2500 
38,000 
2600 
43,000 
3100 
42,000 
11,600 
89,000 
18,800 
187,000 
180,000 
900,000 
450,000 
4,000,000 
48 hours. 
2100 
27,000 
3600 
56,000 
3600 
210,000 
12,000 
360,000 
540,000 
1,940,000 
3,400,000 
38,000,000 
28,000,000 
168,000,000 
500,000,000 
1,000,000,000= 
96 hours. 
1850 
24,000 
218,000 
4,300,000 
500,000 
5,760,000 
1,480,000 
12,200,000 
300,000,000 
1,000,000,000= 
168 hours. 
1400 
19,000 
4,209,000 
38,000,000 
11,200,000 
120,000,000 
80,000,000 
300,000,000 
1,000,000,0002 
1 The figures referring to tests of the second sample are printed in heavy-face type. 
2 These figures signify the maximum growth and estimates only. 622 
APPLIED MICROBIOLOGY 
Observations on Bacterial Multiplication in Milk at 90° F., a Temperature 
Common in New York in Hot Summer Weather. 
Milk I. 
Fresh and of good 
quality. 
Milk II. 
Fair quality from 
store. 
Milk III. 
Bad quality from 
store. 
Original number . 
5200 
92,000 
2,600,000 
After two hours . 
8400 
184,000 
4,220,000 
After four hours . 
12,400 
470,000 
19,000,000 
After six hours 
68,500 
1,260,000 
39,000,000 
After eight hours 
654,000 
6,800,000 
124,000,000 
Table II.-Number oe Bacteria per c.c. 
A sample of milk No. I removed after six hours and cooled to 50° F. contained 145,000,- 
000 at the end of twenty-four hours. Some of this milk, kept cool from the beginning, 
contained but 12,800 bacteria per cubic centimeter at the end of twenty-four hours. 
Pasteurization of Milk.-The two dominant factors which control 
the temperature and time at which the milk' should be heated are 
(1) the thermal death points of pathogenic bacteria, and (2) the 
thermolabile food constituents of the milk. The first factor is al- 
most equally important for milk used by persons of all ages, while 
the second factor is only important for milk used in very young children. 
The exposure of bacteria for a short time at a high temperature 
is equivalent to a longer time at a lower temperature. The ferments 
and other labile food constituents, on the other hand, are altered much 
more by the higher temperature. It is well, therefore, to choose the 
lowest possible temperature which will kill the non-spore-bearing patho- 
genic bacteria in a practicable length of time. Such an exposure is 
60° C. (140° F.) for 30 minutes, 70° C. (158° F.) for 5 minutes. Very 
much shorter exposures, as one minute at 70° C., will kill the great ma- 
jority of pathogenic and other bacteria in the milk and add much of 
safety as seen in the tables below, but it is better to be on the safe side. 
Table Showing Effect of Heat Upon Tubercle Bacilli in Milk Heated 
Instantly. 
Degree of heat. 
Time exposed. 
Amount milk. 
Result in guinea-pigs. 
60° C. 
15 min. 
1 c.c. 
Infection 
60° C. 
20 min. 
1 c.c. 
No infection 
60° C. 
30 min. 
1 c.c. 
No infection 
70° C. 
0.51 min. 
1 c.c. 
Infection1 
70° C. 
1 min. 
1 c.c. 
No infection 
70° C. 
2 min. 
1 c.c. 
No infection 
Control not heated 
.001 
Infection 
This milk was infected by adding 
bacilli. 
one-fifth of its quantity of 
sputum rich in tubercle 
Development of Bacteria in Milk Which has been Heated.-There is 
a common idea that bacteria develop much more rapidly in milk that 
has been heated than in raw milk. This is only true for freshly drawn 
milk which has slight bactericidal power. 
The table below shows the effect on bacteria in milk of heating to 
70° C. for one-half and one minute. Not only the immediate reduction 
in number is seen to be great, but the difference continues when the 
milk is kept cold for two days. 
1 Most of the guinea-pigs were not infected by the milk heated for one-half minute. PASTEURIZATION OF MILK 
623 
Two Samples Mixed from 100 Samples of Inspectors. Pasteurized at 160° F. 
Plates Made Same Day. 
Sample I. 
Sample II. 
Before heating . . . 600,000 
J m 2000 
1 m 1000 
Control .... 5,400,000 
| m 7400 
Im 600 
Same Samples Kept in Ice-box Twenty-four Hours at 45° F. (7° C.). 
Control ....'. 6,300,000 
2 m 18,000 
1 m ...... . 900 
Control .... 21,600,000 
| m 12,000 
Im 3600 
Control 16,200,000 
J m 120,000 
Im 10,000 
In Ice-box Forty-eight Hours at 45° F. (7° C.). 
Control .... 63,000,000 
Im 276,000 
1 m 90,000 
Control 36,600,000 
Im 5,460,000 
Im...... 5,400,000 
In Room at 71°. F. (22° C.). 
Control .... 150,000,000 
i m 4,500,000 
Im 3,600,000 
Number of Bacteria in Milk Produced under Different Conditions. 
1. The number of bacteria present at the time of milking and twenty-four, 
forty-eight, and seventy-two hours afterward in milk obtained and kept under 
correct conditions. 
No preservatives were present in any of the following specimens: 
Pure milk obtained where every reasonable means was taken to ensure cleanli- 
ness. The long hairs on the udder were clipped; the cows roughly cleaned and 
placed in clean barns before milking; the udders were wiped off just previous 
to milking; the hands of the men were washed and dried; the pails used had small 
(six-inch) openings, and were thoroughly cleaned and sterilized by steam before 
use. Milk cooled within one hour after milking to 45° F., and subsequently kept 
at that temperature. The first six specimens were obtained from individual 
cows; the last six from mixed milk as it flowed at different times from the 
cooler. Temperature of barns 55° F. 
N umber or Bacteria in 1 c.c. of Milk. 
From Six Individual Cows. 
5 hours after milking. 
After 24 hours. 
After 48 hours. 
After 72 hours. 
500 
700 
12,500 
Not counted. 
700 
700 
29,400 
Not counted. 
19,900 
5200 
24,200 
Not counted. 
400 
200 
8600 
Not counted. 
900 
1600 
12,700 
Not counted. 
13,600 
3200 
19,500 
Not counted. 
Average 6000 
1933 
17,816 
From Samples of Mixed Milk of Entire 
Herd. 
6900 
12,000 
19,800 
494,000 
6100 
2200 
20,200 
550,000 
4100 
700 
7900 
361,000 
1200 
400 
7100 
355,000 
6000 
900 
9800 
445,000 
1700 
400 
8700 
389,000 
Average 
4333 
2766 
10,583 
329,000 
Twenty-five samples taken separately from individual cows on another day 
and tested immediately averaged 4550 bacteria per cubic centimeter and 4500 624 
APPLIED MICROBIOLOGY 
after twenty-four hours. These twenty-five specimens were kept at between 
45° and 50° F. 
2. Milk taken during winter in well-ventilated, fairly clean, but dusty barns. 
Visible dirt was cleaned off the hair about the udder before milking. Milkers' 
hands were wiped off, but not washed. Milk pails and cans were clean, but the 
straining cloths dusty. Milk cooled within two hours after milking to 45° F. 
Number 
of Bacteria in 1 c.c. of 
Milk. 
At time of milking. 
After 24 hours. 
After 48 hours. 
12,000 
14,000 
57,000 
13,000 
20,000 
65,000 
21,500 
31,000 
106,000 
Average 15,500 
21,666 
76,000 
Number in City Milk. 
3. The condition of the average raw city milk is very different, and is shown 
in the following tables. 
The twelve samples were taken late in March, 1912, by Inspectors of the 
Department of Health of New York City from cans of milk immediately upon 
their arrival in the city. Raw milk at present gives similar counts. 
The temperature of the atmosphere averaged 50° F. during the previous 
twenty-four hours. The temperature of the milk when taken from the cans aver- 
aged 45 ° F. Much of this milk had been carried over two hundred miles. From 
the time of its removal from the cans, which was about 2 a.m., until its distribu- 
tion in nutrient agar, at 10 a.m., the milk was kept at about 45° F. 
From New York and Hudson River Railroad. From Harlem Railroad. 
No. of bacteria 
No. of bacteria 
No. of sample. 
in 1 c.c. 
No. of sample. 
in 1 c.c. 
50 .... 
35,200,000 
48 . . . 
6,200,000 
51 .... 
13,000,000 
49 . . . 
2,200,000 
52 .... 
2,500,000 
50 . . . 
15,000,000 
53 .... 
1,400,000 
51 . . . 
70,000 
54 .... 
200,000 
52 . . . 
80,000 
55 .... 
600,000 
53 . . . 
320,000 
The results of the examination of many thousands of specimens last 
year indicated that most of the milk of grade A was of fair quality, 
but that the raw milk of the other grades contained excessive numbers 
of bacteria before pasteurization. 
It must be kept in mind that milk averaging 3,000,000 bacteria 
per cubic centimeter will, when kept at the temperature common in 
the homes of the poor who comprise the larger part of the population, 
soon contain very largely increased numbers and show its dangerous 
condition by turning sour and curdling. 
Cleanliness Used in Obtaining Milk, and Its Influence.-The present 
conditions under which much of the milk is obtained are not pleasant 
to consider. In winter, and to a less extent at other seasons of the 
year, the cows in many stables stand or lie down in stalls in the rear 
portion of which there is altogether too much of manure and urine. 
When milked the hands of the milkers are not cleansed, nor are the 
under portions of the cows, only visible masses of manure adhering 
to the hair about the udder being removed. Some milkers even moisten 
their hands with milk, to lessen friction, and thus wash off the dirt of CLEANLINESS USED IN OBTAINING MILK 
625 
their hands and the cow's teats into the milk in the pails. Some may 
regard it as an unnecessary refinement to ask that farmers should 
roughly clean the floors of their stalls once each day, that no sweeping 
should be done just before milking, and that the udders should be wiped 
with a clean damp cloth and the milkers should thoroughly wash and 
wipe their hands before commencing milking. The pails and cans 
should not only be carefully cleansed, but afterward scalded out with 
boiling water. The washing of the hands would lessen the number 
of ordinary filth bacteria in the milk, and diminish risk of transmitting 
to milk human infectious diseases, like scarlet fever, diphtheria, and 
enteric fever, by the direct washing off of the disease germs from 
infected hands. It would also inculcate general ideas of the necessity 
of cleanliness and of the danger of transmitting disease through milk. 
The value of cleanliness in limiting the number of bacteria is demon- 
strated by the figures contained in the tables. 
General Conclusions.-Because of its location and its hairy covering, 
the cow's udder is always more or less soiled with dirt and manure 
unless cleaned. On account of the position of the pail and the access 
of dust-laden air it is impossible to obtain milk by the usual methods 
without mingling with it a considerable number of bacteria. With 
suitable cleanliness, however, the number is far less than when filthy 
methods are used, there being no reason why fresh milk should contain 
in each cubic centimeter, on the average, more than 12,000 bacteria 
per cubic centimeter in warm weather and 5000 in cold weather. Such 
milk, if quickly cooled to 46° F., and kept at that temperature, will at 
the end of thirty-six hours contain on the average less than 50,000 
bacteria per cubic centimeter, and if cooled to 40° F. will average less 
than its original number. 
With only moderate cleanliness such as can be employed by any 
one without adding appreciably to his expense, namely, clean pails, with 
small openings, straining cloths, cans or bottles, and hands, a clean 
place for milking, and a decent condition of the cow's udder and 
belly, milk when first drawn will not average in hot weather over 
30,000, and in cold weather not over 25,000 bacteria per cubic centi- 
meter. Such milk, if cooled and kept at 50° F., will not contain at 
the end of twenty-four hours over 100,000 bacteria per cubic centi- 
meter. If kept at 40° F. the number of bacteria will not be over 
100,000 per cubic centimeter after forty-eight hours. 
If, however, the hands, cattle, and barns are filthy and the pails 
are not clean, the milk obtained under these conditions will, when 
taken from the pail, contain very large numbers of bacteria, even up 
to a million or more per cubic centimeter. 
Freshly drawn milk contains a slight and variable amount of bac- 
tericidal substances which are capable of inhibiting bacterial growth. 
At temperatures under 50° F. these substances act efficiently (unless 
the milk is filthy) for from twelve to twenty-four hours, but at higher 
temperatures their effect is very soon completely exhausted, and the 
bacteria in such milk will then rapidly increase. Thus the bacteria 626 
APPLIED MICROBIOLOGY 
in fresh milk which originally numbered 5000 per cubic centimeter 
decreased to 2400 in the portion kept at 42° F. for twenty-four hours, 
but rose to 7000 in that kept at 50° F., to 280,000 in that kept at 65° 
F., and to 12,500,000,000 in the portion kept at 95° F. 
As we have seen, the milk in New York City is found on bacterio- 
logical examination to contain, as a rule, excessive numbers of bac- 
teria. During the cold weather the raw milk in the shops averages 
over 300,000 bacteria per cubic centimeter, during cool weather about 
1,000,000, and during hot weather about 2,000,000. The milk in 
other large cities is, from all accounts, in about the same condition. 
The above statement holds for milk sold at the ordinary shops, 
and not that of the best of the special dairies, where, as previously 
stated, the'milk contains only from 1000 to 30,000 bacteria, accord- 
ing to the season of the year. 
The question might be raised, Are even these enormous numbers 
of bacteria often found in milk during hot weather harmful? 
Our knowledge is probably as yet insufficient to state just how 
many bacteria must accumulate to make them noticeably dangerous 
in milk. Some varieties are undoubtedly more Harmful than others, 
and we have no way of restricting the kinds that will fall into milk, 
except by enforcing cleanliness. We have also to consider that milk 
is not entirely used for some twelve hours after being purchased, and 
that during all this time bacteria are rapidly multiplying, especially 
where, as among the poor, no provision for cooling it is made. Slight 
changes in the milk which to one child would be harmless, would in 
another produce disturbances which might lead to serious disease. 
A safe conclusion is that no more bacterial contamination should be 
allowed than it is practicable to avoid. Any intelligent farmer can 
use sufficient cleanliness and apply sufficient cold, with almost no 
increase in expense, | cent per quart, to supply milk twenty-four to 
thirty-six hours old which will not contain in each cubic centimeter 
over 50,000 to 100,000 bacteria, and no milk containing more bacteria 
should be sold. 
The most deleterious changes which occur in milk during its trans- 
portation are now known not to be due to skimming off the cream or to 
the addition of water, but to the changes produced in the milk by mul- 
tiplication of bacteria. During this multiplication, acids and distinctly 
poisonous bacterial products are added to the milk, to such an extent 
that much of it has become distinctly deleterious to infants and invalids. 
It is the duty of health authorities to prevent the sale of milk rendered 
unfit for use through excessive numbers of bacteria and their products. 
The culture tests to determine the number of bacteria present in 
any sample of milk require at least forty-eight hours; so that the sale 
of milk found impure cannot be prevented. It will, however, be the 
purpose of the authorities gradually to force the farmers and the middle- 
men to use cleanliness, cold, and dispatch in the handling of their milk, 
rather than to prevent the use of the small amount tested on any one 
day. TRANSMISSION OF CONTAGIOUS DISEASES THROUGH MILK 
627 
If the milk on the train or at the dealer's were found to contain 
excessive numbers of bacteria, the farmers would be cautioned and 
instructed to carry out the simple necessary rules, which would be 
furnished. 
Transmission of Contagious Diseases through Milk. - Pathogenic 
Bacteria in Milk.-Tuberculosis, typhoid fever, septic sore throat, 
scarlet fever, and diphtheria are the chief diseases transmitted by 
means of milk in this locality. In other countries cholera, malta fever, 
and possibly other diseases may be due at times to milk infection 
The obscure disease trembles is also believed to be due to milk. 
The tubercle bacilli are in the majority of cases derived from the cow, 
but may come from human sources, the typhoid bacilli are entirely from 
man, the contagion of true scarlet fever conveyed in milk is probably 
always from man, but the contagion of a disease closely allied to it is 
certainly given off by cows suffering from certain septic diseases as yet 
not fully identified. Diphtheria bacilli are probably always of human 
origin, as animals, except cats, practically never suffer from the disease 
and these only under exceptional conditions. The streptococci exciting 
septic throats are probably usually from human cases of septic inflam- 
mation but may at times come from septic cows. As milk is usually 
kept below 60° F. the typhoid bacilli and the streptococci are the only 
pathogenic germs that we believe increase in any appreciable extent. 
The following epidemics and cases have been recorded in the bulletin 
of the Marine Hospital Service, as produced by cow's milk: 
Epidemics. 
Cases. 
Typhoid fever 
. . . . 179 
6900 
Scarlet fever 
. . . . 51 
2400 
Sore throat 
. . . . 7 
1100 
Diphtheria 
Tuberculosis . . 
. . . . 23 
960 
No case of measles, smallpox, chickenpox, whooping cough, or mumps 
has been clearly traced to milk. 
The Relation of the Typhoid Carrier to Milk Infection.-Many epi- 
demics of typhoid fever have until recently puzzled investigators be- 
cause, though evidently milk-borne, yet no case of typhoid fever could 
be found. The discovery that about 2 per cent, of those who have 
recovered from typhoid fever remain infected and continue during the 
rest of their lives to pass typhoid bacilli has cleared up the mystery. 
Epidemics due to these carriers have already been traced both in New 
York City and elsewhere. Many observers have already discussed the 
relation of typhoid cases to milk infection. Hands, water, flies, etc., 
may all aid in the transfer of the bacilli from the dejecta to the milk. 
Recently we traced over four hundred cases to infection of a milk supply 
by a typhoid carrier who had the disease forty-seven years ago. 
The Conveyance of Scarlet Fever by Means of Milk.-As we do not 
know the organism which excites scarlet fever, we are not as clear as to 
the means by which it is spread as we are in the case of tuberculosis, 
typhoid fever, and diphtheria. We know, however, that the throat 628 
APPLIED MICROBIOLOGY 
secretions are dangerous. Where the infection has been traced it has 
usually been found that the milker has suffered from an unrecognized 
case or is convalescent. It seems as if the contagion must either 
increase in milk or be capable of infecting when greatly diluted, for 
cases have developed from milk after great dilution. A small number 
of epidemics have appeared to come from the milk of diseased cows. 
Many are skeptical about this, but after personal experience we cannot 
doubt it. The history of one case was as follows: The milk from a 
septic cow was delivered to two schools. About thirty of the boys who 
drank the milk developed the disease while none of the day scholars 
who went home to lunch did. Some of the pases developed at first 
only sore throats, others only the rash. On the second day the cases 
resembled very closely scarlet fever. There was no scarlet fever in the 
town. The milk contained immense numbers of long-chained strepto- 
cocci. 
Diphtheria and septic sore throats are occasionally produced by milk. 
The diphtheria bacilli usually originate from a mild case, the nature of 
which is not detected. Septic sore throats produced by milk are usually 
traced to contamination from a human source, but like cases develop- 
ing the scarlet rash the infection may come from cows suffering from 
some acute septic udder disease. 
The Grading of Milk.-The appreciation of the importance of the 
bacteria in milk has led to the pasteurization of all milk entering New 
York City, except that produced and transported under the very best 
conditions. Together with the farm conditions the bacterial content 
of milk is used to grade it. All milk in New York City is divided into 
grades A, B, and C. 
Grade A is raw and pasteurized. The raw is from cows which 
have successfully passed the tuberculin test. 
Grade B is all pasteurized. 
Grade C is all pasteurized. 
The grade A may contain 60,000 bacteria per cubic centimeter. 
The grade B may contain 1,500,000 when raw and 50,000 after pas- 
teurization. 
The grade C may contain any number before and 100,000 after pas- 
teurization. It is supposed to be used for cooking purposes only. 
The State of New York has adopted a similar grading except that it 
allows raw and pasteurized in all three grades. Many cities and states 
are adopting such grades. A powerful influence in this direction was 
the report of the national committee of experts on milk standards. 
This was a commission appointed by the New' York Milk Committee. CHAPTER XLVIII. ' 
THE SOIL BACTERIA AND THEIR FUNCTIONS. SEWAGE 
BACTERIA. BACTERIA IN INDUSTRIES. 
The bacteria1 in the soil belong to many varieties. Some varieties 
are only accidentally present, being dne to the contamination of the 
earth with the bacteria contained in animal feces and other waste 
products. The majority, however, pass their life and reproduce them- 
selves chiefly or wholly in the soil. Many of these varieties have most 
important functions to perform in continuing the earth's food supply. 
Without them plant food, and, therefore, animal food, would cease 
to exist. Some make available for plants the carbon, nitrogen, hy- 
drogen, and other compounds locked up in the dead bodies of animals 
and plants. Others construct food for plants from the gases of the air 
and the inorganic elements of the earth which in their simpler forms 
were not available. 
The bacteria, together with the other somewhat less important 
microscopic plants and animals, thus form a vital link in the earth's 
life cycle, plants and animals. The bacteria in the soil require for 
their activities food, moisture, and a proper temperature. They may 
be present to the extent of many millions in a single gram of rich loam, 
while in an equal quantity of sand they may be almost absent. 
The various species associated together in the soil flora influence 
each other. Thus anaerobic bacteria are enabled to grow because of 
associated aerobes using up the free oxygen, while other species make 
assimilable substances not usable by others. 
The Splitting up of Carbon Compounds.-The plants form starch, 
and from it cellulose, wood, fats, and sugar. These substances once 
formed cannot be utilized by other generations of plants. Some of 
these are transformed in the bodies of animals, but the largest per- 
centage await the activities of the microorganisms. The sugars and 
starches usually undergo an alcoholic fermentation excited by the 
yeasts and molds with the production of alcohol and carbon dioxide, 
or an acid fermentation excited by bacteria with the production of 
acids and frequently of carbon dioxide. 
Cellulose which is so resistant to decay is attacked by certain varie- 
ties of bacteria which are abundant in the soil. They act both in the 
presence and absence of free oxygen. Molds also act on cellulose. 
Carbon dioxide, marsh gas, and other products are produced. Wood 
is apparently first attacked by the fungi and only later by the micro- 
organisms. These bacteria are carried into the intestines and act 
upon cellulose and other substances. 
1 L. H. Bailey. "Bacteria in Relation to Country Life." 630 
APPLIED MICROBIOLOGY 
The Decomposition of Nitrogenous Compounds.-Plants obtain 
their nitrogen chiefly in the form of nitrates. The small amount of 
usable nitrogen in the soil must be constantly replenished. This 
must either come from the nitrogen forming a part of proteid materials 
or from the free nitrogen in the air. 
The animals utilize the plant proteids and reduce them to much 
simpler compounds, such as urea, but even these are not suitable for 
plant use. We now know that microorganisms are employed to break 
compounds into simpler compounds and also to utilize the nitrogen 
of the air. 
