PLATE I 
ANTHONY VON LEEUWENHOEK 
Who first saw bacteria THE FUNDAMENTALS 
OF 
BACTERIOLOGY 
BY 
CHARLES BRADFIELD MORREY, B.A., M.D. 
PROFESSOR OF BACTERIOLOGY AND HEAD OF THE DEPARTMENT IN THE 
OHIO STATE UNIVERSITY, COLUMBUS, OHIO 
ILLUSTRATED WITH 171 ENGRAVINGS AND 6 PLATES 
Second Edition, thoroughly Revised S' 
LEA & FEBIGER 
PHILADELPHIA AND NEW YORK 
1921 Copyright 
LEA & FEBIGER 
1921 TO 
GRACE HAMILTON MORREY 
AMERICAN PIANIST PREFACE TO SECOND EDITION 
The first edition seems to have fulfilled a need for a general 
text-book on the subject of bacteriology. The original method 
of presentation is preserved. The text-book idea is adhered to, 
so that the individual instructor may have full liberty to ex- 
pand on topics in which he is especially interested. A number 
of illustrations have been added, the text has been improved 
in many instances by the addition of further explanatory 
matter and the most recent general advances in the Science. 
Examples are the System of Classification of the Society of 
American Bacteriologists, which is used throughout the text, 
their Key to the Genera of Bacteria, a discussion of the H-ion 
concentration method of standardization, the selective action 
of anilin dyes, the mechanism of entrance of pathogenic or- 
ganisms into the body, a more detailed explanation of the 
origin of antibodies, the nature of antigens and a table of 
antigens and antibodies. 
Professor Vera McCoy Masters has assisted in the revision 
by aiding in the preparation of manuscript and the reading 
of proof and in the making of the index, for which services 
the author's thanks are hereby expressed. 
C. B. M. 
Columbus, Ohio, 1921. PREFACE TO FIRST EDITION 
An experience of nearly twenty years in the teaching of 
Bacteriology has convinced the author that students of this 
subject need a comprehensive grasp of the entire field and 
special training in fundamental technic before specializing 
in any particular line of work. Courses at the University 
are arranged on this basis. One semester is devoted to Gen- 
eral Bacteriology. During the second semester the student 
has a choice of special work in Pathogenic, Dairy, Soil, Water, 
or Chemical Bacteriology. A second year may be devoted 
to advanced work in any of the above lines, to Immunity 
and Serum Therapy, or to Pathogenic Protozoa. 
This text-book is intended to cover the first or introductory 
semester's work, and requires two classroom periods per week. 
Each student is compelled to take two laboratory periods 
of three hours per week along with the class work. The out- 
line of the laboratory work is given at the end of the text. 
Results attained seem to justify this plan. A text-book is 
but one of many pedagogical mechanisms and is not intended 
to be an encyclopedia of the subject. 
The author makes no claim to originality of content, since 
the facts presented are well known to every bacteriologist, 
though the method of presentation is somewhat different from 
texts in general. During the preparation of this work he 
has made a thorough review of the literature of Bacteriology, 
covering the standard text-books as well as works of reference 
and the leading periodicals dealing with the subject. Thus 
the latest information has been incorporated. 
No attempt has been made to give detailed references in a 
work of this character. 
VII VIII 
PREFACE 
The photomicrographs are original except where otherwise 
indicated and are all of a magnification of one thousand diam- 
eters where no statement to the contrary appears. These 
photographs were made with a Bausch & Lomb Projection 
Microscope fitted with a home-made camera box. Direct cur- 
rent arc light was used and exposures were five to ten seconds. 
Photographs of cultures are also original with a few indicated 
exceptions. All temperatures are indicated in degrees centi- 
grade. 
For use of electrotypes or for prints furnished the author 
is indebted to the following: A. P. Barber Creamery Supply 
Company, Chicago, Ill.; Bausch & Lomb Optical Company, 
Rochester, N. Y.; Creamery Package Manufacturing Com- 
pany, Chicago, Ill.; Davis Milk Machinery Company, North 
Chicago, Ill.; Mr. C. B. Hoover, Superintendent of Sewage 
Disposal Plant, Columbus, O.; Mr. C. P. Hoover, Super- 
intendent of Water Filtration Plant, Columbus, 0.; The Hy- 
draulic Press Manufacturing Company, Mt. Gilead, 0.; Loew 
Manufacturing Company, Cleveland, O.; Metric Metal Works, 
Erie, Pa.; Sprague Canning Machine Company, Chicago, Ill.; 
U. S. Marine Hospital Service; Wallace and Tiernan Com- 
pany, New York City, N. Y. 
For the preparation of many cultures and slides, for great 
assistance in the reading of proof and in the preparation of 
the index, Miss Vera M. McCoy, Instructor in Bacteriology, 
deserves the author's thanks. 
The author trusts that the book will find a place in College 
and University courses in Bacteriology. 
C. B. M. CONTENTS 
Historical Introduction-Spontaneous Generation-Causation of 
Disease-Putrefaction and Fermentation-Study of Forms- 
Chronological Table 17 
CHAPTER I. 
Position of Bacteria-Relationships to Algae--Yeasts-Molds- 
Protozoa , 37 
PART I. 
MORPHOLOGY. 
CHAPTER II. 
Cell Structures-Cell Wall-Protoplasm--Plasmolysis-Plasmop- 
tysis-Nucleus-Vacuoles-Capsules-Metachromatic Granules 
-Flagella-Spores 41 
CHAPTER III. 
Cell Forms-Coccus-Bacillus-Spirillum-Involution Forms . 52 
CHAPTER IV. 
Cell Groupings 55 
CHAPTER V. 
Classification-Migula's-Society of American Bacteriologists'- 
Key to the Latter 59 X 
CONTENTS 
PART II. 
PHYSIOLOGY. 
CHAPTER VI. 
Occurrence-General Conditions for Growth-Moisture-Tem- 
perature-Light-Oxygen-Osmotic Pressure-Electricity - 
X-rays and Radium Emanations-Pressure-Mechanical Vi- 
bration 71 
CHAPTER VII. 
Chemical Environment-Reaction of Medium-Chemical Com- 
position • . 81 
CHAPTER VIII. 
Chemical Environment (Continued)-General Food Relation- 
ships-Metabolism of Elements 86 
CHAPTER IX. 
Physiological Activities-Fermentation of Carbohydrates-Split- 
ting of Fats 93 
CHAPTER X. 
Physiological Activities (Continued)-Putrefaction of Proteins- 
Cycles of Nitrogen, Carbon, Sulphur, Phosphorus .... 102 
CHAPTER XI. 
Physiological Activities (Continued)--Production of Acids, Gases, 
Esters, Alcohols, Aldehydes, Aromatic Compounds-Phosphor- 
escence-Chromogenesis-Reduction-Oxidation-Production 
of Heat-Absorption of Free Nitrogen-Nitrogen Nutrition of 
Green Plants 110 
CHAPTER XII. 
Physiological Activities (Continued)-Production of Enzymes- 
Discussion on Enzymes-Toxins-Causation of Disease . . 121 
CHAPTER XIII. 
Disinfection - Steril ization - Disinfectants - Physical Agents- 
Pasteurization 130 CONTENTS 
XI 
CHAPTER XIV. 
Disinfection and Sterilization (Continued)-Chemical Agents- 
Anilin Dyes 156 
CHAPTER XV. 
Disinfection and Sterilization (Continued)-Choice of Agent- . 
Standardization of Disinfectants-Phenol Coefficient-Prac- 
tical Sterilization and Disinfection 164 
PART in. 
THE STUDY OF BACTERIA. 
CHAPTER XVI. 
Culture Media-Broth, Milk, Gelatin, Agar, Potatoes, Blood 
Serum-Standardization of Media-H-ion Concentration Meth- 
od-Synthetic Media 171 
CHAPTER XVII. 
Methods of Using Culture Media - Culture Tubes - Plates - 
Anaerobic Cultures-Vignal Tubes-Fermentation Tubes- 
Deep Culture Tubes-Novy Jars-Inoculation of Culture 
Media 184 
CHAPTER XVIII. 
Isolation of Bacteria in Pure Culture-Dilution-Plating-Streak- 
ing-Barber Apparatus-Aids in Isolation-Heat-Selective 
Antiseptics-Selective Food-Indicators-Animal Inoculation 194 
CHAPTER XIX. 
Study of the Morphology of Bacteria-Bacteriological Microscope 
-Hanging Drop Slides-Staining-Gram's Method-Spores- 
Acid-fast Bacilli-Capsules-Flagella-Metachromatic Gran- 
ules • 200 XII 
CONTENTS 
CHAPTER XX. 
Study of the Physiology of Bacteria-Temperature-Incubators- 
Thermal Death Point-Oxygen Relationships-Study of Physi- 
ological Acticities-Appearance of Growth on Culture Media- 
Appearance of Molds on Plate Cultures 213 
CHAPTER XXI. 
Animal Inoculation-Material for Bacteriological Examination . 227 
PART IV. 
GENERAL PATHOGENIC BACTERIOLOGY. 
CHAPTER XXII. 
Introduction - Infection - Acute Infection - Chronic Infection 
-Specific-Non-specific-Koch's Postulates-Virulence-Sus- 
ceptibility . . 231 
CHAPTER XXIII. 
Pathogenic Bacteria Outside the Body-As Saprophytes-As 
Facultative Saprophytes-Latent-Carriers-Universal Car- 
riers-Accidental Carriers-Necessary Intermediate Hosts . 237 
CHAPTER XXIV. 
Channels of Infection-Skin-Mucosae-Respiratory Tract-Ali- 
mentary Tract-Mechanism of Entrance of Organisms-Dis- 
semination in the Body-Paths of Elimination-Specificity of 
Location 243 
CHAPTER XXV. 
Immunity-Natural-Artificial-Active-Passive-Production of 
Immunity-Vaccine-Antiserum-Practical Applications of 
Immunity Reactions 250 
CHAPTER XXVI. 
Theories of Immunity-Pasteur-Chauveau-Baumgartner- 
Metchnikoff-Ehrlich-Principles of Ehrlich's Theory . . . 256 XIII 
CONTENTS 
CHAPTER XXVII. 
Ehrlich's Theory (Continued)-Receptors of the First Order- 
Antitoxin-Antienzyme-Preparation of Antitoxins-Units . 261 
CHAPTER XXVIII. 
Ehrlich's Theory (Continued)-Receptors of the Second Order- 
Agglutinins-Agglutination Reaction-Precipitins-Precipitin 
Test 265 
CHAPTER XXIX. 
Ehrlich's Theory (Continued)-Receptors of the Third Order- 
Cytolysins - Amboceptor - Complement - Anti-amboceptors 
-Antisnake Venoms-Failure of Cytolytic Serums in Practice 
-Complement-fixation Test 271 
CHAPTER XXX. 
Phagocytosis-Opsonins-Opsonic Index-Bacterial Vaccines- 
Preparation of-Use of-Lipovaccines-Aggressins .... 280 
CHAPTER XXXI. 
Anaphylaxis-Author's Theory-Tuberculin Test-Table of Anti- 
gens and Antibodies-Summary of Immunity as Applied to 
Protection from Disease 289 BACTERIOLOGY. 
HISTORICAL INTRODUCTION. 