Decomposition.-This process is to some extent carried out through 
the agency of yeasts, molds, and fungi, but it is chiefly due to the 
activities of bacteria. When this process is carried on in the absence 
of oxygen it is incomplete giving rise to substances with unpleasant 
odors, such as H2S, NH3 and CH4. This is called putrefaction. When 
oxygen is freely accessible more complete decomposition occurs with 
such end products as CO2, N and H2O. These two processes, putre- 
faction and complete decay, cannot be sharply separated, as the second 
usually follows the first. The varieties of organisms causing these 
changes are many. Some groups will be found chiefly in decaying 
vegetable substances, others in animal tissues. They include all mor- 
phologic forms of bacteria as well as yeasts and higher fungi. These 
forms exist everywhere in nature, although in various degrees, so that 
every bit of dead organic matter is sure to be decomposed if only mois- 
ture and warmth are present. B. subtilis and B. proteus vulgaris 
are well-known laboratory bacteria that are commonly found among 
decomposing materials. B. proteus is described under Pathogenic 
Bacteria. B. subtilis (hay bacillus) has the following characteristics 
(Fig. 208): 
Source and Habitat.-Hay, straw, soil, dust, milk, etc. 
Morphology.--Short, thick rods with round ends, sometimes form threads; 
sometimes also chains of long rods, short rods, and coccus forms. 0.8p to 1.2M 
broad, 1.3^ to 3/2 long. Often united in strings and threads. 
Staining Reaction.--Stains by Gram's method. 
Capsule, Flagella, Motility.-Bacilli possess a thin capsule and many flagella 
which are long and numerous; short forms actively motile; threads iinmotile. 
Spore Formation.-Oval spores formed in presence of air germinating at 
right angles to long diameter. Spores are set free in about 24 hours, size 1.2M 
by 0.6/z; widely distributed in nature, dust, air, excreta, etc. (Plate III, Fig. 23). 
Biology: Cultural Characters (Including Biochemical Features).-Bouillon.- 
Uniformly cloudy growth with marked pellicle, wrinkled and thick; copious 
spore formation. 
Gelatin Plates and Tubes.-Saucer-like depressions; colonies have granu- 
lar centres and folded margins. Surface growth in stab cultures is whitish- 
gray; colonies sink on liquefaction of medium; liquefaction progresses in a 
cylindric form, and a thick white scum is formed. 
Agar Plates and Tubes.-Small, irregular, grayish-white colonies; moist 
glistening growth along needle track in stab cultures. 
The bacteria in taking certain atoms from the molecules utilized 
in their growth leave the other atoms to enter into new relations and DENI TRIFICA TION 
631 
form new compounds. The actual products will depend on the decaying 
substance, the variety of bacteria and the conditions present. 
Nitrification.-This is a process of oxidation by which through 
bacterial activities ammonia compounds are changed to nitrates and 
thus rendered utilizable by plants. This change is accomplished in 
two stages: first, the ammonia is oxidized to nitrite and, second, to 
nitrate. The nitrates are taken up by the plant roots from the soil. 
The bacterial nature of these changes were discovered in 1877 by 
two French investigators, Schlosing and Muntz. They noted that 
fermenting sewage after a time lost its ammonia and gained in nitrates, 
but that if the sewage was treated with antiseptics, so that fermentation 
ceased, no such change occurred. Warrington first and Winogradsky 
later more thoroughly investigated the bacterial cause of these changes. 
The latter by means of silica jelly, which contained no organic matter, 
was able to isolate two varieties of cocci, one in Europe and the other 
in America, which were able to change ammonia to nitrites. He called 
Fig. 208 
Bacillus subtilis with spores. Agar culture. Stained with gentian violet. X 1000 
diameters. (Franke.) 
the one nitrosomonas and the other nitrosococcus. They are capable 
of acting on almost any ammonia salt. One variety of organisms 
capable of changing nitrites to nitrates was isolated, and this bacillus 
he called nitrobacter. These are small, slightly elongated bacilli. These 
bacteria are remarkable in that in pure cultures very small amounts 
of organic matter in the media act as antiseptics. They appear to be 
able to depend on mineral substances for their food. These bacteria 
are extremely important, for the plants take up most of their nitrogen 
in the form of nitrates. These changes are mostly produced in the 
surface soil. If the reaction of the soil becomes acid growth ceases. 
Soil bacteriologists are studying the nitrifying power of different types 
of soil under identical conditions. The process being one of oxidation, 
the access of air is necessary. 
Denitrification.-This is a reducing process. The nitrate is made 
to yield up a part or all of its oxygen and thus becomes changed to 
nitrites and to ammonia and even to free nitrogen. The partial change 632 
APPLIED MICROBIOLOGY 
does not rob the soil of its available nitrogen as does the total change, 
for the nitrites and ammonia may be changed by the nitrifying bacteria 
to nitrates. These bacteria exist normally in most soils and are especially 
abundant in manure. There are three different types of nitrogen 
reduction. (1) The reduction of nitrates to nitrites and ammonia. 
(2) The reduction of nitrates and nitrites to gaseous oxides of nitrogen. 
(3) The reduction of nitrites with the development of free nitrogen 
gas.. 
Nitrogen Fixing Bacteria.--Helbrigel in 1886 demonstrated that 
certain plants were able to use the nitrogen of the air and this ap- 
parently through the aid of bacteria growing in their roots. These 
root bacteria are named B. radicicola. They produce enlargements 
(tubercles) on the roots. 
According to Ball,1 there is no reasonable doubt but that B. radicicola 
can and usually does remain active for very long periods in soil devoid 
of leguminous vegetation. Furthermore, the bacterium diffuses at a 
very considerable rate through soils that are in proper condition; 
therefore, if a soil should be found lacking the organism, it is illogical 
to attempt to introduce it artificially without having first made the 
soil fit for the development of the bacteria. 
It has not been shown by anyone that increased powers of resistance 
to unfavorable conditions of certain varieties are at all correlated 
with their enhanced "greed for nitrogen." Moreover, it is far from 
being proven that any one race or "physiologic species" is really more 
virile than another. Greig-Smith2 has shown that as many as three 
races are sometimes present in one and the same tubercle. Possibly, 
therefore, fixation of nitrogen may occur most rapidly only when two 
or more of these races are growing together. 
Buchanan3 has recently made a minute morphologic study of B. 
radicicola. Some of his conclusions are as follows: 
1. Considerable variation in the morphology of B. radicicola may 
be induced in artificial media by the use of appropriate nutrients. 
Of the salts of the organic acids, sodium succinate brings about the 
most luxuriant development and the production of the greatest variety 
of bacteroids. 
2. B. radicicola in the roots of the legumes shows the same type of 
bacteroids as may be found in suitable culture media. On the other 
hand, there is little or no correspondence between the type of bacteroid 
produced in culture media by a certain organisrh and that produced 
in the nodule by the same form. 
3. It is probable that the term B. radicicola includes an entire group 
of closely related varieties or species, which differ from each other to 
some degree in morphologic characters. 
1 Ball, O. M. A contribution to the Life History of B. radicicola Beij., Centralbl. f. 
Bakt., etc., 1909, II Abt., xxiii, 47. 
2 Greig-Smith. Jour. Soc. Chern. Indust., 1907, No. 7. 
3 Buchanan, R. E. The Bacteroids of Bacillus radicicola. Centralbl. f. Bakt., etc., 
1909, II Abt., xxiii, 59. BACTERIA IN SEWAGE 
633 
4. The nodule organism resembles morphologically both the yeasts 
and the bacteria. The difference between this form and those ordi- 
narily included under the terms Bacillus and Pseudomonas justify the 
use of a separate generic name, Rhizobium. 
In 1893 Winogradsky furnished proof that there are in the soil bac- 
teria which are outside of the plant roots performing the same function 
as those within the roots. These bacilli he called Clostridium pasteur- 
ianum. They are anaerobic and produce spores. Their power to fix 
nitrogen is increased in presence of sugar and lessened in presence 
of nitrogenous substances. 
Beyerinck in 1901 described two aerobic species of nitrogen-fixing 
bacteria. Later Bailey described three additional species. These were 
called Azotobacter. These studies have already led to the inoculation 
of soils and to the investigation of the kind of soils and crops best 
fitted for the growth of these bacteria. Many impoverished soils have 
already been greatly improved. There are probably many other 
varieties of bacteria capable of fixing nitrogen, because one can hardly 
examine the roots of any leguminous plants with outfinding tubercles 
different. The use of seed inoculated with the special variety of bacteria 
suitable for the plant and the soil is already largely practised. 
Bacteria and Soil Minerals.-Some of the bacterial products act 
upon the inorganic constituents of the soil. The carbonic dioxide and 
the organic acids act upon compounds of lime and magnesia, practically 
insoluble in water, to form more soluble substances. The same is true 
of the rock phosphates, the silicate of potassium, sulphates, etc. 
Scientific farming is beginning to make use of the knowledge already 
acquired, and there is reason to hope that great practical advantages 
will flow from the investigation of the relation of bacteria to soil 
exhaustion and replenishment. 
The effect of excessive bacterial development appears at times to 
be harmful to the soil. Each crop seems to favor the growth of certain 
varieties, and the exhaustion of the soil which follows the constant 
raising of the same crop is now suspected to be due in part at least 
to the continuance of a few restricted species of bacteria in the 
soil, which failing to produce all the necessary substances for the 
nutrition of the special crop, vegetation suffers, or again the bacteria 
finally entirely dissipate substances already in the soil necessary to 
growth. 
The application of manure not only adds food for plant life, but also 
countless numbers of bacteria which make the food more available. 
The greatest number of bacteria are contained a little below the sur- 
face of the soil, where they are protected from drying and sunlight and 
are in contact with oxygen and with the roots and other food of the 
superficial soil. 
Bacteria in Sewage.-The materials which flow from our sewers are 
a menace to public health, mainly because they so frequently contain 
pathogenic bacteria. The other products of men and animals are 
offensive, but rarely concentrated enough in drinking water to be 634 
APPLIED MICROBIOLOGY 
appreciably deleterious. Sewage can be made harmless by being 
sterilized, but can be freed from offense only by the destruction of 
organic matter. This, except when chemical precipitants are used, is 
almost wholly obtained through bacterial processes. The putrefying 
value of soil has long been recognized. This is largely due to the action 
of the soil bacteria. 
In 1895, the Englishman, Cameron, introduced the "septic tank," 
which was a covered cemented pit. The sewage admitted at the bottom 
flowed out at the top, after about twenty-four hours' subjection to 
anaerobic conditions. The anaerobic bacteria during this time ferment 
the organic matter energetically, liquefy it, and develop abundant gas. 
The knowledge that soil and sand filters act not only mechanically 
but also and perhaps chiefly bacteriologically, having been acquired, 
intermittent soil filtration was established as one of the best means of 
bacteriologically purifying sewage. The sewage is conducted to the 
beds, allowed to pass through, and then after a few hours again poured 
on. This purification is based chiefly on the action of the aerobic 
bacteria in the upper layers of the soil or sand. The best practical 
results are obtained by combining the two processes; first the anaerobic 
treatment is used to break down the solid materials, and then the 
intermittent sand filtration, to oxidize the compounds and render 
these products harmless. With low temperatures the chemical changes 
are very much lessened and the filter beds act more like pure 
mechanical filters. 
The anaerobic bacteria change the proteid substances into simple 
chemical compounds, among which is ammonia. The carbohydrates 
are changed into gaseous compounds, acids, etc. The gases are mainly 
nitrogen, carbon dioxide, and marsh gas. The bacterial changes pro- 
duced in sewage poured on contact beds made of coarse coke, 
clinkers, or other material act much as in the sand filters after the 
filtration. 
Varieties of Bacteria in Filter Beds and Septic Tanks.-The septic 
tanks all contain spore-bearing bacilli which destroy cellulose, others 
that attack nitrogenous compounds. The cocci are in a minority. 
The filter beds have a number of small, non-spore-bearing bacilli; some 
of these change ammonia into nitrites and nitrates. There are also 
denitrifying bacteria. As before mentioned, the bacterial efficiency 
of the bed is increased with suitable temperature and much lessened 
with low temperature. 
Sewage Farming.-The action of bacteria is availed of in disposing 
of sewage over fields. The amount of sewage which can be poured on 
a certain area is limited. One acre of land can usually take care of 
the sewage from one hundred persons. If too much is poured on, it 
runs off impurified or clogs the soils and prevents the access of oxygen 
to aerobic bacteria. In warm weather evaporation and bacterial 
activities are much greater than in cold weather. So far as experience 
shows, those who eat vegetables from these small farms contract no 
disease from them. BACTERIAL FERMENTATION 
635 
The Curing of Tobacco.-The curing of tobacco is apparently due 
partly to bacterial processes and partly to the action of leaf enzymes. 
The preservation of foods against decomposition by bacteria, yeasts, 
molds, and higher fungi is obtained by using processes which will 
prevent the growth of microorganisms. Drying, exposure to wood 
smoke with consequent absorption of creosote, the addition of salt 
and sugar, of acids such as vinegar, spices, germicides such as boracic 
acid, formaldehyde, all are familiar methods of making foods unsuitable 
for bacterial growth. 
Instead of using food preserved by drying or chemicals, products 
may be kept at temperatures too low for bacterial growth. Cold 
storage of meats, eggs, vegetables, etc., is now common. 
The sterilization of food substances by heat with protection from 
infection afterward is made use of extensively in the canning of fruits 
and vegetables. Care must be taken that absolutely all bacteria are 
killed, for otherwise decomposition will finally occur. 
Vinegar Making.-Vinegar is made from some weak alcoholic solution 
by the union of alcohol with oxygen. This oxidation can be- brought 
about by a purely chemical process. When vinegar is formed in the 
usual way bacteria are essential. The scum on the surface of the 
fermenting alcohol is a mass of microorganisms. The mother of vinegar 
was named. mycoderma by Pearson. Kutzing showed that this was 
composed of living cells. Hansen proved these to be bacteria. We 
now know there are many varieties of bacilli capable of producing 
this fermentation. Each variety has its .own optimum temperature and 
differs in the amount of acid it produces. Most of these have the 
peculiarity of growing at high temperatures into long threads without 
any traces of division. At low temperatures they produce long threads 
with swollen centres. The usual vinegar is made by using the variety 
of bacilli prevalent in the surroundings, but the custom is growing of 
adding to the pasteurized alcoholic solution the special variety desired 
in pure culture. 
Sauerkraut.-This is cabbage leaves shredded, slightly fermented, 
and. prevented from decay by the lactic acid bacteria. At first both 
yeasts and bacteria increase together, but with the increase in acidity 
all growth ceases. Putrefaction is prevented by the same cause. The 
lactic acid bacteria are the same as those found in sour milk. 
Ensilage.-The fermentation here is believed to be due partly to 
enzymes in the corn tissues and partly to bacterial action. The first 
changes are due chiefly to the enzymes. 
The Bacterial Diseases of Plants.-These are probably as serious and 
varied for plants as for animals. The pear blight, the wilt disease 
of melons, the brown rot of tomatoes, the black rot of cabbages are 
examples. These plant diseases can be communicated by means of 
the pathogenic pure cultures of bacteria experimentally just as readily 
as animal diseases by their specific bacteria. 
Bacterial Fermentation in Relation to Miscellaneous Products.- 
Pasteur in 1857 explained the process of fermentation as due to the 636 
APPLIED MICROBIOLOGY 
action of microorganisms. He demonstrated that the change of sugar 
into lactic acid only occurred when living bacilli were present. If 
the fluid was sterilized the fermentation ceased. lie stated that "or- 
ganic liquids do not alter until a living germ is introduced into them." 
When the action is direct we speak of an organized ferment; when it 
is indirect, that is, due to the cell product, we call it an unorganized 
soluble ferment or enzyme. Similar enzymes are produced by the 
cells of the animal tissues, such as ptyalin, pepsin, and trypsin. Pas- 
teur's work led to the conclusion that the different fermentations were 
due to different varieties of organisms. The major part of fermenta- 
tion is due to yeast. Some important fermentations are due to bacteria 
and a few to the molds. 
Wines and Beers.-Alcoholic Fermentation.-If there is a develop- 
ment of the yeast cells in a solution of grape-sugar we have a fermenta- 
tion of the sugar with a final development of alcohol and carbon dioxide. 
It is thus that beers and wines are developed. When the carbohydrate 
is in the form of starch this is first converted into sugar and then later 
into the final products. If the sugar is in the form of saccharose, it is 
first changed by the yeast ferments to glucose. In all these three 
forms of fermentation the sugar is changed into alcohol and carbonic 
acid. When the alcohol reaches about 13 per cent, it stops further 
fermentation. These yeasts called saccharomyces comprise a number 
of distinct varieties, some of which are cultivated while others, called 
"wild yeasts," propagate themselves. The distillery, brewery, and 
wine industries each make use of special yeasts and special conditions. 
The rising of bread is one of the most common uses of fermentation 
by yeast. The yeast acts upon the sugar made by the diastase from 
the starch. The resulting CO2 and alcohol create myriads of little 
bubbles in the dough. 
Diseases in Beer and Wines.-Hansen, Pasteur, and others demon- 
strated that the spoiling of beers and wines was due to the development 
of varieties of bacteria and yeasts which produce different kinds of 
fermentation from that desired. These produce alterations in flavor, 
bitterness, acidity. CHAPTER XL IX. 
THE DESTRUCTION OE BACTERIA BY CHEMICALS. 
PRACTICAL USE OF DISINFECTANTS. 
Many substances, when brought in contact with bacteria, combine 
with their cell substance and destroy the life of the bacteria. While 
in the vegetative stage bacteria are much more easily killed than when 
in the spore form, and their life processes are inhibited by substances 
less deleterious than those required to destroy them. 
Bacteria, both in the vegetative and in the spore form, differ 
among themselves considerably in their resistance to the poisonous 
effectsof chemicals. The reason for this is not wholly clear, but it 
is connected with the structure and chemical nature of their cell 
substance. 
Chemicals in sufficient amount to destroy life are more poisonous 
at temperature's suitable for the best growth of bacteria than at lower 
temperatures, and act more quickly upon bacteria when they are sus- 
pended in fluids singly than when in clumps, and in pure water rather 
than in solutions containing organic matter. The increased energy of 
disinfectants at higher temperatures indicates in itself that a true 
chemical reaction takes place. In estimating the extent of the de- 
structive or inhibitive action of chemicals the following degrees are 
usually distinguished: 
1. The growth is not permanently interfered with, but the patho- 
genic and zymogenic functions of the organism are diminished- 
attenuation. This loss of function is usually quickly recovered. 
2. The organisms are not able to multiply, but they are not destroyed 
-antiseptic action. When transferred to a suitable culture fluid free 
of the disinfectant these bacteria are capable of reproduction. 
3. The vegetative development of the organisms is destroyed, but 
not the spores-incomplete or complete sterilization or disinfection, 
according as to whether spores are present in the organisms exposed 
and as to whether these spores are capable of causing infection. 
4. Vegetative and spore formation are destroyed. This is complete 
sterilization or disinfection.1 
The methods employed for the determination of the germicidal 
action of chemical agents on bacteria are, briefly as follows: 
1 Disinfection strictly defined is the destruction of all organisms and their products 
which are capable of producing disease. Sterilization is the destruction of all saprophytic 
as well as parasitic bacteria. It is not necessary in most cases to require disinfectants 
to be capable of sterilizing infected materials containing spores, for there are but few 
varieties of pathogenic bacteria which produce spores. 638 
APPLIED MICROBIOLOGY 
If it is desired to determine the minimum concentration of the chemi- 
cal substance required to produce complete inhibition of growth we 
proceed thus: A 10 per cent, solution of the disinfectant is prepared 
and 1 c.c., 0.5 c.c., 0.3 c.c., 0.1 c.c., etc., of this is added to 10 c.c. of 
liquefied gelatin, agar, or bouillon, or, more accurately 10 c.c. minus 
the amount of solution added, in so many tubes. The tubes then 
contain 1 per cent., 0.5 per cent., 0.3 per cent., and 0.1 per cent, of 
the disinfectant. The fluid media in the tubes are then inoculated with 
a platinum loopful of the test bacteria. The melted agar and gelatin 
may be simply shaken and allowed to remain in the tubes, and watched 
as to whether any growth takes place, or the contents of the tubes 
may be poured into Petri dishes, where the development or lack of 
development of colonies and the number can be observed. If no 
growth occurs in any of the dilutions, higher dilutions are tested. 
Bacteria that have been previously injured in any way will be inhib- 
ited by much weaker solutions of chemicals than will vigorous cells. 
The same test can be made with material containing only spores. 
If it is desired to determine the degree of concentration required 
for the destruction of vegetative development, the organism to be 
used is cultivated in bouillon, and into each of a series of tubes is 
placed a definite amount of diluted culture from which all clumps of 
bacteria have been filtered; to these a definite amount of watery solution 
of different percentages of the disinfectant is added. At intervals of 
one, five, ten, fifteen, and thirty minutes, one hour, and so on a small 
platinum loopful of the mixture is taken from each tube and inoculated 
into 10 c.c. of fluid agar or gelatin, from which plate cultures are made. 
Whenever it is possible that the antiseptic power of the disinfectant 
approaches somewhat the germicidal, it is necessary to inoculate a second 
series of tubes from the first so as to decrease still further the amount 
of antiseptic carried over. The results obtained are signified as follows: 
x per cent, of the disinfectant in watery solution and at x temperature 
kills the organism in twenty minutes, y per cent, kills in one minute, 
and so on. If there be any doubt whether the trace of the disinfectant 
carried over with the platinum loops may have rendered the gelatin 
unsuitable for growth, thus falsifying results, control cultures should 
be made by adding bacteria which have been somewhat enfeebled by 
slight contact with the disinfectant to fluid to which a similar trace of 
the disinfectant has been added. If the strength of the disinfectant 
is to be tested for different substances it must be tested in these sub- 
stances or their equivalent, and not in water. 
The disinfectant to be examined should always be dissolved in an 
inert fluid, such as water; if, on account of its being difficultly solu- 
ble in water, it is necessary to use alcohol for its solution, control experi- 
ments may be required to determine the action of the alcohol on the 
organism. Sometimes, as in the case of corrosive sublimate, the chemi- 
cal unites with the cell substance to form an unstable compound, which 
inhibits the growth of the organism for a time before destroying it. 
If this compound is not broken up in the media, it will probably not THE STANDARDIZATION OF DISINFECTANTS 
639 
be in the body. In some tests it is of interest to break up this union 
and note then whether the organism is alive or dead. With corrosive 
sublimate the bacteria die in fifteen to thirty minutes after the union 
occurs. 
In the above determinations the absolute strength of the disinfect- 
ant required is considerably less when culture media poor in albumin 
are employed than when the opposite is the case. Cholera spirilla 
grown in bouillon containing no peptone or only 0.5 per cent, of pep- 
tone are destroyed in half an hour by 0.1 per cent, of hydrochloric 
acid; grown in 2 per cent, peptone-bouillon, their vitality is destroyed 
in the same time on the addition of 0.4 per cent. HC1. In any case 
the organisms to be tested should all be treated in exactly the same 
way and the results accompanied by a statement of the conditions 
under which the tests were made. It is becoming the custom to 
state the power of a disinfectant in terms of comparison with pure 
carbolic acid. A substance which had the same destructive power 
in a 1 to 1000 solution as carbolic acid in a 1 to 100 solution would 
be rated as of a strength ten times that of carbolic acid. 
The following table gives the results and methods used in an actual 
experiment to test the effect of blood-serum upon the disinfecting 
action of bichloride of mercury and carbolic acid upon bacteria: 
Test for the Difference of Effect of Bichloride of Mercury and Carbolic Acid 
Solutions on Typhoid Bacilli in Serum and in Bouillon. 