Bacteriology as a science is a development of the latter 
half of the nineteenth century. It may be said to have 
begun in the decade between 1870 and 1880, due largely 
to the wide circulation given to Koch's work in proving 
that Bacillus anthracis is the cause of Anthrax in 1876, 
in devising new culture methods and in demonstrating that 
wound infections are due to microorganisms, 1878. Asso- 
ciated with this work were the great improvements in the 
microscope by Abbe and the introduction of anilin dyes 
for staining bacteria by Weigert. These results attracted 
workers throughout the world to the " new science. " Never- 
theless, this work of Koch's was preceded by numerous 
observations and experiments which led up to it. Cer- 
tainly the most important discoveries immediately respon- 
sible were those of Pasteur. He must be considered as the 
greatest of the pioneer bacteriologists since he worked in 
all fields of the subject. Some of the antecedent 
work was done in attempting to disprove the old " spon- 
taneous generation" theory as to the origin of organisms; 
some in searching for the causes of disease and some in the 
study of fermentation and putrefaction. 
SPONTANEOUS GENERATION. 
Speculation as to the first origin of life is as old as history 
and doubtless older. Every people of antiquity had its own 
legends, as for example, the account in Genesis. This ques- 
tion never can be definitely settled, even though living 
matter should be made in the laboratory. 18 
HIS TO RIC AL IN TROD UC TION 
The doctrine of the "spontaneous origin" of particular 
animals or plants from dead material under man's own 
observation is a somewhat different proposition and may be 
subjected to experimental test. The old Greek philosophers 
believed it. Anaximander (b.c. 610-547) taught that some 
animals are derived from moisture. Even Aristotle (b. c. 
384-322) said that "animals sometimes arise in soil, in 
plants, or in other animals," i. e., spontaneously. It can be 
stated that this belief was general from his day down through 
the Dark and Middle Ages and later. Cardano (a_.d. 1501- 
1576) wrote that water gives rise to fish and animals and is 
also the cause of fermentation. Van Helmont (1578-1644) 
gives directions for making artificial mice. Kircher (1602- 
1680) describes and figures animals produced under his own 
eyes by water on plant stems. 
However, many thinkers of the seventeenth century 
doubted the truth of this long-established belief. Francesco 
Redi (1626-1698) made a number of experiments which 
tended to prove that maggots did not arise spontaneously 
in meat, as was generally believed, but developed only when 
flies had an opportunity to deposit their eggs on the meat. 
It seems that by the latter part of this century the idea 
that organisms large enough to be seen with the naked eye 
could originate spontaneously was generally abandoned by 
learned men. 
The work of Leeuwenhoek served to suspend for a time 
the subject of spontaneous generation, only to have it revived 
more vigorously later on. He is usually called "The Father 
of the Microscope," though the compound microscope was 
invented probably by Hans Zansz or his son Zacharias, of 
Holland, about 1590. Leeuwenhoek used a simple lens, but 
his instruments were so much more powerful that they 
opened up an entirely new and unknown world. (Fig. 1.) 
Anthony van Leeuwenhoek (1632-1723) was apprenticed 
to a linen draper and accumulated a comfortable fortune 
in this business. He became interested in the grinding of 
spectacle lenses, then an important industry in Delft, 
Holland, where he lived, and did a great deal of experimental 
work in this line, mainly for his own enjoyment. Finally he SPON TAN EO US GENERA TION 
19 
succeeded in making a lens so powerful that he could see 
in water and various infusions very minute living bodies 
never before observed. Leeuwenhoek contributed 112 pa- 
pers to the Royal Society of Great Britain, the first in 1673, 
many of them accompanied by such accurate descriptions 
and drawings, for example a paper submitted September 12, 
1683, that there is no doubt that he really saw bacteria and 
was the first to do so (Fig. 2). Rightly may he be styled 
"The Father of Bacteriology," if not of the microscope. He 
Fig. 1.-Leeuwenhoek's Microscope. A is the simple bi-convex lens held 
firmly in place. In front of this is the small table, B, with the support, C, 
on the tip of which the object to be examined was held. This support could 
be brought nearer to or removed further away from the lens and held firmly 
in place by the screw, D. E is a second screw for raising or lowering the entire- 
table. A concave mirror that Leeuwenhoek sometimes used to focus more 
light on the object under examination, is shown at the right. 
says in one paper: "With the greatest astonishment I 
observed that everywhere through the material I was- 
examining were distributed animalcules of the most micro- 
scopic dimness which moved themselves about in a remark- 
ably energetic way." Thus he considered these living 
objects to be animals, from their motion, and this belief 
held sway for nearly two hundred years. 
Leeuwenhoek was a pure observer of facts and made no 
attempt at speculation, but his discoveries soon started the 20 
HISTORICAL INTRODUCTION 
theorists to discussing the origin of these minute organisms. 
Most observers, as was probably to be expected, believed 
that they arose spontaneously. Needham, in 1749, described 
the development of microorganisms around grains of barley 
in water. Bonnet, in 1768, suggested that probably Need- 
ham's animalcules came from ova in the liquid. The Abbot 
Spallanzani, in 1769, called attention to the crudeness of 
Needham's methods and later, in 1776, attempted to dis- 
prove spontaneous origin by heating infusions of organic 
material in flasks and then sealing them. His critics raised 
the objections that heating the liquids destroyed their 
Fig. 2.-The first drawings of bacteria by Leeuwenhoek. The dotted line 
C-D indicates the movement of the organism. 
ability to support life, and that sealing prevented the access 
of fresh air which was also necessary. The first objection 
was disproved by the accidental cracking of some of the 
flasks which thereafter showed an abundant growth. This 
accident seemed also to support the second objection, and 
Spallanzani did not answer it. Though Spallanzani's experi- 
ments failed to convince his opponents, they led to important 
practical results, since Frangois Appert, in 1810, applied 
them to the preserving of fruits, meats, etc., and in a sense 
started the modern canning industry. 
From Spallanzani to Schultze, there were no further 
experiments to prove or disprove spontaneous generation. SPON TAN EO US GENERA TION 
21 
Schultze, in 1836, attempted to meet the second objection 
to Spallanzani's experiment, i. e., the exclusion of air, by 
drawing air through his boiled infusions, first causing it to 
bubble through concentrated sulphuric acid to kill the 
"germs" (Fig. 3.). His flasks fortunately showed no 
growths, but his critics claimed that the strong acid changed 
the properties of the air so that it would not support life. 