Time. 
1' 
3' 
5' 
10' 
20' 
30' 
45' 
1 
hr. 
1 2 
hrs. 
2 
hrs. 
A. Serum . 2.5 C-C.! 
HgCh sol. 1:1000 2.5c.cJ 
Typhoid broth culture. J 
+ 
+ 
+ 
- 
- 
- 
- 
- 
- 
- 
f Solution 
equals 1: 2000 
[ bichloride. 
B. Bouillon . 2.5 c.c.l 
HgChsol. 1:1000 2.5c.c.> 
Typhoid broth culture. J 
- 
Same. 
C. Serum . 2.5 c.c.l 
Carbolicsol. 5% 2.5 c.c. f 
Typhoid broth culture. J 
+ 
+ 
- 
(Solution 
equals 2|% 
[ carbolic acid. 
D. Bouillon . 2.5 c.c.) 
Carbolicsol. 5% 2.5c.c. > 
Typhoid broth culture. J 
+ 
- 
Same. 
- Indicates total destruction of bacteria with no growth in media 
+ Indicates lack of destruction of bacteria with growth in media. 
THE STANDARDIZATION OF DISINFECTANTS. 
Rideal and Walker were the first to urge a useful method for doing 
this. 
In carrying out the test the various factors must be carefully con- 
trolled, thus: Time: this should be constant, the strength of the 
disinfectant being the variant. Test organisms: a standard culture of 640 
APPLIED MICROBIOLOGY 
the typhoid bacillus (Hopkins' strain) is used to avoid any variations 
due to the different degrees of resistance of various strains. The 
culture should be subinoculated three days before used. Medium: 
a standard meat-extract broth 1.5 per cent, acid to phenolphthalein; 
10 c.c. to a tube is employed. T emperature: this test is done at 
20° C. This is important, as the germicidal activity increases with the 
temperature. Constant amount of culture used: 0.1 c.c. of the twenty- 
four-hour broth culture is added to 5 c.c. of the disinfectant solution. 
This is more accurate than the drop method. Amount inoculated: 
it is essential that the same amount be inoculated from each dilution. 
Platinum loops made of 23 United States gauge wire, the loops being 
4 mm. in diameter are employed. Several are used, being left on a 
rack after sterilization so as to be cold when needed. The loop is bent 
at an angle of 45 to the shank. 
The actual test is carried out as follows: A 5 per cent, carbolic solu- 
tion (phenol C. P.), is prepared and standardized by bromine titration. 
From this freshly prepared 1 to 90 to 1 to 100 and 1 to 110 dilutions 
are made as needed. The necessary dilutions of germicide are then 
prepared. Wide jumps in the dilutions are made and then narrowed 
as the limits of the disinfectant to be tested are determined. 
Five test-tubes are arranged in a row in a water bath at 20° C. and 
the solutions added in 5 c.c. amounts. Time must be allowed for the 
solution to reach 20° C. If the bath be large enough, little attention 
is needed to keep the temperature constant. The culture having been 
brought to 20° C. is then added in 0.1 c.c. amounts and the tubes shaken, 
an interval of 30 seconds allowed between each tube. Subinoculation 
of the first tube is then made after thirty seconds, which gives an interval 
two and one-half minutes after inoculation. The tubes are then sub- 
inoculated in order at thirty-second intervals, giving an interval for 
each of two and one-half minutes after inoculation, and starting at the 
first, gives an interval of five minutes, etc. 
It is possible to use ten tubes, as each step can be done in less than 
fifteen seconds if properly arranged, allowing a much wider range of 
each test. It is not necessary to keep the cotton plugs in the tubes 
during the operation nor to remove the tubes from the bath to obtain 
the loopful for inoculation. The loop is plunged to the bottom, care 
being taken not to touch the sides of the tubes, and care should also be 
taken that a loopful is carried away each time. The broth tubes are 
incubated for forty-eight hours and then examined for growth. 
The following are given as two examples: 
Time of 
exposure: 
2.5 
5 
7.5 
10 
12.5 
15 
Phenol 
Sample. 
Dilution. 
min. 
min. 
min. 
min. 
min. 
min. 
coefficient. 
Phenol 
1 
to 90 
+ 
- 
- 
- 
- 
- 
1 
to 100 
+ 
+ 
+ 
- 
- 
- 
100)550 
5.5 
Disinfectant A. 
1 
to 450 
+ 
+ 
- 
- 
- 
- 
1 
to 550 
+ 
+ 
+ 
- 
-- 
- 
1 
to 600 
+ 
+ 
+ 
+ 
- 
- THE STANDARDIZATION OF DISINFECTANTS 
641 
Sample. 
Dilution. 
2.5 
min. 
Time of exposure: 
15 
min. 
Phenol 
coefficient. 
min. 
7.5 
min. 
10 
min. 
12.5 
min. 
Phenol .... 
1 to 100 
+ 
+ 
+ 
- 
- 
- 
110)650 
.1 to 110 
+ 
+ 
+ 
+ 
+ 
- 
5.191 
Disinfectant A. 
1 to 600 
+ 
+ 
+ 
+ 
- 
- 
1 to 650 
+ 
+ 
+ 
+ 
+ 
- 
1 to 700 
+ 
+ 
+ 
+ 
+ 
+ 
These tables not only serve as an example, but also show that 
except many repetitions of the tests are made and averaged consider- 
able variations in the results may be obtained. A report of 5.5 or 
5.1 is equally accurate in the test here reported of the Rideal-Walker 
method, even with certain improvements added. With practice, and 
by selection of the dilutions to be employed, the operator evidently 
can regulate the time factor so that fairly uniform results are obtain- 
able. On the other hand it not infrequently happens that if more 
than one carbolic dilution is employed, more than one time period is 
open to comparison. For these reasons Anderson and McClintic have 
modified the test by setting two time limits two and one-half and fif- 
teen minutes and taking the average. The following is an example: 
Time of exposure: 
2.5 
5 
7.5 
10 
12.5 
15 
Phenol 
Sample. 
Dilution. 
min. 
min. 
min. 
min. 
min. 
min. 
coefficient. 
Phenol 
1 
to 80 
- 
- 
- 
- 
- 
- 
375 
+ 650 
1 
to 90 
+ 
- 
- 
- 
- 
- 
- 
1 
to 100 
+ 
+ 
+ . 
- 
- 
- 
80 
110 
1 
to 110 
4- 
+. 
+ 
+ 
+ 
- 
- 
= 
2 
Disinfectant A. 
1 
to 350 
- 
- 
- 
1 
1 
to 375 
to 400 
+ 
- 
- 
4.69 
+ 5.91 
1 
to 425 
+ 
+ 
- 
- 
- 
- 
2 
1 
to 450 
+ 
+ 
- 
- 
- 
- 
1 
to 500 
+ 
+ 
- 
- 
- 
- 
5 
.30 
1 
to 550 
+ 
+ 
+ 
- 
- 
- 
1 
to 600 
+ 
+ 
+ 
+ 
- 
- 
1 
to 650 
+ 
+ 
+ 
+ 
+ 
1 
to 700 
+ 
+ 
+ 
+ 
+ 
+ 
1 
to 750 
+ 
+ 
. + 
+ 
+ 
+ 
Disinfectants vary widely in their germicidal properties, depending 
on whether organic matter is present or not. As under practical condi- 
tions organic matter is usually present, it is of some importance to 
know how far organic matter decreases the efficiency. 
For the purpose of obtaining comparable results, Anderson and 
McClintic have, suggested the use of peptone 10 per cent, and gelatin 
5 per cent, in distilled water. One part of the culture is mixed with 
10 parts of the organic solution, 1.1 c.c. being then added to a series 
of dilution tubes containing 4 c.c. In determining the coefficient 
allowance must be made for the added amount of organic matter. 
The modified methods of Anderson and McClintic1 are called the 
1 See Hygienic Laboratory Bulletin No. 82, for further details and apparatus for 
simplifying the steps of the test. 642 
APPLIED MICROBIOLOGY 
" hygienic laboratory phenol coefficient," with or without organic 
matter. Any organic matter may be used in the test to approach the 
special conditions under which a disinfectant is to be used. 
In comparing the value of disinfectants the cost as well as the 
coefficient must be considered. This is best stated in terms of the 
relative cost of 100 units of efficiency as compared with pure 
phenol = 100, thus: 
Cost of disinfectant per gallon 
Cost of phenol per gallon 
(Cost ratio) -s- 
Coefficient of disinfectant. 
Coefficient of phenol (1). 
= the efficiency, X 100 = relative cost per 100 units. 
Antiseptic Value.-With certain disinfectants there is sufficient of 
the disinfectant carried over by the loop to exert antiseptic action 
and growth does not occur. If this is not taken in consideration a 
disinfectant will be given an excessively high coefficient. No satis- 
factory method has been devised to avoid this difficulty. The inocu- 
lated broth tube may be shaken and a loop or more inoculated from it 
to second broth tube, in this way diluting the disinfectant still farther. 
Chick1 has attempted to overcome the difficulty in the case of 
mercury-containing disinfectants by adding 0.2 c.c. of a saturated 
watery solution of hydrogen sulphide to each tube of broth. 
Many substances which are strong disinfectants become altered 
under the conditions in which they are used, so that they lose a portion 
or all of their germicidal properties; thus, quicklime and milk of lime 
act by means of their alkali and are disinfecting agents only so long 
as sufficient calcium hydroxide is present. If this is changed by the 
carbon dioxide of the air into carbonate of lime it becomes harmless. 
Bichloride of mercury and many other chemicals form compounds 
with many organic and inorganic substances, which, though still 
germicidal, are much less so than the original substances. Solutions of 
chlorine, peroxides, etc., when in contact with an excess of organic 
matter soon become inert because of the chemical compounds formed. 
The Disinfecting Properties of Inorganic Compounds.-Bichloride of 
Mercury.-This substance, which dissolves in 16 parts of cold water, 
when present in 1 part in 100,000 in nutrient gelatin or bouillon, 
inhibits the development of most forms of bacteria. In water 1 part 
in 50,000 will kill many varieties in a few minutes, but in bouillon 
twenty-four hours may be needed. With organic substances its power 
is lessened, so that 1 part to 1000 may be required. Most spores are 
killed in 1: 500 watery solution within one hour. Corrosive sublimate 
is less effective as a germicide of alkaline fluids containing much 
albuminous substance than in watery solution. In such fluids, besides 
loss in other ways, precipitates of albuminate of mercury are formed 
which are at first insoluble, so that a part of the mercuric salt does 
not really exert any action. In alkaline solutions, such as blood, blood- 
serum, pus, sputum, tissue fluids, etc., the soluble compounds of 
mercury are converted into oxides or hydroxides. 
1 Journal of Hygiene, 1908, viii, 654. THE STANDARDIZATION OF DISINFECTANTS 
643 
For ordinary use, where corrosive sublimate is employed, solutions 
of 1 to 500 and 1 to 2000 will suffice, when brought in contact with 
bacteria in that strength, to kill the vegetative forms within from one 
to twenty minutes, the stronger solution to be used when much organic 
matter is present. 
Mercuric chloride volatilizes slowly and it is better to wash off 
walls after use of bichloride solutions. Solutions of this salt should 
not be kept in metal receptacles. Mercuric chloride solution has dis- 
advantages in that it corrodes metals, irritates the skin, and forms 
almost inert compounds with albuminous matter. In order to avoid 
accidents, solutions of this odorless disinfectant should be colored by 
some dye. 
Biniodide of Mercury.-This salt is very similar in its effect to the 
bichloride. 
Nitrate of Silver.-Nitrate of silver in watery solution has about 
one-fourth the value of the bichloride of mercury as a disinfectant, but 
nearly the same value in inhibiting growth. In albuminous solutions 
it is equal to bichloride of mercury. Compounds of silver nitrate and 
albuminous substances have been used because of the absence of 
irritative properties combined with moderate antiseptic power. 
Sulphate of Copper.-This salt has about 50 per cent, of the value 
of mercuric chloride. It has a quite remarkable affinity for many 
species of algae, so that when in water 1 to 1,000,000 it destroys many 
forms; 1 to 400,000 destroys typhoid bacilli in twenty-four hours when 
the water has no excessive amount of organic material. It is not known 
to be poisonous in this strength, so that it can be temporarily added 
to water supplies. 
Sulphate of Iron.-This is a much less powerful disinfectant than 
sulphate of copper. A 5 per cent, solution requires several days to 
kill the typhoid bacilli. It can only be considered as a mild antiseptic 
and deodorant. 
Zinc Chloride.-This is very soluble in water, but is a still weaker 
disinfectant than copper sulphate. 
Sodium Compounds.-A 30 per cent, solution of NaOH kills anthrax 
spores in about ten minutes, and in 4 per cent, in about forty-five 
minutes. One per cent, kills vegetative forms in a few minutes. 
Sodium carbonate kills spores with difficulty even in concentrated 
solution, but at 85° C. it kills spores in from eight to ten minutes. 
It is used frequently to cover metallic instruments. A 5 per cent, 
solution kills in a short time the vegetative forms of bacteria. Even 
ordinary soapsuds have a slight bactericidal as well as a marked 
cleansing effect. The bicarbonate has almost no destructive effect on 
bacteria. 
Calcium Compounds.-Calcium hydroxide, Ca(OH)2, is a powerful 
disinfectant; the carbonate, on the other hand, is almost without effect. 
The former is prepared by adding one pint of water to two pounds 
of lime (quicklime, CaO). Exposed to the air the calcium hydrate 
slowly becomes the inert carbonate. A 1 per cent, watery solution 644 
APPLIED MICROBIOLOGY 
of the hydroxide kills bacteria which are not in the spore form within 
a few hours. A 3 per cent, solution kills typhoid bacilli in one 
hour. A 20 per cent, solution added to equal parts of feces or other 
filth and mixed with them will completely sterilize them within two 
hours. 
Effect of Acids.-An amount of acid which equals 40 c.c. of normal 
hydrochloric acid per liter is sufficient to prevent the growth of all 
varieties of bacteria and to kill many. Twice this amount destroys 
most bacteria within a short time. The variety of acid makes little 
difference. Bulk for bulk, the mineral acids are more germicidal than 
the vegetable acids, but that is because their molecular weight is so 
much less. A 1 to 500 solution of sulphuric acid kills typhoid bacilli 
within one hour. Hydrochloric acid is about one-third weaker, and 
acetic acid somewhat weaker still. Citric, tartaric, malic, formic, 
and salicylic acids are similar to acetic acid. Boric acid destroys 
the less resistant bacteria in 2 per cent, solution and inhibits the 
others. 
Gaseous Disinfectants.-The germicidal action of gases is much 
more active in the presence of moisture than in a dry condition. 
Sulphur Dioxide (SO2).-Numerous experiments have been made 
with this gas owing to the fact that it has been so extensively used for 
the disinfection of hospitals, ships, apartments, clothing, etc. This gas 
is a much more active germicide in a moist than in a dry condition; 
due, no doubt, to the formation of the more active disinfecting agent- 
sulphurous acid (H2SO3). In a pure state anhydrous sulphur dioxide 
does not destroy spores, and is not certain to destroy bacteria in the 
vegetative form. Sternberg has shown that the spores of the Bacillus 
anthracis and Bacillus subtilis are not killed by contact for some time 
with liquid SO2 (liquefied by pressure). Koch found that various 
species of spore-bearing bacilli exposed for ninety-six hours in a dis- 
infecting chamber to the action of SO2, in the proportion of from 4 to 
6 per cent, by volume, were not destroyed. In the absence of spores, 
however, the anthrax bacillus in a moist condition, attached to silk 
threads, was found by Sternberg to be destroyed in thirty minutes in 
an atmosphere containing 1 volume per cent. 
As the result of a large number of experiments with SO2 as a disin- 
fectant it has been determined that an " exposure for eight hours to 
an atmosphere containing at least 4 volumes per cent, of this gas in 
the presence of moisture" will destroy most, if not all, of the patho- 
genic bacteria in the absence of spores. Four pounds of sulphur 
burned for each 1000 cubic feet will give an excess of gas. 
Peroxide of Hydrogen (H2O2).-This is an energetic disinfectant, and 
in 2 per cent, solution (about 40 per cent, of the ordinary commercial 
article) will kill the spores of anthrax in from two to three hours. A 
20 per cent, solution of a good commercial hydrogen peroxide solution 
will quickly destroy the pyogenic cocci and other spore-free bacteria. 
It combines with organic matter, becoming inert. It is prompt in its 
action and not poisonous, but apt to deteriorate if not properly kept. THE STANDARDIZATION OF DISINFECTANTS 
645 
Chlorine.-Chlorine is a powerful gaseous germicide, owing its 
activity to its affinity for hydrogen and the consequent release of 
nascent oxygen when it comes in contact with microorganisms in 
moist condition. It is, therefore, a much more active germicide in 
the presence of moisture than in a dry condition. Thus, Fischer and 
Proskauer found that dried anthrax spores exposed for an hour in 
an atmosphere containing 44.7 per cent, of dry chlorine were not 
destroyed; but if the spores were previously moistened and w'ere 
exposed in a moist atmosphere for the same time, 4 per cent, was 
effective, and when the time was extended to three hours 1 per cent, 
destroyed their vitality. The anthrax bacillus, in the absence of 
spores, was killed by exposure in a moist atmosphere containing 1 part 
to 2500 for twenty-four hours. 
In watery solutions 0.2 per cent, kills spores within five minutes and 
the vegetative forms almost immediately. One part in one million is 
sufficient to destroy typhoid bacilli in a water containing little organic 
matter in the course of a few hours. In water with much organic matter 
a much greater amount is required. 
Chlorinated Lime (Called "Chloride of Lime"). - Chlorinated lime 
is made by passing nascent chlorine gas over unslaked lime. It should 
not contain less than 10 per cent, of available chlorine, and can now be 
obtained containing 30 per cent. It should have a strong odor of 
chlorine. Its efficacy depends on the chlorine it contains in the form of 
hypochlorites. The calcium hypochlorite is readily broken up into 
hypochlorous acid. A solution in water of 0.5 to 1 per cent, of chlorin- 
ated lime will kill most bacteria in one to five minutes, and 1 part 
in 100,000 will destroy typhoid bacilli in twenty-four hours. A 5 per 
cent, solution usually destroys spores within one hour. Chlorinated 
lime not only bleaches, but destroys fabrics. 
The Hypochlorites (Labarraque's Solution). - Solutions of hypo- 
chlorites are practically the same as solutions of chlorinated lime and 
are much more expensive. 
Bromine and iodine are of about the same value as chlorine for 
gaseous disinfectants, in the moist condition; but, like chlorine, they 
are not applicable for general use in house disinfection, owing to their 
poisonous and destructive properties; they have a use in sewers and 
similar places. 
Trichloride of iodine in 0.5 per cent, solution destroys the vegetative 
forms of bacteria in five minutes. 
Organic Disinfectants.-Alcohol in 10 per cent, solution inhibits 
the growth of bacteria; absolute alcohol kills bacteria in the vegetative 
form in from several to twenty-four hours. According to Epstein, 50 
per cent, alcohol (in water) has more germicidal power than any other 
strength, the power gradually diminishing with both stronger and 
weaker solutions. 
Formaldehyde.-Formaldehyde, or formic aldehyde, was isolated 
by von Hoffmann in 1867, who obtained it by passing the vapors of 
methyl-alcohol mixed with air over finely divided platinum heated to 646 
APPLIED MICROBIOLOGY 
redness. The methyl-alcohol is oxidized and produces formaldehyde 
as follows: 
ch3oh + o = ch2o + h2o. 
Formaldehyde is a gaseous compound possessed of an extremely 
irritating odor. At a temperature of 68° F. the gas is polymerized- 
that is to say, a second body is formed, composed of a union of two 
molecules of CH2O. This is known as a paraformaldehyde, and is a 
white, soapy body, soluble in boiling water and in alcohol. Formal- 
dehyde is sold in commerce as a clear, watery liquid containing from 
33 to 40 per cent, of the gas and 10 to 20 per cent, of methyl-alcohol, 
its chief impurity. If the commercial solution-ordinarily known in 
the trade as "formalin"-is evaporated or concentrated above 40 per 
cent., paraformaldehyde results; and when this is dried in vacuo over 
sulphuric acid a third body-trioxymethylene-is produced, consisting 
of three molecules of CH2O. This is a white powder, almost soluble 
in water or alcohol, and giving off a strong odor of formaldehyde. 
The solid polymers of formaldehyde, when heated, are again reduced 
to the gaseous condition; ignited, they finally take fire and burn with 
a blue flame, leaving but little ash. When burned they have no 
germicidal properties. 
Formaldehyde has an active affinity for many organic substances, 
and forms with some of them definite chemical combinations. It 
combines readily with ammonia to produce a compound called hexa- 
methylene tetramine, which possesses neither the odor nor the anti- 
septic properties of formaldehyde. This action is made use of in 
neutralizing the odor of formaldehyde when it is desired to dispel it 
rapidly after disinfection. Formaldehyde also forms combinations 
with certain aniline colors-viz., fuchsin and safranin-the shades of 
which are thereby changed or intensified. These dyes are tests for 
aldehydes. These are the only colors, however, which are thus af- 
fected, and as they are seldeom used in dyeing, owing to their liability 
to fade, this effect is of little practical significance. The most deli- 
cate fabrics of silk, wool, cotton, fur, leather, etc., are unaffected in 
texture or color by formaldehyde. Iron and steel are attacked, after 
long exposure, by the gas in combination with watery vapor; but copper, 
brass, nickel, zinc, silver, and gilt work are not at all acted upon. For- 
maldehyde unites with nitrogenous products of decay-fermentation 
or decomposition-forming true chemical compounds, which are odor- 
less and sterile. It is thus a true deodorizer in that it does not replace 
one odor by another more powerful, but forms new chemical compounds 
which are odorless. Formaldehyde lias a peculiar action upon albu- 
min, which it transforms into an insoluble and indecomposable sub- 
stance. It renders gelatin insoluble in boiling water and most acids and 
alkalies. It is from the property of combining chemically with the 
albuminoids forming the protoplasm of bacteria that formaldehyde is 
supposed to derive its bactericidal powers. Formaldehyde is an excel- 
lent preservative of organic products. It has been proposed to make THE STANDARDIZATION OF DISINFECTANTS 
647 
use of this action for the preservation of meat, milk, and other food 
products; but, according to Trillat and other investigators, formalde- 
hyde renders these substances indigestible and unfit for food. It has 
been successfully employed as a preservative of pathologic and histologic 
specimens. 
There are no exact experiments recorded of the physiologic action 
of formaldehyde on the human subject when taken internally. A 1 
per cent, solution has been taken in considerable quantity without 
serious results; and trioxymethylene has been given in doses up to 
90 grains as an intestinal antiseptic. The vapors of formaldehyde 
are extremely irritating to the mucous membrane of the eyes, nose, 
and mouth, causing profuse lacrimation, coryza, and flow of saliva. 
Aronson reports that in many of his experiments rabbits and guinea- 
pigs allowed to remain for twelve and twenty-four hours in rooms 
which were being disinfected with formaldehyde gas were found to 
be perfectly well when the rooms were opened. On autopsy the ani- 
mals showed no injurious effects of the gas. Others have noticed that 
animals, such as dogs and cats, which have accidentally been confined 
for any length of time in rooms undergoing formaldehyde disinfection 
occasionally died from the effects of the gas. Many observers, how- 
ever, have reported that insects, such as roaches, flies, and bedbugs, 
are not, as a rule, affected. The result of these observations would seem 
to indicate that although formaldehyde is comparatively non-toxic to 
the higher forms of animal life, nevertheless a certain degree of caution 
should be observed in the use of this agent. It is important to remem- 
ber that formaldehyde is practically inert as an insecticide except in 
extremely great concentrations. 
The researches of Pottevin and Trillat have shown that the germi- 
cidal power of the gas depends not only upon its concentration, but 
also upon the temperature and the condition of the objects to be steril- 
ized. As with other gaseous disinfectants-viz., sulphur dioxide and 
chlorine-it has been found that the action is more rapid and complete 
at high temperatures-i. e., at 35° to 45° C. (95° to 120° F.)-and 
when the test objects are moist than at lower temperatures and when 
the objects are dry. Still, it has been repeatedly demonstrated by 
actual experiment in rooms that it is possible to disinfect the surface 
of apartments and articles contained in them, under the conditions of 
temperature and moisture ordinarily existing in rooms even in winter, 
by an exposure of a few hours to a saturated atmosphere of formalde- 
hyde gas. The results of numerous experiments have shown that in 
the air 2.5 per cent, by volume of the aqueous solution, or 1 per cent, 
by volume of the gas, are sufficient to destroy fresh virulent cultures of 
the common non-spore-bearing pathogenic bacteria in a few minutes. 
Stahl has shown that bandages and iodoform gauze can be kept 
well sterilized by placing in the jars containing them pieces of a prepa- 
ration of paraformaldehyde in tablet form containing 50 per cent, 
of formaldehyde. The same experimenter has also succeeded in 
making carpets and articles of clothing germ-free by spraying them 648 
APPLIED MICROBIOLOGY 
with 0.5 to 2 per cent, solution of formaldehyde for fifteen to twenty 
minutes without the color of the fabrics being in any way affected. 
The investigations of Trillat, Aronson, Pottevin, and others have 
shown that a concentration of tu^o o of the aqueous solution (40 per 
cent.), equal to YyffTtf °f pure formaldehyde, was safe and sufficiently 
powerful to retard bacterial growth. 
A 2 per cent, watery solution of formalin destroys the vegetative 
forms of bacteria within five to thirty minutes. In our experiments 
formalin has upon the vegetative forms about one-half the strength of 
pure carbolic acid. 
Chloroform (CHC13).-This substance, even in pure form, does not 
destroy spores, although it kills bacteria in vegetative form, even in 
1 per cent, solution.. Chloroform is used practically as an antiseptic 
in antitoxic sera and in blood-serum for culture purposes. The chloro- 
form is expelled from the serum by heating it to 55° C. 
Iodoform (CHI3).-This substance has but very little destructive 
action upon bacteria; indeed, upon most varieties it has no appreciable 
effect whatever. When mixed with putrefying matter, wound dis- 
charges, etc., the iodoform is reduced into soluble iodine compounds, 
which act partly destructively upon the bacteria and partly by uniting 
with poisons already produced. 
Carbolic Acid (C6H5OH).-Pure phenol crystallizes in long, colorless 
crystals. In contact with air it deliquesces. It has a penetrating 
odor, a burning taste, and is a corrosive poison. It is soluble at 
ordinary temperatures in about 15 parts of water. Carbolic acid 
dissolves in water with some difficulty and should be therefore thor- 
oughly mixed. It is not destructive to fabrics, colors, metals, or wood, 
and does not combine as actively with albuminous matters as bichloride 
of mercury. It is therefore more suitable for the disinfection of feces, 
etc. A solution having 1 part to 1000 inhibits the growth of bacteria; 
1 part to 400 kills the less resistant bacteria, and 1 part to 100 kills 
the remainder. A 5 per cent, solution kills the less resistant spores 
within a few hours and the more resistant in from one day to four 
weeks. A slight increase in temperature aids the destructive action; 
thus, even at 37.5° spores are killed in three hours. A 3 per cent, 
solution kills streptococci, staphylococci, anthrax bacilli, etc., within 
one minute. Carbolic acid loses much of its value when in solution in 
alcohol or ether. An addition of 0.5 HC1 aids its activity. Carbolic 
acid is so permanent and so comparatively little influenced by albumin 
that it is rightly widely used in practical disinfection even in place of 
more powerful substances. 
Cresol. - Cresol [C6H4(CH3)OH] is the chief ingredient of the 
so-called "crude carbolic acid." This is almost insoluble in water, 
and therefore of restricted value. Many methods are used for bring- 
ing it into solution so as to make use of its powerful disinfectipg 
properties. With equal parts of crude sulphuric acid it is a powerful 
disinfectant, but it is, of course, strongly corrosive. An alkaline 
emulsion of the cresols and other products contained in "crude" car- THE STANDARDIZATION OF DISINFECTANTS 
649 
bolic acid with soap is called creolin. It is used in 1 to 5 per cent, 
emulsions. It is fully as powerful as pure carbolic acid. Lysol is 
similar to creolin, except that it has more of the cresols and less of the 
other products. It and creolin are of about the same value. 
Tricresol.-Tricresol is a refined mixture of the three cresols (meta- 
cresol, paracresol, and orthocresol). It is soluble in water to the extent 
of 2.5 per cent, and its disinfecting power is about three times as great 
as that of carbolic acid. 
Creolin.-Creolin contains 10 per cent, of cresols held in solution by 
soap. 
Lysol.-Lysol contains about 50 per cent, of cresols. It mixes with 
water in all dilutions. 
Oil of Turpentine, 1 to 200, prevents the growth of bacteria. 
Camphor has very slight antiseptic action. 
Creosote in 1 to 200 kills many bacteria in ten minutes; 1 to 100 failed 
to kill tubercle bacilli in tw'elve hours. 
Essential Oils.-Cardeac and Meumir found that the essences of 
cinnamon, cloves, thyme, and others killed typhoid bacilli within one 
hour. Sandalwood required twelve hours. 
Thymol and eucalyptol have about one-fourth the strength of car- 
bolic acid (Behring). 
Oil of peppermint in 1 to 100 solution prevents the growth of bacteria. 
Tables of Antiseptic Values.1 
Alum 1 to 222 
Aluminum acetate . 1 to 6000 
Ammonium chloride . 1 to 9 
Boric acid 1 to 143 
Calcium chloride . 1 to 25 
Calcium hypochlorite . . 1 to 1000 
Carbolic acid 1 to 333 
Chloral hydrate . 1 to 107 
Cupric sulphate .... 1 to 2000 
Ferrous sulphate . . 1 to 200 
Formaldehyde (40%) 1 to 10,000 
Hydrogen peroxide 1 to 20,000 
Mercuric chloride . . 1 to 14,300 
Mercuric iodide . 1 to 40,000 
Potassium bromide . . . 1 to 10 
Potassium iodide . . 1 to 10 
Potassium permanganate . 1 to 300 
Pure formaldehyde . 1 to 25,000 
Quinine sulphate . . 1 to 800 
Silver nitrate .... 1 to 12,500 
Sodium borate 1 to 14 
Sodium chloride . . . 1 to 6 
Zinc chloride 1 to 500 
Zinc sulphate . . 1 to 20 
1 These figures are approximately correct, and represent the percentage of disinfection 
required to be added to a fluid containing considerable organic material, in order perma- 
nently to prevent any bacterial growth. Solutions of half the given strength will inhibit 
the growth of most bacteria and prevent the growth of many varieties. CHAPTER L. 
PRACTICAL DISINFECTION AND STERILIZATION (HOUSE, 
PERSON, INSTRUMENTS, AND FOOD). STERILIZATION 
OF MILK FOR FEEDING INFANTS. 
DISINFECTANTS AND METHODS OF DISINFECTION EMPLOYED 
IN THE HOUSE AND SICK-ROOM. 
Disinfection and Disinfectants.-Sunlight, pure air, and cleanliness 
are always very important agents in maintaining health and in protect- 
ing the body against many forms of illness. When, however, it 
becomes necessary to guard against such special dangers as infectious 
material from communicable diseases the additional protection of 
disinfection becomes essential. In order that disinfection shall afford 
complete protection it must be thorough; and perfect cleanliness is 
better, even in the presence of contagious disease, than filth with 
incomplete disinfection. 
In order that as few articles as possible shall be exposed to the germs 
causing the communicable diseases, and thus become possible carriers 
of infection, it is important when conditions allow of it that all articles 
not necessary for the care and comfort of the sick person, especially 
upholstered furniture, carpets, and curtains, should be removed from 
the room before placing the sick person in it. 
Agents for Cleansing and Disinfection.-Too much emphasis cannot 
be placed upon the importance of cleanliness, both as regards the 
person and the dwelling, in protecting the body from all kinds of 
infectious disease. Sunlight and fresh air should be freely admitted, 
and personal cleanliness should be attained by frequently washing the 
hands and body, replacing fabrics infected by expectoration, bowel dis- 
charges, etc. By these means most of the bacteria are removed or 
discharged. 
Cleanliness in dwellings, and in all places where men go, may, under 
ordinary circumstances, be well maintained by the use of the two 
following solutions: 
1. Soapsuds Solution.-For simple cleansing, or for cleansing after 
the method of disinfection by chemicals described below, one ounce 
of common soda should be added to twelve quarts of hot soapsuds 
(soft soap and water). 
2. Strong Soda Solution.--This, which is a stronger and more effective 
cleansing solution and also a fairly efficient disinfectant, is made by 
dissolving one-half pound of common soda in three gallons of hot water. 
The solution thus obtained should be applied by scrubbing with a 
hard brush. DISINFECTION IN THE HOUSE AND SICK-ROOM 
651 
When it becomes necessary to prevent the spread of communicable 
diseases by surely killing the living germs which cause them, more 
powerful agents must be employed than those required for simple 
cleanliness, and these are commonly called disinfectants. The following 
are some of the most reliable ones: 
1. Heat.--Complete destruction by fire is an absolutely safe method 
of disposing of infected articles of small value, but continued high 
temperatures not as great as that of fire will destroy all forms of life; 
thus, boiling or steaming in closed vessels for ten minutes will absolutely 
destroy all disease germs except spores. 
2. Carbolic Acid Solution.-Dissolve six ounces of carbolic acid in 
one gallon of hot water (200 grams in 4000 c.c.). This makes approxi- 
mately a 5 per cent, solution of carbolic acid, which, for most purposes, 
may be diluted with an equal quantity of water. The commercial 
"soluble crude carbolic acid" which is cheaper and twice as effective 
as the carbolic acid, can be used for privies and drains.1 It makes 
a white emulsion on account of its not entering readily into solution. 
Care must be taken that the pure acid does not come in contact with 
the skin. 
3. Bichloride Solution (bichloride of mercury or corrosive sublimate).-- 
Dissolve sixty grains of pulverized corrosive sublimate and two table- 
spoonfuls of common salt in one gallon of hot water. This solution, 
which is approximately 1:1000, must be kept in glass, earthen, or 
wooden vessels (not in metal vessels). For safety it is well to color 
the solution. 
The carbolic and bichloride solutions are very poisonous when 
taken by the mouth, but are harmless when used externally. 
4. Milk of Lime.-This mixture is made by adding one quart of 
dry, freshly slaked lime to four or five quarts of water. (Lime is slaked 
by pouring a small quantity of water on a lump of quicklime. The 
lime becomes hot, crumbles, and as the slaking is completed a white 
powder results. The powder is used to make milk of lime.) Air-slaked 
lime (the carbonate) has no value as a disinfectant. 
5. Dry Chlorinated Lime, "Chloride of Lime."-This must be fresh 
and kept in closed vessels or packages. It should have the strong, 
pungent odor of chlorine (see page 643). 
6. Formalin (this is a watery solution containing 40 per cent, of 
formaldehyde).-Add 1 part of formalin to 10 of water. This equals 
in value the 5 per cent, carbolic acid solution. 
7. Creolin, Tricresol, and Lysol.-The first is of about the same value 
as pure carbolic acid, the latter two about three times as powerful. 
The proprietary disinfectants, which are so often widely advertised 
and whose composition is kept secret, are relatively expensive and often 
1 The cost of the pure carbolic acid solution is much greater than that of most of the 
other solutions, but except for the disinfection of the skin, which in some persons it irritates, 
and of woodwork, it is generally much to be preferred by those not thoroughly familiar 
with disinfectants, as it does not deteriorate, and is rather more uniform in its action 
than some of the other- disinfectants. 652 
APPLIED MICROBIOLOGY 
unreliable and inefficient. It is important to remember that substances 
which destroy or disguise bad odors are not necessarily disinfectants, 
and that there are very few disinfectants that are not poisonous when 
taken internally. Their value should be stated in the circular in 
comparison with pure carbolic acid, so that their strength may be 
known. 
Methods of Disinfection in Infectious and Contagious Diseases.- 
The diseases to be commonly guarded against, outside of surgery, by 
disinfection are scarlet fever, measles, diphtheria, tuberculosis, small- 
pox, typhoid, bubonic plague, and cholera. 
1. Hands and Person.-Dilute the 5 per cent, carbolic solution with 
an equal amount of water or use the 1:1000 bichloride solution without 
dilution. Hands soiled in caring for persons suffering from contagious 
diseases, or soiled portions of the patient's body, should be immediately 
and thoroughly soaked with one of these solutions and then washed 
with soap and water, and finally immersed again in the solutions. The 
nails should, always be kept clean. Before eating, the hands should 
be first washed in one of the above solutions, and then thoroughly 
scrubbed with soap and water by means of a brush. 
2. Soiled clothing, towels, napkins, bedding, etc., should, on removal, 
be immediately immersed in the 2.5 per cent, carbolic solution, in the 
sick-room, and soaked for one or more hours. Articles such as beds, 
woollen clothing, etc., which cannot be washed, should be thoroughly 
exposed to formaldehyde gas, as noted later (see page 654). This is 
not necessary after measles. 
3. Food and Drink.-Food thoroughly cooked and drinks that have 
been boiled are free from disease germs. Food and drinks, after cooking 
or boiling, if not immediately used, should be placed when cool in clean 
dishes or vessels and covered. In the presence of an epidemic of cholera 
or typhoid fever, milk and water used for drinking, cooking, washing 
dishes, etc., should be boiled before using, and all persons should avoid 
eating uncooked fruit and fresh vegetables. 
4. Discharges of all kinds from the mouth, nose, bladder, and bowels 
of patients suffering from contagious diseases should be received into 
glass, metal, or earthen vessels containing the carbolic solution, or 
milk of lime, or they should be removed on pieces of cloth, which are 
immediately immersed in one of these solutions or boiled or destroyed 
by fire. Special care should be observed to disinfect at once the vomited 
matter and the intestinal discharges from cholera patients. In typhoid 
fever the urine and the intestinal discharges, and in diphtheria, measles, 
and scarlet fever the discharges from the throat and nose all carry 
infection and should be treated in the same manner. The volume of the 
solution used to disinfect discharges should be at least twice as great as 
that of the discharge, and should completely mix with it and cover 
it. After standing for an hour or more the discharges with the exception 
of the feces may be thrown into the water-closet. 
Masses of feces are extremely difficult to disinfect except on the 
surface, for it takes disinfectants such as the carbolic acid solution DISINFECTION IN THE HOUSE AND SICK-ROOM 
653 
some twelve hours to penetrate to their interior. If fecal masses are 
to be thrown into places where the disinfectant solution covering 
them will be washed off, it will be necessary to be certain that the 
disinfectant has previously penetrated to all portions and destroyed 
the disease germs.. This can be brought about by stirring them with 
the disinfectant and allowing the mixture to stand for one hour, or 
by washing them into a pot holding soda solution which is already 
at the boiling temperature, or later will be brought to it. 
5. Sputum from Consumptives.-The importance of the proper dis- 
infection of the sputum is still underestimated. Consumption is an 
infectious disease, and is always the result of transmission from the 
sick to the healthy or from animals to man. The sputum contains the 
germs which cause the disease, and in a large proportion of cases is the 
source of infection. After being discharged, unless properly disposed 
of, it may become dry and pulverized and float in the air as dust. This 
dust contains the germs, and is a common cause of the disease, through 
inhalation. In all cases, therefore, the sputum should be disinfected 
when discharged. It should be received in covered cups containing 
the carbolic or milk-of-lime solution. Handkerchiefs soiled by it should 
be soaked in the carbolic solution and then boiled. Dust from the 
walls, mouldings, pictures, etc., in rooms that have been occupied by 
consumptive patients, where the rules of cleanliness have not been 
carried out, contain the germs and will produce tuberculosis in ani- 
mals when used for their inoculation; therefore, rooms should be 
thoroughly renovated or disinfected before they are again occupied. 
If the sputum of all consumptive patients were destroyed at once 
when discharged a large proportion of the cases of the disease wrould 
be prevented. 
6. Closets, Kitchen and Hallway Sinks, etc.-The closet should never 
be used for infected discharges until they have been thoroughly dis- 
infected; when done, one quart of carbolic solution or of 5 per cent, 
solution of formalin should be poured into the pan (after it is emptied) 
and allowed to remain there. Sinks should be flushed at least once 
daily. 
7. Dishes, knives, forks, spoons, etc., used by a patient should, as a 
rule, be kept for his exclusive use and not removed from the room. 
They should be washed first in the carbolic solution, then in boiling- 
hot soapsuds, and finally rinsed in hot water. The remains of the 
patient's meals may be burned or thrown into a vessel containing the 
carbolic solution or milk of lime, and allowed to stand for one hour 
before being thrown away. 
8. Rooms and Their Contents.-Rooms which have been occupied 
by persons suffering from contagious disease should not be again occu- 
pied until they have been thoroughly disinfected. It is true that when 
the patient is freed from isolation probably the disease germs have 
already died, but a few may have survived. The danger from infection 
is much greater when cases are removed during the acute illness. For 
disinfecting rooms careful fumigation with formaldehyde gas should 654 
APPLIED MICROBIOLOGY 
be employed unless vermin are to be killed, when sulphur fumes should 
be substituted. Carpets, curtains, and upholstered furniture which 
have been soiled by discharges, or which have been exposed to infection 
in the room during the illness, may be removed for disinfection to cham- 
bers where they can be exposed to formaldehyde gas and moderate 
warmth for twelve to twenty-four hours, or to steam. Some carpets, 
such as many Wiltons, are discolored by moist steam. These must be 
put in the formaldehyde chamber. This difficult procedure may be 
safely omitted, for even if a few germs remain alive in the depth of the 
fabrics, they probably cannot escape. Wood-work, floors, and plain 
furniture will be thoroughly washed with the soapsuds and bichloride 
solutions. After the disinfection is finished it is well to remove the 
dried bichloride of mercury from the walls. 
9. Rags, cloths, and articles of small value, which have been soiled by 
discharges or infected in other ways, should be boiled or burned. 
10. In case of death the body should be completely wrapped in several 
thicknesses of cloth wrung out of the carbolic or bichloride solution, 
and when possible placed in an hermetically sealed coffin. 
It is important to remember that an abundance of fresh air, sunlight, 
and absolute cleanliness not only helps protect the attendants from 
infection and aids in the recovery of the sick, but directly destroys the 
bacteria which cause disease. 
Methods of Cleanliness and Disinfection to Prevent the Occurrence 
of Illness.-1. Water-closet bowls and all receptacles for human excre- 
ment should be kept perfectly clean by frequent flushing with a large 
quantity of water, and as often as necessary disinfected with the crude 
carbolic, or other efficient solutions. The wood-work around and 
beneath them should be frequently scrubbed with the hot soapsuds 
solution. 
2. Cesspools and Privy Vaults.-An abundance of milk of lime or 
chloride of lime should be thrown into these daily, and their contents 
should be frequently removed. 
3. The wood-work in school-houses should be scrubbed daily with hot 
soapsuds. This refers to floors, doors, door-handles, and all wood-work 
touched by the scholars' hands. 
4. Spittoons in all public places should be emptied daily and washed 
with the hot soapsuds solution, after which a small quantity of the 
carbolic solution or milk of lime should be put in the vessel to receive 
the expectoration. This prevents flies conveying infection. 
5. Cars, Ferry-boats, and Public Conveyances. - The floors, door- 
handles, railings, and all parts touched by the hands of passengers 
should be washed frequently with the hot soapsuds solution. Slat- 
mats from cars, etc., should be cleaned by scrubbing with a stiff brush 
in the hot soapsuds solution. 
Telephone receiver mouth-pieces should also be frequently cleansed. 
The Practical Employment of Formaldehyde Gas in the Surface 
Disinfection of Rooms and the Disinfection of Goods which would 
be Injured by Heat.-Formaldehyde gas has come into such general DISINFECTION IN THE HOUSE AND SICK-ROOM 
655 
use, and is for many purposes so valuable, that the description of 
methods employed to generate and use it will be given in detail. 
If we consider now the practical application of formaldehyde gas 
for purposes of disinfection we find that its destructive action on 
microorganisms depends upon a number of factors, the chief of which 
are its concentration in the surrounding atmosphere, the length of the 
contact, the existing temperature, the accompanying moisture, and 
the nature of the organism. It is not an insecticide-like sulphur dioxide. 
Concentration.-The necessary concentration of gas in the surrounding 
atmosphere to kill the microorganisms varies with each species, for 
some resist chemical agents much more than others, and also with the 
freedom of access of the gas to the bacteria, for if they are under cover 
or within fabrics a greater amount of gas must be generated than if 
they are freely exposed. 
For purely surface disinfection, when the common pathogenic bacteria 
are to be destroyed, there will be required, according to the method 
used, ten to twelve ounces of formalin of full strength, or its equivalent, 
to 1000 cubic feet of air space. 
The gas penetrates through fabrics with difficulty, and to pass 
through heavy goods the concentration of the gas must be doubled 
and moderate heat added (45° C. or above). 
Although- formaldehyde gas does destroy bacteria with the amount 
of moisture usually present in the air, it acts much more powerfully 
and certainly when additional moisture is present, and best when 
present up to the point of saturation. The actual spraying with water 
of walls and goods to be disinfected is even more efficacious. 
Temperature.-A fairly high temperature-but one still below that 
which would injure delicate fabrics-increases not only the activity of 
formaldehyde gas, but also its penetrative power, and for heavy goods 
it is essential. The production of a partial vacuum in the chambers 
before the introduction of the formaldehyde gas still further assists its 
penetration. 
Length of Exposure.-This depends upon the nature of the disease 
for which it is carried out-the penetration required, the concentration 
of the gas used, the amount of moisture in the air, the temperature of 
the air, and the size and shape of the room. For surface disinfection 
in rooms, when as much as twelve ounces of formalin are used for each 
1000 cubic feet, five hours' exposure is amply sufficient, most bacteria 
being killed within the first thirty minutes. For the destruction of 
microorganisms protected by even a layer of thin covering, double 
the formalin and double the time of exposure should be allowed, and 
even then the killing of many species of non-spore-bearing bacteria 
cannot be counted upon in ordinary rooms. When absolutely complete 
disinfection is demanded, where penetration of gas is required, the 
goods must be placed in chambers where moderate heat can be added 
and all leakage of gas prevented. 