This experiment of Schultze's, though devised for a differ- 
ent purpose, was really the first experiment in the use of 
chemical disinfectants, though Thaer (page 31) had used 
Fig. 3.-Schultze's experiment. The set of bulbs next to the face con- 
tained KOH and the other set concentrated H«SOi. Air was drawn through 
at frequent intervals from May until August but no growth developed in 
the boiled infusion. 
chemicals in a practical way. Schwann, in 1837, modified 
this experiment by drawing the air through a tube heated 
to destroy the living germs (Fig. 4). His experiments were 
successful but the "spontaneous generation" theorists 
raised the same objection, i. e., the change in the air by heat- 
ting. This was the first experiment in'which the principle 
of "dry heat" or "hot air" sterilization was used. Similar 
arguments were brought forward, also to the use of cotton 
plugs as filters by Schroeder and Dusch in 1859 (Fig. 5). 
This was the first use of the principle of sterilization by filtra- 
tion. It remained for Chevreuil and Pasteur to overcome 
this objection in 1861 by the use of flasks with long necks 22 
HISTORICAL INTRODUCTION 
drawn out to a point and bent over. These permitted a 
full access of air by diffusion but kept out living germs. 
Fig. 4.-Schwann's experiment. After boiling, as shown in the diagram, 
and cooling, air was drawn into the flask by aspiration while the coiled tube 
was kept hot with the flame. 
Fig. 5.-Schroeder and Dusch's experiment. The aspirating bottle drew 
the air through the flask after it had been filtered by the cotton in the tube. SPON TA NEO US GENER A TION 
23 
Fig. 6.-Pasteur's flask. 
Fig. 7.-Tyndall's box. One side is removed to show the construction. 
The bent tubes at the top are to permit a free circulation of air into the in- 
terior. The window at the back has one corresponding in the front (removed). 
Through these the beam of light sent through from the lamp at the side was 
observed. The three tubes received the infusion and were then boiled in 
an oil bath. The pipette was for filling the tubes. (Popular Science Month- 
ly, April, 1877.). 24 
HIS TO RIC AL IN TROD UC TION 
since these cannot fly but are carried mechanically by air 
currents or fall of their own weight (Fig. 6.). Hoffman, 
the year before (1860), had made similar experiments but 
these remained unnoticed. The Pasteur flasks convinced 
most scientists that "spontaneous generation" has never 
been observed by man, though some few, notably Dr. 
Charlton Bastian, of England, vigorously supported the 
theory from the early seventies until his death in November, 
1915. 
John Tyndall, in combating Bastian's views showed that 
boiled infusions left open to the air in a closed box through 
which air circulated did not show any growth of organisms 
provided the air was so free of particles that the path of a 
ray of light sent through it from side to side could not be 
seen (Fig. 7). Or if such sterilized infusions were exposed 
to dust-free air, as in the high Alps, the majority showed no 
growth, while all infusions in dusty air did show an abun- 
dance of organisms. Tyndall's experiments confirmed those 
of Pasteur and his predecessors and showed that the organ- 
isms developed from "germs" present in the air falling into 
the liquids and not spontaneously. 
While Tyndall's experiments were of great value as in- 
dicated, they probably were harmful in another way. 
These "germs in the air" were considered by bacteriologists 
as well as laymen to include necessarily many disease germs 
and to indicate the very general, if not universal, presence 
of these latter in the air. This idea led to many erroneous 
practices in sanitation and disinfection which even to this 
day are not eliminated. 
CAUSATION OF DISEASE. 
The transmission of disease from person to person was 
recognized by the ancients of European and Asiatic coun- 
tries. Inoculation of smallpox was practiced in China and 
India probably several thousand years ago and was intro- 
duced by Lady Mary Wortley Montague into England in 
1721, from Constantinople. These beliefs and practices do 
not seem to have been associated with any speculations or 
theories as to the cause of the disease. CAUSATION OF DISEASE 
25 
Apparently the first writer on this subject was Varo, about 
b.c. 70, who suggested that fevers in swampy places were 
due to invisible organisms. The treatment of wounds dur- 
ing the thirteenth and fourteenth centuries by hot wine 
fomentations and by the application of plasters was based 
on the theory that the air brought about conditions in the 
wounds which led to suppuration. These practices were 
indeed primitive antisepsis, yet were not based on a germ 
theory of the conditions which were partially prevented. 
Fracastorius (1484-1553), in a work published in 1546, 
elaborated a theory of "disease germs" and "direct and in- 
direct contagion" very similar to modern views, though 
based on no direct pathological knowledge. Nevertheless 
Kircher (mentioned already) is usually given undeserved 
credit for the "contagium vivum" theory. In 1657 by the 
use of simple lenses he observed "worms" in decaying sub- 
stances, in blood and in the pus from bubonic plague patients 
(probably rouleaux of corpuscles in the blood, certainly not 
bacteria in any case). Based on these observations and 
possibly also on reading the work of Fracastorius, his theory 
of a "living cause" for various diseases was published in 
1671, but received little support. > 
The discoveries of Leeuwenhoek which proved the exist- 
ence of microscopic organisms soon revived the " contagium 
vivum" idea of Kircher. Nicolas Andry in a work pub- 
lished in 1701 upheld this view. Lancisi in 1718 advanced 
the idea that "animalcules" were responsible for malaria, a. 
view not proved until Laveran discovered the malarial para- 
site in 1880.1 Physicians ascribed the plague which visited 
Southern France in 1721 to the same cause, and many even 
went so far as to attribute all disease to animalcules, which 
brought the theory into ridicule. Nevertheless the "con- 
tagium vivum" theory survived, and even Linnaeus in his 
Systema Naturae (1753-6) recognized it by placing the 
organisms of Leeuwenhoek, the contagia of diseases and the 
causes of putrefaction and fermentation in one class called 
"Chaos." 