In order to insure complete sterilization of the articles they should 
be so placed as to allow of a free circulation of the gas around them 656 
APPLIED MICROBIOLOGY 
-that is, in the case of bedding, clothing, etc., these should either 
be spread out on perforated wire shelves or loosely suspended in the 
chamber. The aid of a partial vacuum facilitates the operation. Up- 
holstered furniture and articles requiring much space should be placed 
in a large chamber, or, better, in a room which can be heated to the 
required temperature. 
The most delicate fabrics, furs, leather, and other articles, which 
are injured by steam, hot air at 230° F., or other disinfectants, are 
unaffected by formaldehyde. 
Disinfection of Books.-Books may be satisfactorily disinfected 
by means of formaldehyde gas in a special room, or in the ordinary 
steam chamber, as above described, and under the same condition 
of volume of gas, temperature, and time of exposure. The books 
should be arranged to stand as widely open as possible upon perforated 
wire shelves, set about one or one and a half feet apart in the chamber. 
A chamber having a capacity of 200 to 250 cubic feet would thus afford 
accommodation for about one hundred books at a time. 
Books, with the exception of their surfaces, cannot be satisfactorily 
disinfected by formaldehyde gas in the bookcases of houses or libraries, 
or anywhere except in special chambers constructed for the purpose, 
because the conditions required for their thorough disinfection cannot 
otherwise be complied with. 
The bindings, illustrations, and print of books are in no way affected 
by the action of formaldehyde gas. 
Disinfection of Carriages, Etc.-Carriages, ambulances, cars, 
etc., can easily be disinfected by having built a small, tight building, 
in which they are enclosed and surrounded with formaldehyde gas. 
Such a building is used for disinfecting ambulances in New York City. 
With the apparatus there employed a large amount of formalin is 
rapidly vaporized, and superficial disinfection is completed in sixty 
minutes. 
Methods of Providing Formaldehyde gas.-Various forms of apparatus 
can be properly employed to liberate formaldehyde gas for purposes 
of disinfection. There are two essentials to any good method-namely, 
that the formaldehyde gas is given off quickly, and that there is no 
great loss by deterioration of the formalin. 
Wood Alcohol.--A number of lamps have been devised, all very much on 
the same principle, though varying somewhat in mechanical construction, 
which bring about the incomplete oxidation of methyl-alcohol by passing the 
vapors mixed with air over the incandescent metal. Although disinfection can 
be carried out by the best of these lamps, in our experience none of them up to 
the present time are satisfactory or economical. They may be very useful as 
deodorizers in the sick-room or other places. 
The same principle is used efficiently in another form. The vapor of wood 
alcohol is passed over the surfaces of asbestos containing particles of finely 
divided platinum. This apparatus has given very good results, and for a given 
amount of disinfection leaves less odor of formaldehyde gas in the room than 
any other. The apparatus is, however, bulky and expensive. 
Formalin by Boiling and Passing the Vapor through a Superheated Coil or 
Chamber.-This system consists in heating the ordinary commercial formalin DISINFECTION IN THE HOUSE AND SICK-ROOM 
657 
to. a high temperature in an incandescent copper coil or chamber, and allowing 
the vapors to pass off freely. It is claimed for this method that the degree of heat 
necessary to break up the polymerized products formed is supplied, and thus a 
loss of formaldehyde is prevented. A further action of the intense heat in the 
copper tube on the solution is partially to convert the methyl-alcohol contained 
in commercial formalin into formaldehyde gas by partial oxidation, thereby 
utilizing a part of the methyl-alcohol and increasing the amount of formaldehyde. 
(Fig. 209.) 
Trioxymethylene or Paraform.-This system consists in heating the 
solid polymer of formaldehyde (trioxymethylene). 
There are several methods for doing this. They are somewhat 
expensive but efficient. The formaldehyde gas must be protected 
from burning. 
Formalin to which Glycerin has been Added. To the formalin is added 
10 per cent, of glycerin, and the mixture is simply boiled in a suitable copper 
vessel, the steam and formaldehyde gas passing off by a tube. This is a very 
serviceable apparatus. When it is attempted to vaporize the formalin too 
rapidly part of it bubbles over in fluid form. 
With 50 per cent, more of formalin than that used in the high temperature 
autoclave and heated tube or chamber methods, the results seem to be equally 
as good. The apparatus is very easy to use, and is not liable to get out of order. 
Similar forms of apparatus are also employed, when, instead of glycerin, 
the formalin is mixed with an equal quantity of water. The water is for the 
purpose of giving additional moisture to the air, and, at the same time, like the 
glycerin, to prevent the change of formaldehyde into inert substances. 
From Formalin in an Open Pan.-A very simple method, devised by Dr. 
R. J. Wilson, is to fill a tin pan with twelve ounces of formalin for each 1000 
cubic feet and put this on an upright sheet of tin, which is cut so as to allow 
of the entrance of air below and yet protect the formalin in the pan from the 
flame. For heating put under it a small tin can filled with asbestos packing which 
has been soaked with wood alcohol. A still simpler method is to pour on folded 
sheets sixteen ounces of formalin per 1000 cubic feet and then stretch them out 
over lines in a room and leave for ten hours. If the room is tightly sealed very 
fair surface disinfection will take place. 
Lime and Permanganate Method of Generating Formaldehyde Gas.- 
Satisfactory results in disinfection have been obtained from the following 
combination of chemicals. Two ounces of a quick-slaking, coarsely granular 
lime (calcium oxide); 5 ounces of permanganate of potash; j gram oxalic acid; 
5 ounces formaldehyde solution, 40 per cent, strength; and 2| ounces of water. 
This is sufficient in quantity to disinfect 1000 cubic feet of space in five hours. 
It is used as follows: The lime and permanganate of potash are mixed together 
in a pan at least 10| inches in diameter and 3| to 4 inches in depth. Over 
this is poured the freshly prepared mixture of formaldehyde solution, oxalic 
acid, and water. A rapid evolution of gas takes place. 
Another combination is: lime, 2.7 ounces; potassium permanganate, 5.5 ounces; 
formaldehyde solution, 7.4 ounces; water, 2.7 ounces. The technique is as 
follows: The lime and permanganate are mixed in a wide, deep pan as above, 
and the freshly prepared formaldehyde and water mixture is poured over it. 
Permanganate of potash method. The following combination will also disin- 
fect 1000 cubic feet of space in five hours; potassium permanganate, 10 ounces; 
formaldehyde solution, 40 per cent., 9 ounces; water, 4.5 ounces. The for- 
maldehyde and water are mixed together and rapidly poured over the permanga- 
nate of potash. The reaction is immediate and violent. This mixture requires a 
deep, wide pan or a pail at least 18 inches deep. The addition of the water is 
believed to increase the liberation of the formaldehyde gas. 
Lime Method of Generating Formaldehyde Gas. -To ten ounces of 40 per 
cent, formaldehyde solution slowly add one ounce of concentrated sulphuric 658 
APPLIED MICROBIOLOGY 
acid; pour this solution on to two pounds of quicklime that had previously been 
cracked into small lumps and placed in a dairy pan not less than twelve inches 
in diameter. The liberation of a large amount of gas in a short time more than 
compensates for the loss by polymerization, and disinfection is effected by a 
quick union of the gas and organisms to be destroyed. Saturated solution of 
aluminum sulphate may be used instead of concentrated sulphuric acid. 
Fig. 209 
Fig. 210 
Formaldehyde apparatus. 
Wilson's formaldehyde generator: A, 
steam chamber; B, formalin chamber; 
C, steam supply; D, drip; E, inlet for for- 
malin; F, outlet for formaldehyde. 
Rapid Generation of Formaldehyde Gas for Large Chambers by the Method 
of Dr. R. J. Wilson.-The generator (Fig. 210) is made of ordinary iron steam 
pipe and can be manufactured in any pipe-cutting establishment in a very few 
hours. It consists of an outer steam jacket of six-inch pipe, two feet long, and 
capped at both ends. Through the upper cap there is a four-inch opening, with 
a thread, through which projects an inner chamber for formalin. This chamber 
consists of a four-inch pipe, twenty-two inches long, capped at the upper end 
and welded or capped at the lower end. The upper end of this pipe is so threaded 
as to permit of its being screwed through the cap of the steam jacket before that 
cap is screwed on. The cap of the formalin chamber is fitted on the same thread 
that passes through the cap of the steam jacket. The in-take for steam is near 
the top of the steam j acket, through a half-inch pipe, and the steam is controlled 
by a globe valve. The outlet for steam or drip is through a half-inch pipe from 
the bottom cap of the chamber and is also controlled by a globe valve. The in- 
take for formalin is through the upper cap of the formalin chamber through a 
half-inch pipe controlled by a globe valve. The outlet for formaldehyde is a 
half-inch pipe through the upper cap of the formalin chamber. 
As a result of the investigations undertaken in the Department of 
Health laboratories on methods for testing the efficiency of room disin- 
fections, we adopted the plan of placing infected threads in the rooms. 
This plan, adopted by us, may be summarized as follows: DISINFECTION IN THE HOUSE AND SICK-ROOM 
659 
Method for Testing Efficacy of Room Disinfection.-The 
following method modified by Dr. Schroder, working in the Research 
Laboratory, is now in use in the Department of Health. 
The main points of the system are as follows: 
No. 36 cotton is cut into inch lengths, placed in a Petri dish, and 
covered with a forty-eight-hour broth culture of pyocyaneus. 
They are left for two or three minutes or until they are thoroughly 
saturated, then removed to filter-paper in another covered Petri dish 
and left to dry. When dry, they are placed in tissue-paper envelopes, 
which are stamped with all necessary data. Each envelope is dated 
and sealed and sent to the disinfector who places it in the room which 
is to be disinfected. 
The driver who calls for the bedding takes up the tests, placing 
them in a manilia envelope and entering them upon his card. The 
envelopes are then returned to the laboratory where the tests and 
receipt card are compared and any discrepancy noted. 
The test envelopes are then stamped with date of receipt, and the 
threads are removed and placed in a modified Ayer's medium, which 
is a synthetic medium and consists of the following: 
Asp ar agin 4 
Neutral sodium phos 2 
Sodium lact 6 
Sodium chlor 5 
Water 1000 
Add enough NaOH to render the medium alkaline to litmus. This 
culture medium may be depended upon to give bright green color 
reaction in twenty-four to forty-eight hours. 
The tubes are incubated for forty-eight hours and the color reac- 
tion noted and entered upon test envelope. 
At the end of the week a bacteriologist's report is compiled which 
shows at a glance the work of each disinfector, the number of cases 
of each disease for which disinfection was performed, the number of 
successful disinfections, the number of tests lost, etc. 
Sulphur Dioxide in House Disinfection.-Four pounds of sulphur 
should be burned for every 1000 cubic feet. The sulphur should be 
broken into small pieces and put into a pan sufficiently large not to 
allow the melted sulphur to over flow. This pan is placed in a much 
larger pan holding a little water. The cracks of the room should be 
carefully pasted up and the door, after closing, also sealed. Upon 
the broken sulphur is poured three to four ounces of alcohol and the 
whole lighted by a match. The alcohol is not only for the purpose of 
aiding the sulphur to ignite, but also to add moisture to the air. An 
exposure of eight to twelve hours should be given. 
Sulphur fumigation carried out as above indicated is not as efficient 
as formaldehyde fumigation, but suffices for surface disinfection for 
diphtheria and the exanthemata. All heavy goods should be removed 
for steam disinfection if there is any possibility of the infection having 
penetrated beneath their surface. If there is no place for steam disin- 
fection their surfaces should be thoroughly exposed to fumigation 660 
APPLIED MICROBIOLOGY 
and then to the air and sunlight. In many cases when cleanliness 
has been observed, surface disinfection of halls, bedding, and furniture 
may be all that will be required. 
There is always a very slight possibility of a deeper penetration of 
infection than that believed to have occurred; it is, therefore, better 
to be more thorough than is considered necessary rather than less. 
Sulphur dioxide without the addition of moisture has, as already 
stated under the consideration of disinfectants, very little germicidal 
value upon dry bacteria. 
An vantages of Formaldehyde Gas over Sulphur Dioxide 
for Disinfection of Dwellings.-Formaldehyde gas is superior 
to sulphur dioxide as a disinfectant for dwellings; first, because it is 
more efficient in its action; second, because it is less injurious in its 
effects on household goods; third, because when necessary it can easily 
be supplied from a generator placed outside of the room and watched 
by an attendant, thus avoiding, in some cases, danger of fire. 
Apart from the cost of the apparatus and the greater time involved, 
formaldehyde gas, generated from commercial formalin, is not much 
more expensive than sulphur dioxide-viz., twelve to twenty cents per 
1000 cubic feet against ten cents with sulphur. Therefore, we believe 
that formaldehyde gas is the best disinfectant at present known for 
the surface disinfection of infected dwellings. For heavy goods it is 
far inferior in penetrative power to steam; but for the disinfection of 
fine wearing apparel, furs, leather, upholstery, books, and the like, 
which are injured by great heat, it is, when properly employed, better 
adapted than any other disinfectant now in use. 
Public Steam Disinfecting Chambers.-These should be of sufficient 
size to receive all necessary -goods, and may be either cylindrical 
or rectangular in shape, and are provided with steam-tight doors 
opening at either end, so that the goods put in at one door may be 
removed at the other. When large the doors are handled by con- 
venient cranes and drawn tight by drop-forged steel eye-bolts swinging 
in and out of slots in the door frames. The chambers should be able 
to withstand a steam pressure of at least one-half an atmosphere, and 
should be constructed-with an inside jacket, either in the form of an 
inner and outer shell or of a coil of pipes. This jacket is filled with 
steam during the entire operation, and is so used as to bring the goods 
in the disinfecting chamber up to the neighborhood of 220° F. before 
allowing the steam to pass in. This heats the goods, so that the 
steam does not condense on coming in contact with them. It is an 
advantage to displace the air in the chamber before throwing in the 
steam, as hot air has far less germicidal value than steam of the same 
temperature. To do this, a vacuum pump is attached to the piping, 
whereby a vacuum of fifteen inches can be obtained in the chamber. 
The steam should be thrown into the chamber in large amount, both 
above and below the goods, and the excess should escape through an 
opening in the bottom of the chamber, so as more readily to carry off 
with it any air still remaining. The live steam in the chamber should be 
under a pressure of two to three pounds so as to increase its action. DISINFECTION IN THE HOUSE AND SICK-ROOM 
661 
To disinfect the goods, we place them in the chamber, close tight 
the doors, and turn the steam into the jacket. After about ten minutes, 
when the goods have become heated, a vacuum of ten to fifteen inches 
is produced, and then the live steam is thrown in for twenty minutes. 
The steam is now turned off, a vacuum is again formed, and the 
chamber again superheated. The goods are now thoroughly disinfected 
and dry. In order to test the thoroughness of any disinfection, or any 
new chamber maximum, thermometers are placed, some free in the 
chamber and others surrounded by the heaviest goods. It will be 
found that, even under a pressure of three pounds, live steam will 
require ten minutes to penetrate heavy goods. 
Practical Points on Heat Disinfection.-In the practical application of 
steam for disinfecting purposes it must be remembered that while 
moist steam under pressure is more effective than streaming steam, it 
is scarcely necessary to give it the preference, in view of the fact that 
most known pathogenic bacteria produce no spores and the spores of the 
few that do develop them are quickly destroyed by the temperature 
of boiling water, and also that "superheated" steam is less effective 
than moist steam. When confined steam in pipes is "superheated" 
after its generation it has about the same germicidal power as hot, dry 
air at the same temperature. Esmarch found that anthrax spores 
were killed in streaming steam in four minutes, but were not killed in 
the same time by superheated steam at a temperature of 114° C. 
It should also be remembered that dry heat has but little penetrating 
power, and that even steam requires time to pass through heavy goods. 
Koch and Wolffhugel found that registering thermometers placed in 
the interior of folded blankets and packages of various kinds did not 
show a temperature capable of killing bacteria after three hours' expo- 
sure in a dry hot-air oven at 133° C. and over. We have put a piece of 
ice in the middle of several mattresses and recovered it after exposing 
the goods to an atmosphere of live steam for ten minutes. 
The Disinfection of Hands, Instruments, Ligatures, and Dressings 
for Surgical Operations. Instruments.-All instruments, except knives, 
after having been thoroughly cleansed, are boiled for three minutes in 
a 1 per cent, solution of washing soda. Knives, after having been 
thoroughly cleansed, are washed in sterile alcohol and wiped with 
sterile gauze and then put into boiling soda solution for one minute. 
This will not injure their edges to any great extent. 
Gauze.-Gauze is sterilized by moist heat either in an Arnold steam 
sterilizer for one hour or in an autoclave for thirty minutes. It is 
placed in a perforated cylinder or -wrapped in clean towels before 
putting in the sterilizer, and only opened at the operation. 
Iodoform gauze is best made by sprinkling sterile iodoform on 
plain gauze sterilized as described above. 
Ligatures-Catgut.-Boil for one hour in alcohol under pressure at 
about 97° C. It is often put in sealed glass tubes, which are boiled 
under pressure. These remain indefinitely sterile. The alcohol does 
not injure the catgut. If desired, the catgut can be washed in ether 
and then soaked a short time in bichloride before heating in alcohol. 662 
APPLIED MICROBIOLOGY 
Bockman, of St. Paul, suggested wrapping the separate strands of 
catgut in paraffin paper and then heating for three hours at 140° C. 
This procedure prevents the drying out of the moisture and fat from 
the catgut, so that it remains unshrivelled and flexible after its exposure. 
Darling, of Boston, tested this method and found it satisfactory. 
Dry formaldehyde gas does not penetrate sufficiently, and is not 
reliable. Silver wire, silk, silkworm gut, rubber tubing, and catheters 
are boiled the same as the instruments. 
Hand Brushes.-These should be boiled in soda solution for ten minutes. 
The Skin of the Patient.-It is impossible absolutely to sterilize 
the deeper portions of the skin, but sufficient bacteria can be removed 
to render infection rare. The skin is washed thoroughly with warm 
green soap solution, then with alcohol, and finally with 1 to 1000 bi- 
chloride. A compress wet with a 25 per cent, solution of green soap 
is now placed on, covered with rubber tissue, and left for three to 
twelve hours; and after its removal the skin is washed with ether, 
alcohol, and bichloride solution, and then covered with a gauze com- 
press previously moistened with a 1 to 1000 bichloride of mercury solu- 
tion. At the operation the skin is again scrubbed with green soap solu- 
tion followed by ether, alcohol, and then with the bichloride of mercury 
solution. In some places the bichloride compress is replaced one hour 
before the operation by a pad wet in 10 per cent, solution of formalin. 
The Hands.-Fiirbinger's method, slightly modified, is now much 
used, and gives good results. The hands are washed in hot soap and 
water for five minutes, using the nail brush. They are then soaked 
in 85 per cent, alcohol for one minute and scrubbed with a sterile brush. 
They are finally soaked in a 1 to 1000 bichloride of mercury solution for 
two minutes. The alcohol and bichloride of mercury are sometimes 
combined and used together. Another method which gives good 
results is as follows: Skin of operator is scrubbed for five minutes 
with green soap and brush, then washed in chlorinated lime and 
carbonate of soda in proportions to make a good lather; washed off 
in sterile water, and then scrubbed with brush in warm bichloride 
solution 1 to 1000. 
Owing to the risk of leaving untouched bacteria under the nails and 
in cracks of the skin, sterilized rubber gloves are now being used 
more and more in operations. Some surgeons prefer sterilized cotton 
gloves frequently changed. The gloves can be sterilized by steam. 
Mucous Membranes.-Here absolute sterilization cannot be achieved 
without serious injury to the tissues. Those of the mouth and throat 
are cleansed by a solution consisting of equal parts of peroxide of hydro- 
gen and lime-water. In the nostrils it is better to employ the milder 
solutions, such as diluted Dobell's or Listerine. These are also used in 
the mouth instead of the peroxide. Wadsworth1 urges the use of prepara- 
tions containing about 30 per cent, of alcohol as being very efficient. 
The vagina is swabbed out thoroughly with sterile warm soap and 
water, and then irrigated with a 2 per cent, carbolic acid or a 1 to 1000 
bichloride of mercury solution. 
1 Mouth Disinfection, Jour. Infect. Dis., 1906, p. 779. DISINFECTION IN THE HOUSE AND SICK-ROOM 
663 
Hypodermic and Other Syringes.-These when not boiled are steril- 
ized by drawing up into them- boiling water a number of times and 
then finally a 5 per cent, solution of carbolic acid, the acid after three 
minutes to be washed out by boiling water. If cold water is used the 
carbolic solution should remain in the barrel for ten minutes. Great 
care should be taken to wash out all possible organic matter before 
using the carbolic acid or boiling to sterilize. Syringes made entirely 
of glass or of glass and asbestos can be boiled in soda solution. 
The Sterilization of Milk.-Complete sterilization destroys all the 
germs in milk, and so, if no new ones gain entrance, prevents per- 
manently fermentative changes. This requires boiling for fifteen 
to forty-five minutes on two or three consecutive days, according to 
the presence or absence of certain spores. 
Milk is best sterilized by heat, for nearly all chemicals, such as boric 
acid, salicylic acid, and formalin, are not only slightly deleterious 
themselves but also make the milk less digestible, and, therfore, less 
fit for food. Formalin is the least objectionable of the three. Milk 
may be sterilized at a high or low temperature-that is, at the boiling 
temperature-or at a lower degree of heat, obtained by modifying the 
steaming process. Milk heated at as high a temperature as 100° C. is 
not altogether desirable for prolonged use for infants, as the high tem- 
perature causes certain changes in the milk which make it less suitable 
as a food for them. 
Pasteurization.-These changes are almost altogether avoided if a 
temperature below 70° C. is used. It is recommended, therefore, that 
the lowest temperature be used for partial sterilization w'hich will keep 
the milk wholesome for twenty-four hours in the warmest weather and 
kill the tubercle, typhoid, and other non-sporebearing bacilli. Raising 
the milk to a temperature of 60° C. for twenty minutes, 65° C. for fifteen, 
70° for five, 75° for two, or 80° for one will accomplish this. Exposure 
for even one minute at 70° destroys 98 per cent, of the bacteria which 
are not in the spore form. Fully 99 per cent, of tubercle bacilli are 
destroyed. This subject is considered more fully in the chapter on 
milk. One of the many forms of apparatus is the following: 
(a) A tin pail or pot, about ten inches deep by nine inches in diameter, 
provided with the ordinary tin cover which has been perforated with 
eight holes each an inch in diameter. 
(6) A wire basket, with eight nursing bottles (as sold for this pur- 
pose in the shops). 
(c) Rubber corks for bottles and a bristle brush for cleaning them. _ 
Directions (Koplik).-Place the milk, pure or diluted (as the physi- 
cian may direct), in the nursing bottles and place the latter in the wire 
basket. Put only sufficient milk for one nursing in each bottle. Do 
not cork the bottles at first. 
Having previously poured about two inches of water in the tin pail 
or pot and brought it to the boiling point, lower the basket of nurs- 
ing bottles slowly into the pot. Do not allow the bottles to touch the 
water or they will crack. Put on the perforated cover and let the 
steaming continue for ten minutes; then remove the cover and firmly 664 
APPLIED MICROBIOLOGY 
cork each bottle. After replacing the cover, allow the steaming to 
continue for fifteen minutes. The steam must be allowed to escape 
freely or the temperature will rise too high. 
The process is now complete. Place the basket of bottles in a 
cool, dark place or in an ice-chest. The bottles must not be opened 
until just before the milk is to be used, and then it may be warmed 
by plunging the bottle in warm water. If properly prepared the milk 
will taste but little like boiled milk. 
The temperature attained under the conditions stated above will 
not exceed in extreme cases 87° C. (188° F.). 
A different but admirable method is the one devised by Dr. Free- 
man.1 Here a pail is filled to a certain mark with water, and then 
placed on the stove until the water boils. It is then removed, and 
immediately a milk-holder, consisting of a series of zinc cylinders, 
is lowered with its milk bottles partially full of milk. The cover is 
again applied. The heat of the outside water raises the temperature 
of the milk in ten minutes to about 65° C. (150° F.), and holds it nearly 
at that point for some time. After twenty minutes the milk is removed, 
placed in cold water, and quickly cooled. The milk is kept in the ice- 
chest until used. When milk is pasteurized in great quantities it should 
always be done by the "holding process'' as "flash" pasteurization is 
unreliable. Milk should be pasteurized when it is as fresh as possible, 
and only sufficient milk for twenty-four hours should be pastuerized at 
one time. If after nursing the infant leaves some milk in the bottle 
this should be thrown away. 
Care of the Bottles.-After nursing, the bottles should be filled with 
a strong solution of washing soda, allowed to stand twenty-four hours, 
and then carefully cleaned with a bristle (bottle) brush. The rubber 
corks and nipples after using should be boiled in strong soda solution 
for fifteen minutes and then rinsed and dried. 
After sterilizing milk should never be put into unsterilized bottles, as 
this will spoil it. 
GENERAL CONCLUSIONS ON DISINFECTION. 
The previous pages have shown that it is comparatively easy to 
destroy microbes by germicides or heat when access to them is attain- 
able. Foods, instruments, clothing, bedding, the excreta, the surface 
of the body, etc., can be readily disinfected, but when we try to disin- 
fect the mucous membranes of the living person we fail. 
The Importance of Disinfection After Recovery or Death from Infec- 
tious Diseases.-Year by year knowledge is accumulating which indi- 
cates that nearly all cases of spread of infection are due to the 
immediate transfer from a living carrier to the person who contracted 
the disease. The carrier may be diseased or may simply harbor the 
germs. If cleanliness is maintained throughout the disease there is, as 
a rule, little need of disinfection after recovery. 
'Agent for Pasteurizer, James Dougherty, 411 W. 59th St. INDEX OF AUTHORS. 
A 
Abbott, 598 
Abel, 354, 470 
Abrami, 237 
Agramonte, 590, 591, 594, 595 
Albrecht, 275 
Ambroz, 34 
Amoss, 492 
Anderson, 224, 492, 493, 639 
Andrews, 249 
Anthony, 252, 494 
Aristotle, 590 
Arloing, 412 
Arning, 423 
Aronson, 645, 646 
Arrhenius, 169, 170 
Arthus, 224 
Ashburn, 492 
Atkinson, 168, 169, 173, 174 
Avery, 340 
Axenfeld, 438 
Ayer. 657 
B 
Babes, 558, 581, 586 
Baelz, 541 
Baeslack, 490 
Bail, 174 
Bailey, 627, 631 
Baker, 503 
Balbiani, 544 
Baldwin, 340 
Balfour, 526 
Ball, 630 
Banzhaf, 146, 168, 173, 174, 227 
Barbagallo, 533 
Barbara, 535 
Bard, 292 
Baroni, 576 
Bass, 106, 553 
Bassi, 22 
Bateman, 508 
Baumgarten, 388 
Beattie, 544 
Beebe, 301, 517 
von Behring, 154, 165, 172, 173, 310, 325 
404, 647 
Beijerinck, 489 
Bendick, 340 
Bentley, 558 
Bergey, 612 
Bernberg-Gossler, 558 
Berry, 103, 576 
Bertarelli, 521, 564 
Bertram, 546 
Besredka, 222, 225, 363 
Beyerinck, 631 
Bienstock, 335, 343 
Biggs, 308 
Bignami, 548, 549 
Billroth, 22 
Binger, 488 
Blanchard, 591 
Blandford, 513 
Blochmann, 530 
Bockman, 660 
Boeckman, 660 
Boerner, 377 
Boinet, 363, 422 
von Bokay, 227 
Bolduan, 265 
Bollinger, 477 
Bolton, 325, 489 
Bonhoff, 276 
Boni, 34 
Bonome, 427 
Booker, 387 
Bordet, 159, 160, 162, 165, 193 
Borget and Gengou, 177, 441, 442 
Borrel, 422, 448, 517, 565 
Bosquillon, 570 
Bossowski, 247 
Bostroem, 477, 478, 480 
Bouchard, 384, 425 
Braun, 64 
Breinl, 512, 526, 559, 560 
Bretonneau, 292 
Brieger, 149, 150, 324 
Brill, 493 
Bioeden, 503 
Brooks, 547 
Brown, 300, 374 
Bruce, 290, 503, 508 
Bruck, 523 
Brunner, 387 
Buchanan, 630 
Buchner, 56, 60, 160, 163 
Buermann, 236 
Bumm, 283 
Bunge, 80 
Burri, 74 
Buschke, 239, 521 
Busse, 30, 237, 239 
Biitschli, 19, 34 
Buxton, 374 
C 
Cahn, 341 
Calkins, 496, 518, 532, 537, 560, 564, 571 
Callison, 363 
Calmette, 408, 448, 564 
Cameron, 632 
Canon, 432 
Capitan, 425 
Cardeac, 647 
Carle, 320 
Carlisle, 526 
Carroll, 590 
Carter, 526 666 
INDEX OF AUTHORS 
Casagrandi, 492, 533 
Castellani, 503, 523 
Catrin, 494 
Celli, 533 
Centanni, 587 
Cernovodeanu, 327 
Chagas, 503, 506 
Chamberland, 456 
Charrin, 57, 193, 384, 386, 425 
Chevreul, 21 
Chick, 640 
Chowning, 494 
Christophers, 500, 558, 562 
Clairmont, 270 
Clausen, 470 
Clegg, 420, 481, 509, 533, 535, 537, 538 
Cohan, 522 
Cohen, 436, 441 
Cohendy, 338 
Cohn, 21, 324, 517 
Cole, 270, 271 
Coley, 255 
Collins, 199, 270 
Conner, 494 
Conradi, 341, 360, 380 
Conte, 501 
Councilman, 532, 533, 548, 564, 566 
Courmont, 412 
Craig, 492, 533, 535, 536, 537, 538, 541 
Cramer, 50 
Cullinan, 364 
Cumming, 577, 585 
Cunningham, 533 
Curtis, 238 
Cushing, 360 
Cygnaeus, 358 
D 
Dale, 57 
Dallera, 227 
Danyz, 377 
Darling, 500, 526, 546, 660 
D'Arsonval, 57 
Davaine, 22, 450, 497 
Davis, 303 
Dean, 212 
Deneke, 464 
Denys, 208 
Dieudonne, 56 
Dochez, 270, 271 
Dock, 541 
Doerr, 173, 226 
Doflein, 496, 547 
Dold, 226 
Donitz, 328 
Donne, 530 
Donovan, 500 
Doptor, 276 
Dorset, 489 
Douglass, 209 
Doutrelepont, 422 
Dreyer, 194 
Drigalski, 360 
Ducrey, 443 
Dujardin, 19 
Dunham, 160, 378, 463 
Durham 193, 374, 513 
von Dusch, 21 
Dutton, 503, 506, 511, 526, 527 
Duval, 420 
E 
Eaton, 363 
Eberth, 355 
Ehlers, 385 
Ehrenberg, 515, 516 
Ehrlich, 22, 160, 161, 162, 165, 166, 169, 
170, 172, 173; 193, 471, 512, 513 
Eichhorn, 513 
Elser, 276 
Emmerich, 52, 53, 245, 384, 469 
Endo, 104 
Engelmann, 19 
Eppinger, 477, 482, 486 
Epstein, 643 
Ernst, 385 
Escherich, 335, 343 
Esmarch, 659 
von Esmarch, 488 
Evans, 503 
Ewing, 492, 517, 521 
F 
Faguet, 487 
Famuleuer, 227 
Fantham, 506, 544, 562 
Fehleisen, 255 
Feinberg, 34 
Fenger, 427 
Fermi, 587 
Ferre, 487 
Field, 33S, 492, 493 
Finger, 522 
Finkler, 464 
Fiocca, 533 
Firth, 364 
Fisch, 325 
Fischer, 34, 521, 643 
Flexner, 83, 276, 277, 378, 482, 491, 492, 
533 
Fliigge, 320, 397 
Foley, 526 
Fontaine, 238 
Ford, 336 
Foulerton, 476 
Fox, 362, 371 
Frankel, 262, 244, 526 
Franklands, 598 
Freeman, 662 
Friedberger, 147, 224, 225, 226, 576 
Friedlander, 262, 353 
Friedmann, 415 
Frosch, 360, 489 
Fullerton, 40 
Fiirbinger, 229, 660 
G 
Gabritchewsky, 260 
Gaffky, 247, 355 
Gage, 73 
Galli-Valerio, 559 
Gamprecht, 326 
Garre, 246 INDEX OF AUTHORS 
667 
Garrison, 495 
Gartner, 374 
Gasperini, 480 
Gasten, 477 
Gay, 224 
Gaylord, 517 
Gengou, 177, 441 
Gerlief, 456 
Ghedini, 434 
Ghon, 275 
Gibson, 168, 174 
Giemsa, 86, 549 
Gilchrist, 238 
Gilliland, 404 
Goldberger, 492, 493, 494 
Goldhorn, 78, 82 
Golgi, 548 
Gomez, 494 
Goodwin, 275 
Gordon, 71, 249 
Gottschlich, 34, 466 
Gougerot, 236, 237 
Grassi, 532, 533, 548, 549 
Greig-Smith, 630 
Gross, 533 
Gruber, 160, 193 
Gruby, 47 
Griinbaum, 193 
Guarnieri, 564 
Guerard, 308 
Guerin, 564 
Guignard, 384 
Guilliermond, 30, 34 
Gwyn, 374 
H 
Hadwen, 561 
Haeckel, 37 
Haffkine, 449, 471 
Halberstaedter, 439 
Hamilton, 303 
Hammerton, 508 
Hanes, 270 
Hansell, 521 
Hansen, 30, 34, 239, 337, 420, 633 
Harris, 534, 537, 538, 540, 573, 584, 585 
Hartmann, 496, 524, 536, 537, 558, 560 
Hartock, 225 
Harz, 477 
Hasse, 238 
Hastings, 291 
Hauser, 385, 386 
Heidenhain, 84 
Heiman, 286, 288, 581 
Hektoen, 217, 236, 492 
Helbrigel, 630 
Heller, 576 
Hellmann, 581 
Henle, 20 
Henni, 327 
Henri jean, 322 
Hermann, 389 
Herter, 334, 336, 343, 462 
Hertwig, 19, 34, 43 
Hess, 23, 399 
Hetsch, 554 
Heubner, 227 
Hewetson, 562 
Higley, 318 
Hill, 34, 37, 73 
Hindle, 559, 560 
Hiss, 217, 246, 265, 378 
Hodenpyl, 390, 395 
Hoffmann, 21, 490, 515, 516, 517, 518, 
521 
Hogyes, 581 
Holt, 617 
Holth, 423 
Horder, 249 
Houston, 602 
Howard, 592, 596 
Huddleston, 566 
Huntoon, 276 
I 
Israel, 477, 481, 564 
J 
Jaeger, 273 
Jansen, 456 
Jehle, 380 
Jenner, 82, 549, 564 
Jesjakoff, 260 
Jobling, 277 
Joblot, 19 
Jochmann, 277, 436 
Johns, 106, 553 
Johnson, 365 
Jordan, 598 
Jullien, 521 
Jurgens, 541 
K 
Kamen, 438 
Kanthack, 513 
Kaposi, 233 
Kartulis, 239, 532, 533, 538, 541 
Kayser, 361, 374 
Kedrowski, 420 
Kempner, 398, 513 
Kerandel, 514 
Kern, 340 
Kessler, 239 
Kilborne, 558, 559, 560 
King, 490 
Kinyoun, 417 
Kircher, 18 
Kisskalt, 529 
Kitasato, 23, 154, 165, 320, 325, 357, 
446, 452 
Kitt, 445 
Klebs, 292, 520, 548 
Klein, 212, 343, 463, 508 
Klimmer, 415 
Knapp, 304, 524 
Knorr, 257, 328 
Knox, 199 
Koch, 21, 23, 175, 219, 249, 257, 388, 390, 
403, 404, 437, 450, 464, 467, 468, 470, 
513, 517, 525, 526, 560, 584, 642, 659 
Koehler, 496 
Kohler, 71 
Kolle, 159, 276, 277, 363, 471 
Kolliker, 19 668 
INDEX OF AUTHORS 
Koplik, 662 
Kossel, 560 
Krambals, 385 
Kraus, 206, 380, 467, 471, 491, 587 
Krause, 249 
Krauss, 575 
Kromer, 277 
Kruse, 378, 380, 385, 533, 538 
Krumwiede, 100, 121 
Kuhne, 22, 78, 260 
Kummel, 603 
Kurpjuweit, 341 
Kurth, 37 
Kutschert, 304 
Kutzing, 633 
L 
Ladenburg, 149 
Ladowski, 428 
Lafleur, 532, 533 
Lambert, 332, 564 
Lambl, 532 
Landsteiner, 491, 522 
Lang, 64 
von Langenbeck, 477 
Larrier-Nallen, 524 
Lartigau, 385 
Latzer, 343 
Laveran, 22, 23, 494, 499, 503, 513, 546, 
548 
Lazear, 590 
Leber, 441 
Leclef, 208 
Lee, 85 
Leeuwenhoeck, 18 
Lehmann, 111 
Leiner, 491 
Leishman, 82, 209, 211, 526 
Lentz, 360, 378 
Lepriere, 276 
Levaditi, 83, 491, 516, 517, 519, 520, 521, 
524 
Levin, 334 
Levy, 361, 387 
Lewis, 464, 491, 503 
Leyden, 541 
Lieberkuhn, 469 
Liebermann, 229 
Lindemann, 543 
Lindner, 30 
Lingard, 503 
von Lingelsheim, 281 
Linnaeus, 18 
Lister, 22 
Loffler, 23, 80, 292, 299, 374, 425, 489 
Losch, 532, 533 
Loew, 384 
Loux, 471 
Lowden, 596 
Loewenthal, 517 
Lowit, 34 
Luhe, 559 
Lustgarten, 424, 518 
Luttle, 537 
Lyall, 249, 250 
M 
McBryde, 489 
McClintic, 639 
McDaniel, 304 
McIntosh, 519 
McKenzie, 217 
Maassen, 62 
MacCallum, 291, 549, 557 • 
MacFadyan, 452, 490 
Mackie, 508, 527 
MacNeal, 81, 287, 495, 497, 509, 513 
Madsen, 170, 
Magendie, 224 
Makkas, 502 
Maklezow, 335 
Mallory, 359, 442, 443, 493, 535 
Malmsten, 546, 547 
Malvos, 163 
Manouelian, 575 
Manson, 503, 541, 548 
Maragliano, 227 
Marchiafava, 548 
Marchoux, 518, 527 
Marie, 222, 326, 328, 581, 586, 587, 588 
Marks, 492 
Markus, 439 
Marmorek, 257 
Marshall, 199, 501 
Martin, 216, 217 
Martini, 378 
Massart, 34 
Massol, 338 
Maupas, 19 
Mayer, 445 
Meader, 361 
Meigen, 591 
Meissner, 430, 433 
Meltzer, 57 
Menicke, 492 
Mesnil, 499, 513, 546 
Metchnikoff, 159, 160, 163, 208, 334, 336, 
337, 338, 468, 471, 520, 525 
Mecthnikoff, Mme., 334 
Meumir, 647 
Meyer, 326, 377, 423 
Michaels, 76 
Miescher, 545 
Migula, 34 
Miller, 464 
Minchin, 544 
Mitchell, 238 
Mitrophanow, 34 
Moeller, 80, 526 
Mohler, 398, 513 
Moore, 65 
Morax, 326, 328, 438 
Morgagni, 570 
Morgenroth, 160, 161 
Moro, 409 
Moser, 260 
Motas, 562 
Muhlens, 516, 520, 524 
Muir, 162, 216 
Muller, 439, 526 
Mulzer, 521 
Musgrave, 481, 509, 533, 535, 537,538,540 INDEX OF AUTHORS 
669 
Prior, 464 
Proskauer, 643 
Protopopoff, 581 
Prowazek, 439, 441, 508, 516, 517, 533, 
565, 571 
Prudden, 244, 372, 390, 395 
Q 
Quincke, 533 
R 
Rabinowitsch, 398, 407, 513 
Ransom, 326, 328 
Rattone, 320 
Raynaud, 154 
Ravenel, 399 
von Recklinghausen, 22 
Reed, 590 
Reichert, 36, 71 
Remak, 233 
Remlinger, 489, 581, 586 
Rhazes, 563 
Ribbert, 245 
Richardson, 363 
Richet, 224 
Ricketts, 493, 494 
Rideal, 637, 639 
Rimpau, 209 
Rindfleisch, 22 
Ritz, 226 
Riviere, 487 
Rixford, 238 
Robinson, 522 
Roger, 193 
Rogers, 499, 501 
Romanowsky, 86 
Romer, 404 
Roos, 533 
Rosenau, 224, 390 
Rosenbach, 241, 246, 249 
Rosenow, 249, 263 
Ross, 499, 548, 557, 562 
Rouget, 503 
Rous, 490 
Roux, 23, 299, 302, 456, 490, 520, 581 
Rowley, 549 
Rudiger, 236 
Rupper, 467 
Russell, 104, 378 
Ruzicka, 34 
S 
Sabouraud, 231, 236 
Sabraces, 487 
Sachs, 226 
Salimbeni, 518, 527 
Salmon, 481, 521 
Sanarelli, 590 
Sanger, 244 
Sauvage, 521 
Schaudinn, 34, 41, 43, 507, 515, 524, 531, 
535, 541, 542, 549, 557, 559 
Schenk, 236 
Schereschewsky, 516, 519 
Schick, 225, 226, 227, 309, 311 
Schimmelbusch, 385 
N 
Nais, 544 
Nakanischi, 34 
zur Nedden, 439 
Negri, 571 
Neisser, 283, 304, 420, 521, 522, 523 
Nelis, 571 
Nepvieu, 503 
Netter, 267 
Neufeld, 208, 209, 216, 270, 271 
Neugebauer 329 
Neumann, 111 
Nichols, 524 
Nicolaier, 320 
Nicoll, 260, 332 
Nicolle, 79, 489, 493, 494, 501, 527 
Nierenstein, 514 
Nocard, 481, 490, 562, 581 
Nocht, 81 
Noguchi, 441, 491, 518, 520, 522 
Norris, 524, 526 
Novy, 490, 497, 498, 501, 509, 513, 515, 
524, 525, 526, 557 
Nuttall, 334, 518, 527, 559, 561, 562 
O 
Obermeier, 524 
Ogston, 241, 249 
Oliver, 238 
Ophuls, 238 . 
Opie, 217, 547 
Orth, 244 
Otto, 596 
P 
Paltauf, 229 
Pappassoterion, 502 
Pappenheimer, 527 
Park, 169. 249, 270, 276, 299, 302, 332, 
372, 378, 564 
Pasquale, 533, 538 
Passet, 245. 249, 385 
Pasteur, 21, 22, 23, 34, 53, 140, 241, 262, 
445, 450, 454, 456, 460, 471, 570, 571, 
580, 586, 633 
Patterson, 539 
Patton, 499, 501 
Pearce, 267 
Pearson, 404, 633 
Perkins, 236 
Permin, 329 
Perrin, 516 
Petruschky, 257, 260, 360 
Pettenkofer, 469 
Pfeiffer, 158, 159, 282, 363, 432, 433, 434, 
435 
Piana, 559 
Pick, 168 
von Pirquet, 225, 226, 227, 228 
Plenciz, 19 
Plotz, 494 
Pollender, 22, 450 
Poor, 489, 577 
Popper, 491 
Porges, 354 
Posner, 541 
Pottevin, 645, 646 670 
INDEX OF AUTHORS 
Schleiden, 19 
Schlosing, and Muntz, 629 
Schmiedelberg, 149 
Schneider, 61, 243 
Schoenlein, 233 
Schlottelius, 334 
Schottmiiller, 249, 374 
Schroeder, 21, 369, 657 
Schroder, 398 
Schultz, 425 
Schulze, 20 
Schurmayer, 385 
Schutz, 430 
Schwann, 20 
de Schweinitz, 489 
Sedgwick, 372 
Seitz, 358 
Seilards, 538 
Sergeant, 526 
Shiga, 378, 382, 533 
von Sholly, 301 
Siler, 495 
Simon, 212, 516, 544, 589 
Smith, 224, 226, 236, 299, 300, 310, 374, 
390, 414, 481, 533, 546, 558, 559, 560, 
561, 562 
Southard, 224 
Spallanzani, 20 
Spencer, 44 
Spronck, 299 
Stahl, 645 
Stanesco, 520 
Starcovici, 558 
Stephen, 506 
Steinhardt, 225, 489, 564, 577 
Stern, 83 
Sternberg, 262, 533, 642 
Stiles, 532, 533 
Stimson, 588 
Straus, 397, 427, 428 
Strong, 378, 380, 533, 535, 537, 538, 540, 
547 
Swellengrebel, 34 
Sydenham, 492 
T 
Talamon, 262 
Thayer, 562 
Theiler, 503 
Theobald, 591 
Thierfelder, 334 > 
Thomas, 468, 512 
Thomassen, 481 
Thompson, 553, 562 
Tissier, 518 
Todd, 492, 511, 527 
Tokishige, 239 
Torrey, 287 
Traum, 513 
Trembley, 19 
Trevisan, 482 
Trillat, 645, 646 
Trudeau, 404 
Tulasse, 22 
Tuttle, 482, 483 
Twort, 420, 423 
Tyzzer, 565, 566 
u 
Uhlenhuth, 521 
Unna, 254 
V 
Van Beneden, 34 
Van Ermengen, 80 
Van Gehuchten, 571 
Van Gieson, 534, 572 
Van Leeuwenhoeck, 18 
Van Sweiten, 570 
Vaughan, 134, 137, 147, 149, 224, 225, 
226 
Viereck, 537 
Villemin, 388 
Vincent, 319 
Voges, 503 
Vosges, 600 
Volpino, 575 
Von Dusch, 21 
Von Hoffmann, 643 
Von Recklinghausen, 22 
W 
Wadsworth, 661 
Waldeyer, 22 
Walker, 493, 536, 538, 637, 639 
Walters, 363, 422 
Warrington, 629 
Wasielewski, 546 
Wassermann, 276, 277, 285, 523 
Weeks, 437 
Weicherdl, 226 
Weichselbaum, 244, 275 
Weigert, 22, 34, 166, 167 
Weisner, 491 
Welch, 246, 247, 462, 463 
Winogradsky, 629, 631 
Wesbrook, 304 
White, 340 
Widal, 193, 237, 365 
Wilder, 494 
Williams, 37, 249, 270, 299, 302, 305, 
435, 532, 533, 535, 536, 571, 575 
Wilson, 56, 63, 122, 204, 304, 449, 494, 
564, 665 
Winogradsky, 63 
Winslow, 243, 372 
Wolbach, 488, 492, 494 
Wolf, 477, 479 
Wolffhugel, 113, 659 
Wollstein, 276, 434, 435, 437 
Wright, 82, 208, 209, 212, 214, 216, 219, 
271, 363, 476, 479, 480, 481, 482, 499, 
511, 535, 549 
Wyssokowitsch, 244 
Y 
Yersin, 299, 302, 446, 448 
Z 
Zettnow, 34 
Zinsser, 218, 336 
Zsigmondy, 70 
Zupink, 327 
Zwinger, 570 GENERAL INDEX. 