!Sir H. A. Blake has called attention to the fact that the "mosquito theory" 
of malaria is mentioned in a Sanscrit manuscript of about the 6th century 
A. D. 26 
HISTORICAL INTRODUCTION 
Plenciz, a prominent physician and professor in the Vienna 
Medical School, published in 1762 a work in which he gave 
strong arguments for the "living cause" theory for trans- 
missable diseases. He taught that the agent is evidently 
transmitted through the air and that there is a certain 
period of incubation pointing to a multiplication within the 
body. He also believed that there was a specific agent 
for each disease. His writings attracted little attention at 
the time and the "contagium vivum" theory seems to have 
been almost lost sight of for more than fifty years. Indeed, 
Oznam, in 1820, said it was no use to waste time in refuting 
hypotheses as to the animal nature of contagium. 
Isolated observers, were, however, keeping the idea alive, 
each in his own locality. In 1787 Wollstein, of Vienna, 
showed that the pus from horses with glanders could infect 
other horses if inoculated into the skin. Abilgaard, of 
Copenhagen, made similar experiments at about the same 
time. In 1797 Eric Viborg, a pupil of Abilgaard's, published 
experiments in which he showed the infectious nature not 
only of the pus but also of the nasal discharges, saliva 
urine, etc., of glandered horses. Jenner in 1795-98 introduced 
vaccination as a method of preventing smallpox. This 
epoch-making discovery attracted world wide attention and 
led to the overcoming of this scourge which had devasted 
Europe for centuries, but contributed little or nothing to 
the question of the causation of disease. Prevost's discovery 
of the cause of grain rust (Puccinia graminis} in 1807 was 
the first instance of an infectious disease of plants shown to 
be due to a microscopic plant organism, though not a bacte- 
rium in this case. 
Doubtless one reason why the work on glanders and grain 
rust attracted little attention among the practitioners of 
human medicine was owing to the prevalent belief in man's 
complete separation from all lower forms of life. The 
evolutionists had not yet paved the way for experimental 
medicine. 
In 1822 Gaspard showed the poisonous nature of material 
from infected wounds by injecting it into animals and caus- 
ing their death. Tiedemann (1822), Peacock (1828) de- CAUSATION OF DISEASE 
27 
scribed "little bodies" in the muscles of human cadavers 
which Hilton (1832) considered to be parasitic in nature. 
Paget (1835) showed that these bodies were round worms 
and Owen (1835) described them more accurately and gave 
the name Trichina spiralis to them. Leidy (1846) found 
organisms in the muscles of hogs which he considered to be 
the same as Owen's Trichina and paved the way for the 
work of Zenker (1860) in showing the pathological relation 
between the Trichina of pork and human Trichinosis. 
Bearing on the "contagium vivum" theory was the redis- 
covery of the "itch mite" (S ar copies scabiei) by Renucci 
(1834), an Italian medical student. This had been declared 
several hundred years before but had been lost sight of. 
Chevreuil and Pasteur, in 1836, showed that putrefaction 
did not occur in meat protected from contamination, and 
suggested that wound infection probably resulted from en- 
trance of germs from without. Bassi, investigating a dis- 
ease of silkworms in Italy, demonstrated that a certain 
mold-like fungus (Botrytis bassiana} was the cause in 1837. 
This was the first instance of a microscopic vegetable organ- 
ism proved to be capable of causing disease in an animal. 
Boehm, in 1838, observed minute organisms in the stools 
of cholera patients and conjectured that they might have a 
causal connection with the disease. Dubini of Milan in 
1838 discovered the Ankylostoma duodenale which later was 
further described by Omodei in 1843 and shown to be the 
cause of Egyptian chlorosis by Griesinger (1851). The 
fungous nature of favuSj a scalp disease, was recognized 
by Schonlein in 1839, and the organism was afterward called 
"Achorion schoenleinii." Berg, in 1839-41, showed that 
thrush is likewise due to a fungus, "Oidium albicans." 
These discoveries led Henle, in 1840, to publish a work in 
which he maintained that all contagious diseases must be 
due to living organisms, and to propound certain postulates 
(afterward restated by Koch and now known as "Koch's 
postulates "p 233) which must be demonstrated before one 
can be sure that a given organism is the specific cause of a 
given disease. The methods then in vogue and the instru- 
ments of that period did not enable Henle to prove his 28 
HISTORICAL INTRODUCTION 
claims, but he must be given the credit for establishing the 
"contagium vivum" theory on a good basis and pointing 
the way for men better equipped to prove its soundness in 
after years. 
In 1842-43 Gruby showed that Herpes tonsurans, a form 
of ringworm, is due to the fungus Trichophyton tonsurans. 
Klencke, in 1843, produced generalized tuberculosis in a 
rabbit by injecting tuberculous material into a vein in the 
ear, but did not carry his researches further. In 1843, 
Doctor Oliver Wendell Holmes wrote a paper in which he 
contended that puerperal fever was qpntagious. Liebert 
identified the Peronospora infestans as the cause of one type 
of potato rot in 1845. The skin disease Pityriasis (tinea) 
versicolor was shown to be due to the Microsporon furfur 
by Eichstedt in 1846. In 1847 Semmelweiss of Vienna 
recommended disinfection of the hands with chloride of 
lime by obstetricians because he believed with Holmes in 
the transmissibility of puerperal fever through poisons 
carried in this way from the dissecting room but his theories 
were ridiculed. 
Pollender, in 1849, and Davaine and Rayer, in 1850, inde- 
pendently observed small rod-like bodies in the blood of 
sheep and cattle which had died of splenic fever (anthrax). 