A 
Abbe condenser, 67, 68 
Abscess, chief producers of, 241 
Absorption methods, 197 
Achorion schoenleinii (favus fungus), 233 
Acid-fast, bacilli, 388, 420-424 
Acids, as disinfectants, 643 
from carbohydrates, 627, 633 
effect of, on bacteria, 643 
Actinomyces, 41, 477 
cultivation, 477 
inoculation into animals, 480 
isolation, 480 
morphology, 479 
occurrence, 481 
Actinomycosis, 41, 481 
Aerobic microbes, 53, 115 
facultatiye, 53 
obligatory, 53 
Agar, nutrient, 96 
Agglutinating strength of a serum, 200, 
201 
Agglutination of bacteria, in hanging 
drops, 201 
group, 195 
nature of substances conerned in, 
193 
relation between agglutinating and 
bactericidal powers, 200 
testing of, 201-206. See also Indi- 
vidual bacteria. 
Agglutinins, 193 
absorption methods for differentia- 
tion of, 197, 199 
characteristics of, 194 
development of, 195, 196 
group, 195, 197 
loss of power of bacteria to absorb, 
199 
relative accumulation of specific and 
group, 197 
specific and group, 195, 197 
Agghitinoids, 194 
Aggressins, 175 
Agitation, influence of, on bacteria, 57 
Air, bacteriologic examination of, 606 
Alcohol as disinfectant, 643 
Alcoholic fermentation, 634 
Alexin, 160 
Amboceptor, 183 
Ammonia in bacterial growths, demon- 
stration of, 91 
Ameba binucleata, 48 
coli (see Entameba coli), 532 
Amebse, 532 
characteristics of, 48, 535, 541 
cultures, 535 
in different diseases, 539, 541 
historical note, 532 
in man, 538 
material and methods of study, 
534 
morphology, 535 
pathogenicity, 537 
reproduction, 536 
sexual phenomena, 537 
sites of, in the human body, 533 
source of, 540 
stains for, 534 
viability, 537 
Amebic dysentery (amebiasis), 538 
diagnosis of, 541 
immunity from, 540 
incidence of, 539 
prognosis of, 540 
symptoms, 539 
tissue changes, 539 
treatment of, 540 
Amebida, 48 
Amphitricha, 35 
Anaerobic bacteria, 53, 115 
culture methods for, 116 
pathogenic forms, 450 
Anaphylactic shock, 225 
Anaphylatoxin, 224 
Anaphylaxis, 224 
dose producing symptoms of, 225 
incubation period for development 
of, 225 
sensitizing dose producing condition 
of, 225 
specificity, 225 
Angina, Vincent's, 319 
Aniline dyes, 75, 99, 104 
oil as mordant, 77, 78 
Animals, inoculation of, 122 
use of, for diagnostic and test pur- 
poses, 122 
Anopheles, 552 
Antagonism between living body and 
microorganisms, 151 
Antagonists, 52 
Anthrax, 450 
bacillus, 450 
biologic characters of, 452 
growth in media, 452 
identification of, 452 
infection, 455 
morphology of, 450 672 
GENERAL INDEX 
Anthrax bacillus, occurrence of, in cattle 
and sheep, 455 ' 
in man, 455 
pathogenicity, 454 
resistance, 454 
spore formation, 453 
staining, 452 
bacteriologic diagnosis of, 457 
prophylaxis, 457 
serum treatment, 457 
symptomatic, 457 
Antianaphylaxis, 225 
Antibacterial sera, 155 
Antibodies in general, 158 
Antigens, 162, 181, 185, 187, 189, 190, 
191, 192 
Antipneumococcus serum, 271 
Antiseptics, table of values, 647 
action, 113 
Antistreptococcus serum, 257 
serums in scarlet fever, 260 
Antitoxic sera, testing power of, 172 
Antitoxins, absorption of, 155 
concentration of, 174 
Ehrlich's theory for production of, 
165-167 
in general (see also under Diph- 
theria and Tetanus), 168 
methods of administration of, 157 
nature of, 165 
other theories as to production of, 
162 
as preventive, 156 
production of, for therapeutic pur- 
poses, 170 
relative development of and bac- 
tericidal substances, 156 
separation from serum, 173 
stability of, in the serum, 149 
unit, 168 
testing of, 168, 172 
Antituberculous serum, 412 
Apparatus, cleansing and sterilization, 86 
Arnold's steam sterilizer, 93 
Aromatic products of decomposition, 62 
Arthrospores, 40 
Ascitic fluid, 101 
Aspergillus, 25 
fumigatus, 229, 231 
glaucus, 28 
Autogenous vaccines, 220 
Autoinfection, 144 
Autopsy of test animals, 124 
Azotobacter, 631 
B 
Babesia, 543, 558. See Piroplasma bi- 
geminum. 
Bacillary dysentery, 378 
Bacilli, acid-fast, 423 
general characters of, 32 
Bacillus, 32 
acidophilus, 341, 342 
aerogenes, 344, 353 
. Bacillus -aerogenes capsulatus, 343, 462 
sporogenes, 343 
alcaligenes, 344, 373 
anthracis, 450 
symptomatic], 457 
avisepticus, 445 
of blue pus, 383 
bifidus, 340 
Bordet-Gengou, 441 
botulinus, 271 
of bubonic plague, 446 
bulgaricus, 338 
butter, 424 
butyricus, 41 
capsulatus, 353 
of chicken cholera, 445 
cholerae suis, 269, 273 
cloacae, 344, 354 
Clostridium pasteurianum, 631 
coli communior, 344, 345 
communis, 344, 349 
colon group, 344 
definition of, 32 
diphtheriae, 292 
diphtheria-like, 302 
of Ducrey, 443 
of dysentery, 344, 378 
enteritidis, 344 
sporogenes, 463 
faecalis alcaligenes, 344 
of Friedlander, 353 
Gaertner, 374 
of glanders, 425 
of green pus, 383 
grass, 424 
of hemorrhagic septicemia group, 445 
hofmanni, 303 
icteroides (in yellow fever), 590, 591 
of influenza, 432 
influenza-like, 436, 437 
of Johne's disease, 423 
of Koch-Weeks, 437 
lactici, 344 
lactis acrogenes, 267 
leprae, 420 
of leprosy, 420 
of Lustgarten, 424 
of malignant oedema group, 460 
mallei, 425 
melitensis. See Micrococcus meliten- 
sis. 
Morax-Axenfeld, 438 
mucosus, 353 
capsulatus, 353 
ozaenae, 354 
paradysentery, 344 
paratyphoid, 344 
perfringens, 343 
pertussis, 441 
pestis, 446 
plague, 446 
plague-like, in rodents, 449 
pneumo-, of Friedlander, 353 
prodigiosus, 50 
proteus vulgaris, 383, 385 
pseudodiphtheriae, 303 GENERAL INDEX 
673 
Bacillus, pseudo-influenza, 436 
putrificus, 343 
pyocyaneus, 383 
radicicola, 630 
rat leprosy, 422 
of rhinoscleroma, 354 
of smegma, 424 
of soft chancre, 443 
subtilis, 96 
suisepticus, 445 
of swine plague, 445 
of tetanus, 320 
of timothy grass, 424 
of tuberculosis, 388 
of typhoid, 340 
welchii, 343, 462 
xerosis, 304 
Bacteria, 30 
adaptation to environment of, 55 
aerobic 53, 115 
anabolic power of, 82 
in air, 606 
anaerobic, 53, 115, 450 
basic forms of, 31 
botanical relationship of, 24 
capsules, 33 
carriers, 143 
characteristics of, 30 
chemical effects of, 59-64, 627-631 
composition of, 50 
classification of, 24, 30 
definition of, 30 
degeneration forms of, 38 
dentrification, 629 
effect of agitation, 57 
electricity, 57 
pressure, 57 
radium, 57 
surrounding forces upon, 51 
x-rays, 57 
examination of, in hanging drops, 
72 
in tissues, 84 
fermenting, 627 
ferments, 60, 634 
general characteristics of, 30 
granules, 35 
growth and reproduction, 37 
habital, 50 
higher forms of, 41, 476 
in Hodgkin's disease, 421 
in industries, 632 
influence of one species upon another, 
52 
of quantity in infection, 137 
intestinal, 334 
prevalent, 340 
regional distribution of, indi- 
gestive tract, 335 
substitution of one variety for 
another, 336 
involution forms, 38 
katabolic power of, 62 
lactic acid, 337-340 
loss of capacity to be agglutinated 
or absorbed, 199 
43 
Bacteria, manner in which they excite 
disease, 132 
membrane, 33 
mesophilic, 54 
morphology and structure of, 30 
motility of, 35, 36 
nitrification by, 63, 629 
non-agglutinable strains, 200 
nuclear substances in, 34 
oxygen and other gas requirements, 
53 
putrefactive, 628 
psychrophilic, 54 
physiologic characteristics of, 36 
products of growth, 59 
reproduction of, 37 
saprophytic, 51 
in sewage, 605, 632 
shape, 31 
size, 31 
in soil, 627 
species, permanence of, 26 
spore formation of, 39 
staining of, 76 
structure of, 33 
substance, 34 
thermic effects of, 59 
thermophilic, 54 
varieties of, in milk, 610 
in water, 600 
Bacterial proteins, 147 
Bactericidal sera, 155, 158 
power, 158, 200 
substances, origin of, 163 
Bacteriolysis, 158 
Bacteriolytic sera, nature of, 160 
Bacterium, caucasicum, 340 
Balantidium, 25, 50 
coli, 546 
minutum, 547 
Nyctootherus faba, 547 
Beggiotoa, 50 
Bichloride of mercury, 640 
Bile media, 105 
Black-leg, 457 
Black-water fever, 558 
Blastomycetes, 25, 28, 29, 229 
classification, 29 
pathogenic, 237 
complement fixation, 237 
cultivation, 109, 240 
staining, 240 
Blepharoplast, 43 
Blister fluid, 201 
Blood, bactericidal properties of, 156 
flagellates, 496 
germicidal properties of, 158 
Blue pus, bacillus of, 383 
Bodo, 528 
lacertae, 528 
urinarius, 528 
Bordet theory of antibodies, 162 
Bordet-Gengou phenomenon, 177 
Brownian movement, 36 
Bubonic plague, 446 
bacillus of, 446 674 
GENERAL INDEX 
Bubonic plague, bacillus of, biology of, 
447 
morphology of, 447 
pathogenicity, 447 
resistance, 449 
staining, 447 
vaccines, 449 
diagnosis of, 449 
immunity against, 448 
C 
Calmette's ophthalmo-tuberculin test, 
408 
Capsules of .bacteria, 33 
staining of, 79 
Carbohydrates, action of bacteria on, 
61, 629 
Carbolic acid as disinfectant, 646 
Carbon dioxides, production of, by colon 
bacillus, 347 
Carcinoma, treatment of, 255 
Carriers, 143, 360, 375, 474, 508, 527 
Cell membrane, 33 
substance, 34 
Cellulitis, streptococci in, 254 
Cellulose, fermentation of, 627 
Centrosome, 43 
Cercomonas hominis,- 497 
Cerebrospinal meningitis, 273 
Chemotaxis, 58 
Chicken sarcoma, 490 
Chlamydozoa, 439 
Chloride of lime, as disinfectant, 643 
Chlorine, as disinfectant, 643 
Chloroform, as disinfectant, 646 
Cholera, Asiatic, 464 
carriers, 474 
lesions in man, 469 
serum therapy, 472 
spirillum, 464 
allied organisms of, 474 
biology of, 465 
distribution in body, 469 
immunity against, 471 
isolation, 472, 474 
morphology of, 464 
pathogenicity of, 468 
prophylaxis, 471 
resistance and vitality of, 467 
staining, 465 
toxin of, 470 
spread of, 469 
Chromidia, 43 
Ciliata, 49, 546 
Cladothrix, 41, 476 
Classification of microorganisms, 24-27 
Claviceps purpurea, 229 
Clostridium pasteurianum, 631 
Cocci, characters of, 31 
enterococcus, 342 
micrococcus tetragenus, 247 
staphylococcus epidermidis (albus) 
(Welch), 246 
pyogenes aureus, 241 
Cocci, staphylococcus, biology, 241 
cultivation, 241 
immunity, 246 
morphology, 241 
occurrence, 245 
pathogenicity, 244 
pigment formation, 243 
staining, 241 
toxin formation, 243 
vaccine therapy, 246 
staphylococcus (pyogenes) albus, 
246 
staphylococcus (pyogenes) citreus, 
247 
streptococcus, pyogenes, 250 
biology, 251 
bacteriologic diagnosis, 260 
complement fixation, 190 
cultural reaction, 251 
development of hemolysis, 252 
of pigment, 252 
effect on tumors, 255 
immunity against, 256 
morphology, 250 
occurrence, 254, 255 
pathogenicity, 253 
resistance, 253 
serum, 257 
Coccidia, 543 
Coccidium bigeminum, 544 
cuniculi, 543 
Coley's streptococcus toxins, 255 
Colon bacillus, 344 
association with other bacteria, 
348 
behavior toward carbohydrates, 
347 
biology of, 346 
chemical activities, 347 
cultivation of, 346 
in cystitis, 351 
in diarrhea, 350 
differential diagnosis from ty- 
phoid bacillus, 352, 356, 373 
as disease producer, 349 
flagella, 346 
gas production, 347 
group of, 344 
growth of, with other bacteria, 
348 
immunity, 352 
immunization against, 352 
in inflammations of bile tract, 
352 
isolation, 352 
morphology of, 345 
occurrence in man and animals, 
349 
in other inflammations, 352 
of pancreas, 351 
passage through walls of intes- 
tines during life, 349 
pathogenicity, 349 
in peritonitis, 350 
as pus formers, 352 
reduction processes, 348 GENERAL INDEX 
675 
Colon bacillus resistance, 348 
in sepsis, 350 
significance of, in water, 600 
staining, 346 
toxins, 348 
treatment, 352 
of urinary tract, 351 
vaccine treatment, 352 
group, 344 
Colonies, characteristics of, 109 
counting of, 110-113 
study of, in plate cultures, 109 
various forms of, 110, 111 
Complement, 161-163, 181 
Bordet-Gengou phenomenon, 177 
fixation of, 177-192 
test for glanders, 190 
gonococcus infection, 189 
meningitis, 192 
pertussis, 190 
streptococcus infection, 190 
syphilis, 187, 523 
tuberculosis, 191 
typhoid, 192 
multiplicity of, 163 
origin of, 163 
Conidia, 28 
Conjunctivitis, influenza-like bacilli in, 
437 
Koch-Weeks bacillus of, 437 
Conradi and Drigalski medium, 104 
Copper sulphate, as germicide, 641 
capsule stain, 79 
Corrosive sublimate, 640 
Cover-glass, preparations of, how made, 
73 
how stained, 74 
how cleaned, 73 
Cowrpox, etiology of, 564 
relation to smallpox, 563 
Creolin, 647 
Creosote, 647 
Crescent bodies in malaria, 552, 556 
Cresol, 646 
Crithidia, 497 
Culex in malaria, 557 
Cultivation of microorganisms, 86 
of protozoa, 106 
Culture, anaerobic, 116 
block and hanging mass, 73 
media, inoculation of, 113, 
preparation of, 91 
reaction of, 88 
special, 103 
plate, making of, 107 
pure, 86, 118 
storage of, 95 
titration of, 88 
Cyst formation, 45 
Cytolytic serum, 160 
D 
Dark ground examination, 70 
Delhi boil, 499 
Dengue, 492 
Dilution methods, 112 
Diphtheria, agglutinin development, 312 
antitoxin, 306 
deleterious effects, 309 
globulin preparation, 311 
neutralizing characteristics of, 
307 
persistence of, in blood, 312 
result of, treatment of, 308 
results of use of refined, 311 
treatment, danger of, 311 
use of, in treatment and im- 
munization, 307 
bacillus, 292-306 
biology of, 296 
causal relationship of, to diph- 
theria, 292 
cultivation, 296-298 
exudate due to, contrasted with 
. that due to other bacteria, 313 
in healthy throats, 301 
morphology, 292, 295 
non-virulent forms of, 302 
pathogenicity, 298 
persistence of,, in throats, 301, 
306 
of characteristics in types 
of, 304 
pseudodiphtheria, 303, 304 
resistance to heat, drying, and 
chemicals, 305 
staining of, 293 
varieties of, 305 
virulence in cases of diphtheria, 
300 
virulent, in healthy throats, 301 
causes of death in, 299 
comparative virulence of different 
cultures, 300 
diagnosis, bacteriologic, 315-319 
relation of bacteriology to, 314 
historic, 292 
-like bacilli, pathogenic for guinea- 
pigs, but not producing diph- 
theria toxin, 302 
mixed infection in, 313 
pseudomembranous exudative in- 
flammations due to bacteria other 
than diphtheria bacillus, 313 
susceptibility to and immunity 
against, 306 
tissue changes in natural human 
infection, 298 
toxin, 299 
as index of virulence, 300 
production of, in culture media 
299 
transmission of, 306 
Diplococcus gonorrhoea. See Gonococcus 
intracellularis meningitidis. See 
Meningococcus. 
of pneumonia. See Pneumococcus. 
Disease in beer and wines, 634 
Disinfectants, 635-647 
gaseous, 642 676 
GENERAL INDEX 
Disinfectants, inorganic, 640 
organic, 643 
standardization of, 637 
Disinfection, 648-663 
agents for, 648-650 
of books, 654 
of clothing, 650, 652 
in contagious diseases, 650 
of discharges, 650 
by formaldehyde, 652, 658 
by heat, 649 
practical, of house, person, instru- 
ments, and food, 650, 651, 652 
657 
preventive, 652 
rooms, etc., 651 
by steam, 658, 659 
by sulphur dioxide, 657, 658 
for surgical operation, 659 
Drigalski and Conradi medium, 104 
Drying, effects of, on bacteria, 58 
Dunham's peptone solution, 96 
Dysentery, amebic, 532. See also Amebae, 
bacillary, 378 
bacilli, group of, 378 
agglutination characteristics, 
382 
differential diagnosis of, 382 
fermentation reaction, 379 
historical note of, 378 
morphology of, 378 
pathogenicity of, 379 
pathology, 380 
resistance, 381 
staining, 378 
toxins, 380 
types of, 378, 382 
E 
Ehrlich's side-chain theory, 165 
Eimeria schubergi, 544 
stiedae, 543 
Elimination of microorganisms through 
milk, 144 
through skin and mucous mem- 
brane, 145 
Endo medium, 104 
Endospores, 40 
Ensilage, 633 
Entamoeba. See also Amebae. 
buccalis, 533 
coli, 532 
histolytica, 532 
Enterococcus, 342 
Enzymes, 60 
Eumycetes. See Hyphomycetes. 
Examination, of air, 606 
of hanging drop, 72 
of soil, 607 
of water, 597 
F 
Farcy, 482 
Favus, 233 
Fermentation, 60 
alcoholic, 634 
by bacteria, 627, 633 
tube, 88 
of urea, 61 
Ferments, characteristics of, 60 
diastatic, 61 
inverting, 61 
proteolytic, 60 
rennin-like, 61 
Film preparations, 73 
Filter beds, 605 
Filters, 91-93, 488 
Filtrable viruses, 488 
Group I, of unknown morphology, 
489 
Group II, visible, 490 
Group III, of questionable filtra- 
bility, 492 
Filtration, technique of, 488 
of water, 604, 605 
Fixatives, 74, 84 
Fixation of complement. See Comple- 
ment. 
Flagella, 35 
staining of, 80 
Flagellata, 25, 496 
classification, 24 
general characteristics of, 45 
life cycle of, 47 
materials and methods, 496 
Flies as carriers, relation to trypanoso- 
miasis, 508 
Foot and mouth disease, 489 
Formaldehyde as disinfectant, 643 
permanganate of potash method, 655 
Wilson's rapid generator, 656 
Framboesia tropica, 523 
Fungi, pathogenic varieties, 25, 28, 229- 
237 
of pityriasis, 234 
of ringworm, 230 
of sporotrichosis, 236 
of thrush (soor), 29, 235 
G 
Gas production by bacteria, 64 
test for, 119 
Gelatin media, 96 
Germicidal action, method of determina- 
tion, 158, 159 
Germination of spore, 41 
Glanders bacillus, 425 
biology of, 425 
cultivation of, 426 
pathogenicity, 426 
staining, 425 
diagnosis of, 190, 428, 429 
by agglutination test, 430 
by complement fixation, 190, 
v 431 
by ophthahno-reaction, 429, 430 
immunity against, 427 
mode of spread, 427 GENERAL INDEX 
677 
Glanders, test for (mallein), 428 
vaccine, 428 
Glassware, preparation of, 86, 179 
Globulin in antitoxic sera, 173, 174 
Glossina in relation to trypanosomiasis, 
508, 510 
Gonococcus, 283-289 
■ agglutination, 287 
bacteria resembling, 289 
bacteriologic diagnosis, 288 
cultivation, 286 
diseases excited by, 286, 287 
in endocarditis, 287 
morphology, 283 
occurrence of, 286 
serum and vaccine therapy, 287 
staining reactions, 284 
toxins of, 286 
Gonorrhea, bacteriologic diagnosis of, 288 
Gram-negative and Gram-positive mi- 
crobes, list of, 128-131 
stain, 78 
Grass bacillus, 628 
Group agglutinins, 195 
Gruber-Widal reaction, 201-206 
H 
Halteridium, 557 
Hanging drop for study of bacteria, 72 
mass or block, 73 
Hemameba (see Plasmodium malariae), 
falciparum, 549 
malariae, 549 
vivax, 549 
Hemoglobinophilic bacilli, 432 
Hemolysins, Ehrlich's studies on, 160 
Hemolysis, 161, 177 
Hemolytic sera, 160, 161 
Hemoproteus, 557 
Herpetomonas, 497 
Histoplasma capsulatum, 500 
Historical sketch of microbiology, 17 
Hodgkin's disease, 421 
Hog cholera, 489 
Hollow slide, 72 
Hydrogen peroxide, 642 
Hydrophobia (see Rabies), 570 
Hypersensitiveness, 224. See also Ana- 
phylaxis. 
Hyphomycetes, 27, 229 
agglutination, 237 
■classification, 27 
complement fixation, 237 
cultivation, 236 
pathogenicity, 237 
I 
Ice, bacteria in, 55 
Immune body, 161 
multiplicity of, 163 
Immunity, active, 151, 219 
passive, 154 
Immunity, specific, 153 
Increase of toxicity and virulence of 
bacteria, 138 
Incubators, 113-115 
low temperatures in, 114 
India-ink method of examining bacteria, 
74 
Indol, 120 
Infection, 134 
influence of quantity in, 137 
localized, vaccine therapy for, 219, 
220 
mixed, 139 
protection afforded by skin and 
mucous membranes, 140, 145 
spread of, 142 
Influence of one species upon growth of 
another, 52 
Influenza bacillus, 432 
agglutination of, 437 
bacteriologic diagnosis, 435 
biology, 433 
complement fixation, 434 
cultivation 433 
distribution in the body, 434 
epidemiology, 435 
examination of sputum for, 436 
immunity to, 434 
morphology of, 432 
pathogenicity, 434 
resistance, 433 
serum therapy, 437 
staining characteristics, 433 
in tuberculosis, 435 
vaccines, 437 
Inoculation, methods of, 122 
Insect carriers, 527. See also Carriers 
and Vermin. 
Intestines, anaerobic conditions in, 335 
development of bacteria in, 334 
methods used in examination of 
feces, 336 
regional distribution of bacteria in, 
335 
significance of bacteria in, 334 
Isolation of microorganisms, 107-109 
K 
Kala-azar, 498 
Koch's original tuberculin, 403, 407 
phenomenon of, 175 
L 
Lamblia, 530 
intestinalis, 531 
Laverania malariae, 549 
Leishmania donovani, 499 
bedbugs as carriers, 501 
cultivation, 501 
morphology, 501 
pathology, 499, 501 
prophylaxis, 502 678 
GENERAL INDEX 
Leprosy bacillus, 420-424 
differential diagnosis of, 423 
morphology of, 420 
pathogenicity of, 422 
Leptomonas, 497 
Leptothrix, 41, 476 
Leukocytes, extract of, 217 
part played by, in immunity, 208 
production of exudates rich in, 217 
for testing phagocytosis, 124 
Light, production of, by bacteria, 59 
influence of, on bacteria, 56 
Litmus, 88 
Lophotricha, 36 
Lyssa (see Rabies), 570 
M 
Madura foot, 481 
Malaria, 548. See also Plasmodium 
malaria?. 
diagnosis 555 
estivo-autumnal, 549 
historical note, 548 
immunity, 555 
infection, how acquired, 549 
-like organisms in other animals, 
557 
materials and methods for study, 549 
mosquitoes in relation to, 552, 554 
parasites, 549 
prophylaxis, 555 
quartan, 549 
technique of blood examination in, 
83, 555 
tropical, 549 
Malignant edema, bacillus of, 460 
Mallei, bacillus, 425. See Glanders. 
Mallein, preparation of, 428 
test for glanders, 429 
Malta fever, 290 
spread by goats' milk, 290 
Material for bacteriologic examination, 
procuring of, from those suf- 
fering from disease, 125 
routine technique of examina- 
tion, 127 
Measles, 492 
Media, preparation and sterilization of. 
86 
reaction of, 52, 66 
special, 103-107 
Bordet-Gengou, 103 
for cholera, 105 
for protozoa, 106 
synthetic, 104 
for toxin production, 105 
for typhoid-colon groups, 104 
for yeasts and molds, 106 
storage of, 95 
various kinds of, 95-107 
Meningitis, bacteriologic diagnosis of, 280 
various organisms exciting, 280 
Meningococcus, 273-281 
agglutination, 275 
Meningococcus, bacteriologic diagnosis, 
280 
morphology, 273 
cocci resembling, 281 
pathogenicity, 274 
presence in nares of both sick and 
healthy persons, 275 
resistance, 274 
serum treatment, 276 
staining, 273 
Metachromatic granules, 35 
Metchnikoff, spirillum of, 475 
Microbiology, applied, 579 
historical sketch, 17 
Micrococcus catarrhalis, 282 
gonorrhoea, 283-289 
intracellularis, 273-281. See Men- 
ingococcus. 
lanceolatus, 262-272. See Pneu- 
mococcus. 
melitensis, 290 
tetragenus, 247 
biology, 248 
growth on media, 248 
morphology, 247 
pathogenicity, 248 
staining, 248 
zymogens, 291 
Microorganisms, chemical composition, 
50 
classification, 24-27 
effects of surrounding forces upon, 
51-59 
general characteristics, 24-65 
group characteristics, 27-50 
products of growth of, 59-64 
Microscope, different parts of, 66 
Microscopic methods, 71 
Microsporon, 234 
Milk, bacterial contamination of, 617 
bacteriology of, in relation to dis- 
ease, 609, 613, 614, 617 
development of bacteria in heated, 
620 
elimination of bacteria through, 
144 
examination of, 609 
grading of, 626 
heated vs. raw, in feeding, 615 
identification of bacteria in, 610 
influence of cleanliness on, 622 
of temperature on growth of 
bacteria in, 609, 618 
lactic acid milk, 337 
number of bacteria in, 609 
pasteurization of, 620, 661 
pathogenic properties of bacteria 
isolated from, 612 
smear method of estimating num- 
ber of bacteria, 609, 610 
sterilization of, 661 
streptococci in, 612 
time required for multiplication of 
bacteria in, 609 
transmission of disease through, 
625 GENERAL INDEX 
679 
Molds, pathogenic, 27, 229. See Hypho- 
mycetes. 
Monotricha, 35 
Mosquitoes as agents of infection in 
malaria, 548, 552, 553 
in yellow fever, 592-595 
trypanosomes in, 497 
Mordants, 77 
Morphology of bacteria, 30 
of molds, 27 
of protozoa, 42 
of yeasts, 29 
Mosaic diseases of tobacco, 489 
Motility of bacteria, 36 
organs of, 35 
of protozoa, 44 
organs of, 42 
Mucor, 229, 230 
Mumps, 494 
Mycetoma, 481 
Mycomycetes, 28 
Myxosporidia, 544 
N 
Nagana, 503 
Negri bodies in rabies, 570, 571, 573 
morphology of, 573 
smear method of demonstrating, 
572 
staining method for, 83 
Neurorycetes hydrophobias, 571 
Nitrification, 629 
Nitrifying bacteria, 629 
Nitrogen fixing bacteria, 630 
Nocardia, 42, 482 
animal inoculation, 486 
cultivation, 485 
morphology, 486 
pathology, 482-485 
treatment of infection, 487 
Nosema, 543 
bombycis, 545 
lophii, 545 
rat virus, 490 
O 
01'dium, 235 
Opsonic index, 211 
accuracy of, 212 
determination of, 212 
diagnostic value, 216 
dilution method, technique, 212 
Simon's method, technique, 212 
test, 211-214 
variation of, during treatment 
by inoculation, 214 
variation in healthy persons, 
215 
Wright's method, technique, 211 
Opsonins, 208, 216, 217 
distribution in body, 217 
p 
Paradysentery bacilli, 379 
Parameningococcus, 276 
Paratyphoid group, 374 
bacilli, 374 
type A, 374 
type B, 374 
type C, 377 
diagnosis, 377 
enteritidis types, 374, 377 
Pasteur treatment of rabies, 580 
Pasteurization, 661, 663 
Pellagra, 495 
Penicillium, 229, 230 
Peritricha, 35 
Pest (bubonic plague), 446 
Phenolphthalein as indicator, 88 
Phycomycetes, 28 
Pigment production by microbes, 61 
Piroplasma (Babesia), 558 
bigeminum, 558 
blood organisms similar to, 562 
morphology, 559 
pathogenicity, 561 
prognosis, 561 
prophylaxis, 561 
symptoms, 561 
ticks as carriers of, 560 
treatment, 561 
canis, 559 
staining, 559 
v. Pirquet cutaneous tuberculin test, 
408 
Pityriasis versicolor, 234 
Plague, bubonic, 446 
bacillus, 447 
biology, 447 
morphology, 447 
pathogenicity, 447 
staining, 447 
bacteriologic diagnosis, 449 
historic, 446 
immunity, 448 
occurrence, 449 
resistance, 449 
vaccines, 449 
Plants, bacterial disease of, 633 
Plasmodium (Hemameba), 543 
malarias, 543, 548, 551 
aestivo-autumnal parasite, 547 
classification, 551 
cultivation, 553 
cycle in man, 549 
in mosquito, 552 
examination of blood for, 549 
pathogenicity, 555 
quartan parasite, 549 
staining methods for, 73, 74, 78, 
81, 82 
tertian parasite, 549 
prophylaxis, 555 
toxin production, 555 
vivax, 547 
Pleuropneumonia, contagious, of cattle, 
490 680 
GENERAL INDEX 
Pneumobacillus. See Bacillus mucosus 
capsulatus. 
Pneumococcus, 262-272 
agglutination reaction, 270 
biologic characteristics, 263 
cultural reactions, 264, 265 
immunity to infection by, 271 
morphology, 262 
mucosus, 270 
special media for cultivation, 265 
staining, 263 
pathogenicity, 266-270 
resistance, 265 
therapeutic experiments, 271 
toxin production, 266 
vaccines, 272 
varieties of, 270 
virulence, 266 
Poisons, nature of microbal, 147 
vegetable and animal, 147 
Poliomyelitis, epidemic, 490 
Polymastigida, 528, 530 
Polyvalent serum, 164 
Precipitins, 206, 207 
Proteins, bacterial, 134, 135 
Proteosoma, 557 
Protista, 24 
Protozoa, 42 
blepharoplast, 42 
centrosome, 43 
chromidia, 43 
chromatin, 43 
classification, 24 
cyst-formation, 45 
cytoplasm, 42 
developmental cycle, 45 
ectoplasm, 42 
entoplasm, 42 
general characteristics of, 42 
groups, 45 
growth, 44 
karyosome, 43 
motility, 44 
nucleus, 42 
locomotor or kinetic, 43 
nutrition, 44 
physiologic characteristics of, 43 
relationship to other microorgan- 
isms, 24, 25 
reproduction, 44 
respiration, 44 
sexual phenomena, 44 
Pseudodiphtheria bacilli, 303 
Ptomains, 149 
Pyogenic cocci, 241 
R 
Rabies, 489 
complement, binding test, 576 
cultivation of organism, 575 
diagnosis of, 575 
fixed virus, 580 
material and methods for study, 572 
methods of immunization, 580 
Rabies, Negri bodies in, 572-575 
Pasteur's treatemnt, 580 
pathogenesis, 577 
preparation of virus, 582, 584, 586 
prevention, 590 
preventive inoculation against, 580 
resistance of virus, 576 
serum, antirabic, 586 
smear method, 572 
symptoms, 578 
treatment, 580, 584, 587, 588, 589 
Rat leprosy, 423 
virus, Novy's, 490 
Reaction of media, correction of, 88 - 
Vosges' and Proskauer's test in, 608 
Relapsing fever, spirillum of, 492, 524 
Rhinosporidium kinealyi, 544 
Rinderpest, 489 
Rocky Mountain spotted fever, 494 
Russell's double sugar medium, 104 
S 
Saccharomyces, 237 
Busse, 237 
Sarcinae, 32 
Sarcocystis, 543 
muris, 546 
Sarcoma, chicken, 490 
Sarcosporidia, 543, 545 
Sarcosporidiosis, 594 
Sauerkraut, 633 
Scarlet fever, 492 
conveyance of, by milk, 625 
streptococci in, 260 
Schizogony, 549 
Schizomycetes, 25 
Sections, preparation of, 84 
Loftier's staining method for, 185 
Sera, antitoxic, 168, 175 
bactericidal, 155, 158 
bacteriolytic, 160 
cytolytic, 160 
demonstration of nature of, 160 
therapeutic value, 155 
multipartial, 164 
polyvalent, 164 
precipitating, production of, 207 
Serum, sickness, 226 
Sewage, bacteria in, 631 
disposal of, 605 
farming, 632 
Schick test, 309 
Silkworm disease, 545 
Sleeping sickness, 503. See Trypano- 
somiasis 
Smallpox, 492, 563 
etiology, 564 
historical, 563 
pathogenicity, 566 
relation to vaccinia, 563 
virus, preparation of, 566 
Smear method in diagnosis of rabies, 572 
for direct examination of milk, 
609 GENERAL INDEX 
681 
Soft chancre, 443 
Soil, bacteria in, 607, 627 
examination of, 607 
South African horse sickness, 490 
Species, permanence of, 26 
Specimens, preservation of, 84 
Spirilla, general characteristics of, 32 
Spirillum of Asiatic cholera, 464. See 
Cholera. 
of Tinkler and Prior, 475 
of Metchnikoff, 475 
of relapsing fever, 524. See Spiro- 
cheta obermeieri. 
Spirocheta, 25, 515 
balantidis, 516 
balbiani, 516 
buccalis, 517 
carteri, 526 
dentium, 517 
duttoni, 526 
gallinarum, 518 
obermeieri (recurrentis), 524 
biologic characteristics, 525 
morphology, 524 
pathogenicity, 525 
pallida, 518. See Treponema pal- 
lidum. 
pertenuis, 523 
refringens, 517 
macrodentium, 518 
microdentium, 518 
vincenti, 517 
Spirochetse, 515 
cultivation, 516, 519 
in framboesia tropica, 523 
in mouth, 517 
methods for study, 515 
miscellaneous, 518 
pathology, 520, 522 
refringens, 517 
in relapsing fever, 524 
staining, 516 
in tick fever, 526 
treponema pallidum, 518 
in tumors, 517 
in Vincent's angina, 517 
Spirochetes from relapsing fever in 
America, 526 
Spores, 39, 41, 49 
arthrospores, 40 
endospores, 40 
formation, 39 
germination of, 41 
resistance to heat, 55 
protozoan, 49 
staining of, 79 
Sporotricha, 236 
Sporozoa, 49 
life cycle, 45 
Spotted fever, Rocky Mountain, 494 
Staining acid-fast bacteria, 79 
capsules, 79 
flagella, 80 
principles underlying, 76 
spirochetes, 82, 83 
spores, 79 
Stains, blue, 75, 77, 78 
brown 75 
eosin-methylene-blue, 83 
Giemsa, 81 
Goldhorn, 82 
Gram, 78 
green, 75 
Heidenhain, 84 
Hiss, 79 
Jenner, 82 
Koch-Ehrlich, 78 
Kuhne methylene-blue, 78 
Leishman, 82 
Loffler's, 77, 80 
MacNeal, 81 
Mallory, 83 
Moeller, 79 
Neisser, 295 
Nocht-Romanowsky, 81 
red, 75 
Ross, 83 
violet, 75 
Van Gieson, 83 
Welch, 79 
Williams, 83 
Wright, 82 
Ziehl-Neelsen, 77 
Staphylococcus, 241, 247. See Cocci. 
Sterilization, 91 
fractional, 93 
intermittent, 94 
of glassware, 86 
of'milk, 661 
Sterilizer, dry heat, 89 
steam, 93 
Stock vaccines, 223 
Streptococci, 249, 261. See Cocci. 
Streptothrix. See Nocardia. 
Sulphur-dioxide gas in house disinfec- 
tion, 642 
Symbioses, 52 
Symptomatic anthrax, 457 
Syphilis, 518, 520, 523. See also Tre- 
ponema pallidum. 
immunity, 522, 523 
Wassermann reaction in, 523 
T 
Tabardillo, 493 
Tetanolysin, 324 
Tetanospasmin, 324 
Tetanus, 320 
antitoxin, 325 
therapeutic value, 332 
method of administration of, 
329 
persistence in blood, 326 
production of, for therapeutic 
purposes, 325 
results of use of, for immuniza- 
tion, 331 
technique of testing, 325 
unit, 325 
bacillus, animal experiments, 322 682 
GENERAL INDEX 
Tetanus bacillus, biology of, 321 
differential diagnosis, 333 
growth in media, 321 
isolation of pure cultures, 322 
morphology, 320 
non-virulent type, 333 
occurrence in soil, 320 
pathogenicity, 322 
spores, resistance of, 321 
staining of, 321 
diagnostic procedures in, 333 
differential diagnosis, 333 
immunization against, 332 
in man, 323 
natural.infection, 323 
toxin, 323 
action of, in body, 324 
neutralization of, 326 
presence in blood, 325 
production of, for immunizing 
horses, 324 
relation to tissues, 326, 327 
theories of mode of action, 326 
treatment with antitoxin, 331, 332 
Texas fever, parasite of, 558 
Thermo-regulator, 115 
Thrush fungus, 235 
Tick fever, 526 
Ticks in relation to disease, 494, 560 
Tinea, 230 
method of examination, 231 
Tissue, examination of bacteria in, 84 
fixing and hardening of, 84 
Titration of culture media, 88 
Tobacco industry, bacteria in, 632 
Toxins, 133, 134, 136, 139, 145 
Ehrlich's theory as to the nature 
of, 148 
extracellular, 145 
ferment characteristics of, 146 
intracellular, 146 
Toxoids, 148 
Toxophore group, 148, 149 
Trachoma, 439, 493 
hemoglobinophilic bacilli in, 439 
inclusions, 440 
Treponema pallidum, 518-524. See 
Syphilis. 
cultivation, 519 
morphology of, 518 
pathogenicity, 520 
staining, 516 
pertenuis, 523 
Trichomonas, 48, 530 
hominis, 530 
vaginalis, 530 
Trichomycetes, 25, 32, 41, 476 
Trichophyton megalosporon, 231 
microsporon, 231 
tonsurans, 231 
Trypanosoma, 47, 503 
comparative characteristics of dif- 
ferent species, 506 
cultivation of, 509 
examination for, 511 
cytoplasm, 507 
Trypanosoma gambiense, 506 
historical note, 503 
insect carriers, 508 
life cycle of, 508 
morphology, 507 
motility, 507 
nuclei, 507 
pathogenicity, 509 
pathogenic forms, 503, 505 
reproduction, 508 
table of pathogenic forms, 504, 505 
Trypanosomiasis, 509-514 
diagnosis, 511 
duration, 511 
methods of examination for, 511 
pathologic changes, 511 
prophylaxis, 512 
serum therapy in, 513 
symptoms, 509 
treatment, 512 
Tubercle bacillus, 388 
agglutination, 412 
attenuation, 402 
avian, 414 
bovine, 413 
chemical constituents of, 389 
cultivation, 391 
diagnosis by animal inoculation, 
415 
differences between human and 
bovine types, 413 
discovery, 388 
distribution, 388 
•examination of material for, 
415 
by animal inoculation, 415, 
419 
of cerebrospinal fluid, 418 
of feces, 418 
of milk, 399 
in mixed infection, 415, 418 
of sputum, 416, 419 
of urine, 418 
growth, 391 
human, 339 
immunization against, 403 
methods of examination for, 
399, 412, 415, 416, 418 
in mixed infection, 402 
morphology, 388 
pathogenicity, 394, 413, 414 
point of entrance, 395 
poisons, 395 
action on tissues, 395 
pure cultures, 393 
resistance, 389 
staining peculiarities, 389 
toxins, 395 
transmissibility, 402 
types, stability of, 415 
viability, 389 
Tuberculin, 403, 405-412, 415 
Calmette's test, 408 
diagnostic use of, 408-411 
Mantoux's test, 408 
Moro test, 409 GENERAL INDEX 
683 
Tuberculin preparation, 407 
subcutaneous test, 409 
therapeutic use of, 403, 410 
theoretical considerations, 409 
von Pirquet's test, 408 
Tuberculosis, 388 
agglutination reaction for diagnosis, 
412 
bovine infection of man, 399 
of different regions, 396 
in domestic animals, 413 
fetal infection, 402 
immunization against, 403 
individual susceptibility, 402 
infection, by ingestion of milk, 
398 
by inhalation, 396 
mixed, 402 
mode of infection, 395, 396 
prophylaxis, 412 
serum treatment of, 412 
U. S. Govt, directions for inspect- 
ing herds for, 411 
Typhoid bacillus, 355 
biological characteristics, 355 
carriers, 360, 361 
relation to milk infection, 
625 
cultivation, 356 
distribution in human subject, 
358, 371 
duration of life outside the 
body, 361 
elimination of, through urine, 
359 
and feces, 369 
in feces, 369 
flagella, 356 
in healthy persons, 360 
identification, 356, 373 
in ice, 372 
importance of, in mixed infec- 
tion, 359 
isolation of, 369-371, 602 
from blood, 369 
from feces, 369 
from urine, 371 
from water, 371, 602 
lesions, 359 
mixed infection, 359 
motility, 356 
morphology, 355 
occurrence in water, oysters, 
and milk, 362 
resistance, 355, 361 
staining, 355 
toxic effects, 358 
in urine, 370 
communicability, 361 
diagnosis by means of serum test, 
365 
due to infected milk, 362, 625 
Gruber-Widal reaction in, 365 
technique of test, 365 
- use of dead cultures, 368 
immunization against, 363 
Typhoid, individual susceptibility, 363 
vaccine therapy, 363 
Typhus fever, 493 
U 
Ultramicroscopic organisms. 70, 489 
Urea, fermentation of, 61 
V 
Vaccination, immunity conferred by, 
563, 566. See Smallpox. 
of calves, 567 
Vaccinia, 563 
bodies in, 564, 565 
Vaccine, bacterial, 219 
autogenous, 223 
as immunizing agents, 223 
bacterial, 219 
preparation of, 221 
sensitized, 222 
stock, 223 
therapy, 181 
dosage in, 222 
results of, 220 
for variola, 566 
bacteria in, 568 
bovine virus, 566 
clinical test for efficiency, 568 
collection, 567 
human virus, 566 
keeping of, 569 
preparation of, 566, 568 
rabbit virus, 566 
seed virus, 566, 567 
Variola, etiology of, 563. See Smallpox. 
Vermin, disinfection for, 117 
in Lieshmania infections, 501 
in typhus fever, 493 
Vibrio. See Spirillum. 
Vincent's angina, 319 
Vinegar, production of, 633 
Virulence, relation between, and build- 
ing of immune bodies, 163 
Vosges and Proskaner reaction, 600, 608 
W 
Wassermann reaction, 187 
Water, bacilli of colon group in, 600 
bacteriologic examination of, 598 
interpretation of results, 
601 
collection of samples of, 598 
contamination and purification of, 
603 
purification of, for domestic pur- 
poses, 605 
on large scale, 495, 604 
quantitative analysis of, 598 
sea, 606 
streptococci in, 602 684 
GENERAL INDEX 
Water, typhoid bacilli in, 602 
Weigert's law of overproduction, 167 
Whooping cough, 441 
Widal reaction. See Gruber-Widal, 201- 
206 
Wilson, apparatus for formaldehyde 
disinfection, 656 
method for anaerobic cultures, 
116 
Wolffhiigel's apparatus, 113 
Wool-sorters' disease, 450. See Anthrax. 
Y 
Yaws, 523 
Yeasts. See Blastomycetes. 
Yellow fever, 489 
etiology, 590, 591 
mosquitoes in, 591