That Egyptian chlorosis, afterward identified with Old 
World "hookworm disease," is caused by the Ankylostoma 
duodenale was shown by Greisinger in 1851. In the same 
year the Schistosomum hematobium was shown to be the 
cause of the "Bilharzia disease" by Bilharz. Kiichen- 
meister discovered the tapeworm, Tomia solium, in 1852, 
Cohn, an infectious disease of flies due to a parasitic fungus 
(Empusa muscoe) in 1855, and Zenker showed the connec- 
tion between trichinosis of pork ("measly pork") and human 
trichinosis (1860) as indicated above. The organisms just 
mentioned are, of course, not bacteria, but these discoveries 
proved conclusively that living things of one kind or another, 
some large, most of them microscopic, could cause disease in 
other organisms and stimulated the search for other " living 
contagiums." In 1863 Davaine, already mentioned, showed 
that anthrax could be transmitted from animal to animal PLATE II 
SIR JOSEPH LISTER PLATE III 
ROBERT KOCH CAUSATION OF DISEASE 
29 
by inoculation of blood, but only if the blood contained the 
minute rods which he believed to be the cause. Davaine 
later abandoned this belief because he transmitted the 
disease with old blood in which he could find no rods. It is 
now known that this was because the bacilli were in the 
"spore" form which Davaine did not recognize. He thus 
missed the definite proof of the bacterial nature of anthrax 
because he was not familiar with the life history of the 
organism which was worked out by Koch thirteen years 
later. In 1865 Villemin repeatedly caused tuberculosis in 
rabbits by subcutaneous injection of tuberculous material 
and showed that this disease must be infectious also. In 
the same year Lord Lister introduced antiseptic methods 
in surgery. He believed that wound infections were due 
to microorganisms getting in from the air, the surgeon's 
fingers, etc., and without proving this, he used carbolic acid 
to kill these germs and prevent the infection. His pioneer 
experiments made modern surgery possible. In this year 
also, Pasteur was sent to investigate a disease, Pebrine, 
which was destroying the silkworms in Southern France. 
He showed the cause to be a protozoan which had been seen 
previously by Cornalia and described by Nageli under the 
name Nosevia bombycis and devised preventive measures. 
This was the first infectious disease shown to be due to a proto- 
zoan. In 1866 Rindfleisch observed small pin-point-like 
bodies in the heart muscle of persons who had died of wound 
infection. Klebs, in 1870-71, published descriptions and 
names of organisms he had found in the material from simi- 
lar wounds, though he did not establish their causal rela- 
tion. Bollinger, in 1872, discovered the spores of anthrax 
and explained the persistence of the disease in certain dis- 
tricts as due to the resistant spores. In 1873 Obermeier 
observed in the blood of patients suffering from recurrent 
fever long, flexible spiral organisms which have been named 
Spirochaeta obermeieri. Losch ascribed tropical dysentery 
to an ameba, named by him Amoeba coli, in 1875. Finally, 
Koch, in 1876, isolated the anthrax bacillus, worked out the 
life history of the organism and reproduced the disease by 
the injection of pure cultures and recovered the organism 30 
HISTORICAL INTRODUCTION 
from the inoculated animals, thus establishing beyond 
reasonable doubt its causal relationship to the disease. 
This was the first instance of a bacterium proved to be the 
cause of a disease in animals. Pasteur, working on the 
disease at the same time, confirmed all of Koch's findings, 
though his results were published the next year, 1877. 
Bollinger determined that the Actinomyces bovis (Strepto- 
thrix bovis) is the cause of actinomycosis in cattle in 1877. 
Woronin in the same year discovered a protozoan (Plasmo- 
diophora brassicoe) to be the cause of a disease in cabbage 
the first proved instance of a unicellular animal causing a 
disease in a plant. In 1878 Koch published his researches 
on wound infection in which he showed beyond question 
that microorganisms are the cause of this condition, though 
Pasteur in 1837, had suggested the same thing and Lister 
had acted on the theory in preventing infection. 
These discoveries, especially those of Koch, immediately 
attracted world-wide attention and stimulated a host of 
workers, so that within the next ten years most of the bac- 
teria which produce disease in men and animals were iso- 
lated and described. It is well to remember that the first 
specific disease of man proved to be caused by a bacterium 
was tuberculosis, by Koch in 1882. 
Progress was greatly assisted by the introduction of anilin 
dyes as suitable stains for organisms by Weigert in 1877, by 
Koch's application of special technic and gelatin cultures 
for isolation and study, 1881, and the great improvements 
in the microscope by Prof. Abbe, of Jena. 
Laveran's discovery of the malarial parasite in 1880 
turned attention to protozoa as the causes of disease and led 
to the discovery of the various piroplasmoses and trypan- 
osomiases in man and the lower animals. 
Pasteur's protective inoculations in chicken cholera and 
anthrax directed attention to the possibility of using bac- 
teria or their products as a specific protective or curative 
means against particular diseases. This finally led to the 
discovery of diphtheria antitoxin by Behring, and independ- 
ently by Roux, in 1890, a discovery which opened up the 
wide field of immunity which is so persistently cultivated at 
the present time. PLATE IV 
LOUIS PASTEUR PUTREFACTION AND FERMENTATION 
31 
While the causation of disease by bacteria has probably 
attracted most attention, especially in the popular mind, it 
should not be forgotten that this is but one of the numerous 
ways in which these organisms manifest their activities, and 
in a sense it is one of their least-important ways, since other 
kinds are essential in many industries (dairying, agriculture) 
and processes (sewage purification) and are even indispen- 
sable for the very existence of all green plants and hence of 
animals, including man himself. 
PUTREFACTION AND FERMENTATION. 
The idea that there is a certain resemblance between 
some infectious diseases and the processes of putrefaction 
ar-d fermentation seems to have originated during the dis- 
cussion on spontaneous generation and the " contagium 
vivum" theory which followed Leeuwenhoek's discoveries. 
Plenciz (1762) appears to have first formulated this belief 
in writing. He considered putrefaction to be due to the 
"animalcules" and said that it occurred only when there 
was a coat of organisms on the material and only when 
they increased and multiplied. Spallanzani's experiments 
tended to support this view since his infusions did not " spoil " 
when boiled and sealed. Appert's practical application 
of this idea has been mentioned. 
Thaer, in his Principles of Rational Agriculture, pub- 
lished in the first quarter of the nineteenth century, expressed 
the belief that the "blue milk fermentation" was probably 
due to a kind of fungus that gets in from the air, and stated 
that he had prevented it by treating the milk cellars and 
vessels, with sulphur fumes or with "oxygenated hydro- 
chloric acid" (hypochlorous acid). 
In 1836 Chevreuil and Pasteur showed that putrefaction 
did not occur in meat protected from contamination. In 
1837 Caignard-Latour, in France, and Schwann, in Germany, 
independently showed that alcoholic fermentation in beer 
and wine is due to the growth of a microscopic plant, the 
yeast, in the fermenting wort. C. J. Fuchs described the 
organism which is commonly called the "blue milk bacillus" 32 
HISTORICAL INTRODUCTION 
in 1841 and conjectured that the souring of milk was prob- 
ably bacterial in origin. It remained for Pasteur to prove 
this in 1857. During the following six or seven years Pas- 
teur also proved that acetic acid fermentation, as in vinegar 
making, butyric acid fermentation (odor of rancid butter 
and old cheese) and the ammoniacal fermentation of urea, 
so noticeable around stables, were each due to different 
species of bacteria. Pasteur also, during the progress of this 
work, discovered the class of organisms which can grow in 
the absence of free oxygen-the anaerobic bacteria. There 
is no question that Pasteur from 1857 on did more to lay the 
foundations of the science of bacteriology than any other 
one man. Influenced by Pasteur's work von Kesseling, in 
1866, stated his belief that the process of cheese ripening, 
like the souring of milk, was associated with the growth of 
fungi, and Martin also, in 1867, stated that cheese ripening 
was a process which was akin to alcoholic, lactic and butryic 
fermentations. Kette, in 1869, asserted the probability of 
Pasteur's researches furnishing a scientific basis for many 
processes of change in the soil. In 1873 Schldsing and 
Muntz showed that nitrification must be due to the action 
of microorganisms, though the discovery of the particular 
ones remained for Winogradsky in 1889. Thus the belief 
that fermentation and putrefaction are due to microorgan- 
isms was as well established by the early eighties of the last 
century as that similar organisms are the causes of infectious 
diseases. 
STUDY OF FORMS. 
An important part of the scientific knowledge of living 
organisms is dependent on a study of their forms and rela- 
tionships. As has been stated, Leeuwenhoek considered 
bacteria to be "animalcules" because they showed inde- 
pendent movement. But little attention was paid to the 
natural history of these animalcules for nearly a hundred 
years after Leeuwenhoek. During the last quarter of the 
eighteenth century, however, workers, busied themselves 
chiefly with the discovery and description of new forms. 
Among these students were Baron Gleichen, Jablot, Lesser, STUDY OF FORMS 
33 
Reaumur, Hill and others. Muller, of Copenhagen, in 1786 
published the first attempt at classification, a most import- 
ant step in the study of these organisms. Muller intro- 
duced the terms Monas, Proteus and Vibrio, which are 
still in use. Ehrenberg, in his work on Infusoria, or the 
organisms found in infusions, published in 1838, introduced 
many generic names in use at present, but still classed the 
bacteria with protozoa. Joseph Leidy, the American natur- 
alist, considered that the "vibrios" of previous writers were 
plants and not "animalcules." He seems to have been the 
first to have made this distinction (1849). Perty (1852) 
recognized the presence of spores in some of his organisms. 
Ferdinand Cohn (1854) classed the bacteria among plants. 
Nageli (1857) proposed the name " Schizomycetes " or 
"fission fungi," which is still retained for the entire class of 
bacteria. Cohn in the years 1872-1875 established classifi- 
cation on a modern basis and added greatly to the knowledge 
of morphology and natural history of bacteria. He de- 
scribed spore formation and the development of spores into 
active bacteria, and showed the close relationships as well 
as differences between the bacteria and the lower algee. 
Robert Koch was a pupil of Cohn. 
An examination of the accompanying chronological table 
will show how the investigations and discoveries in con- 
nection with "spontaneous generation," the "contagium 
vivum" theory and putrefaction and fermentation must 
have been mutually suggestive: 
1546. Fracastorius, disease germs theory and direct and 
indirect contagion. 
1671. Kircher, " contagium vivum " theory. 
1675. Leeuwenhoek, first saw bacteria, "animalcules." 
1701. Andry, "animalcules" cause of diseases. 
1718. Lancisi, "animalcules" cause of malaria. 
1749. Needham, described development of organisms in 
water around barley grains. 
1762. Plenciz, arguments for "living cause" theory and 
that "animalcules" cause putrefaction. 
1768. Bonnet, suggested that probably Needham's organ- 
isms came from germs in the liquid. 34 
HISTORICAL INTRODUCTION 
1776. Spallanzani, boiled and sealed infusions. 
1786. Muller, first classified "animalcules." 
1787. Wollstein, glanders pus infectious. 
1795-1798. Jenner, vaccination against smallpox. 
1797. Viborg, transmitted glanders repeatedly. 
1807. Prevost, grain rust, Puccinia graminis. The first 
instance of a microscopic plant organism shown to be the cause 
of a disease in a higher plant. 
1810. Appert, directions for "canning." 
1822. Gaspard, infectiousness of material from wounds. 
1834. Renucci, itch-itch mite (Sarcoptes scabieifi 
1835. Paget and Owen, Trichina spiralis 
1836. Schultze, air through acid to kill "germs." 
1837. Chevreuil and Pasteur, protected meat did not 
putrefy; suggested wound infection due to en- 
trance of germs from without. 
1837. Caignard-Latour, Schwann, alcoholic fermenta- 
tion-yeast. 
1837. Schwann, air through heated tubes to kill germs. 
1837. Bassi, muscardine of silkworms, Botrytis bassiana. 
The first instance of a microscopic plant organism 
shown to be the cause of a disease in an animal. 
1838. Boehm, cholera, saw organisms in stools (not the 
cause). 
1838. Dubini discovered Ankylostoma duodenale. 
1838. Ehrenberg, study of forms. 
1839. Schonlein, Favus, Achorion schoenleinii. 
1839-41. Berg, Thrush, Oidium albicans. 
1840. Henle, theory of contagious diseases. 
1841. Fuchs, bacterial cause of blue milk. 
1842-43. Gruby, Herpes tonsurans, Trichophyton ton- 
surans. 
1843. Klencke, inoculations of tuberculous material into 
rabbit. 
1843. Holmes, puerperal fever contagious. 
1845. Liebert, a potato rot, Peronospora infestans. 
1846. Leidy, Joseph (American Naturalist), Trichina 
spiralis in pork. 
1846. Eichstedt, Pityriasis versicolor, Microsporon fur- 
fur. STUDY OF FORMS 
35 
1847. Semmelweiss, recommended disinfection to pre- 
vent puerperal fever. Not followed. 
1849. Leidy, considered "vibrios" to be plants. 
1849. Pollender, Anthrax, saw rods in blood. 
1850. Davaine and Rayer, Anthrax, saw rods in blood. 
1851. Griesinger, Egyptian chlorosis, Ankylostoma duo- 
denale. 
1851. Bilharz, Bilharzia disease, Schistosomum hemato- 
bium. 
1852. Kiickenmeister, tapeworm, Toenia solium. 
1852. Perty, saw spores in bacteria. 
1854. Cohn, classed bacteria as plants. 
1855. Cohn, disease of flies, Empusa muscoe. 
1857. Nageli, named bacteria, Schizomycetes. 
1857. Pasteur, lactic, acetic, butyric acid fermentation. 
1860. Zenker, Trichinosis, Trichinella spiralis. 
1861. Pasteur, disproof of spontaneous generation. 
1863. Davaine, transmitted anthrax by blood injections. 
1865. Pasteur, Pebrine of silkworms, Nosema bombycis. 
The first instance of a protozoan shown to be the 
cause of a disease in a higher animal. 
1865. Villemin, repeatedly transmitted tuberculosis to 
rabbits. 
1865. Lister, introduced antisepsis in surgery. 
1866. Rindfleisch, Pyemia, organisms in the pus. 
1866. Von Kesseling, cheese ripening. 
1867. De Martin, cheese ripening akin to alcoholic fer- 
mentation. 
1869. Kette, Pasteur's researches scientific basis for 
many processes in the soil. 
1871. Klebs, Pyemia, organisms in the pus. 
1872. Bollinger, spores in anthrax. 
1872-75. Cohn, definite classification. 
1873. Obermeier, recurrent fever, Spirochaeta obermeieri. 
1873. Schlosing and Miinz, nitrification due to organ- 
isms. 
1875. Losch, amebic dysentery, Amoeba coli. 
1875-76. Tyndall, germs in the air. 36 
HISTORICAL INTRODUCTION 
1876. Robert Koch, anthrax, Bacillus anthracis. The 
first instance of a bacterium shown to be the cause 
of disease in an animal. 
1877. Bollinger, actinomycosis, Actinomyces bovis (Strep- 
tothrix bods). 
1877. Weigert, used anilin dyes for staining. 
1877. Wofonin, cabbage disease, Plasmodiophora brass- 
icoe The first instance of a protozoan shown to be 
the cause of a disease in a plant. 
1878. Koch, wound infections, bacterial in origin. 
1881. Koch, gelatin plate cultures, Abbe, improvements 
in the microscope. CHAPTER I. 
POSITION-RELATIONSHIPS. 
Bacteria are considered to belong to the plant kingdom 
not because of any one character they possess, but because 
they most nearly resemble organisms which are generally 
recognized as plants. While it is not difficult to distinguish 
between the higher plants and higher animals, it becomes 
almost, if not quite, impossible to separate the lowest forms 
of life. It is only by the method of resemblances above 
mentioned that a decision is finally reached. It has even 
been proposed to make a third class of organisms neither 
plants nor animals but midway between in which the bac- 
teria are included, but such a classification has not as yet 
been adopted. 
In many respects the bacteria are most nearly related to 
the lowest algoe, since both are unicellular organisms, both 
reproduce by transverse division and the forms of the cell 
are strikingly similar. The bacteria differ in one important 
respect, that is, they do not contain chlorophyl, the green 
coloring matter which enables all plants possessing it to 
absorb and break up carbon dioxide in the light, and hence 
belong among the fungi. Bacteria average much smaller 
than even the smallest algse. 
Bacteria are closely connected with the fission yeasts and 
the yeasts and toruloe. All are unicellular and without 
chlorophyl. The bacteria, as has been stated, reproduce 
by division but the others characteristically by budding or 
gemmation, though the fission yeasts also by division. 
There is a certain resemblance to the molds in their 
absence of chlorophyl. But the molds grow as branching 
threads and also have special fruiting organs for producing 
spores as a means of reproduction, neither of which charac- 
teristics is found among the true bacteria. The higher 38 
POSITION-RELA T ION SHIP 
thread bacteria do show true branching and rudimentary 
fruiting bodies (Streptothrix) and appear to be a link con- 
necting the true bacteria and the molds. 
Fig. 8.-A thread of blue-green 
algae. 
Fig. 9.-A thread of small blue- 
green algae. 
Fig. 10.-A' thread of bacteria. 
■Compare with Figs. 8 and 9. 
Fig. 11.-A chain of spherical blue- 
green algae. 
Fig. 12.-A chain of spherical 
bacteria. 
Fig. 13.-A pair of spherical blue- 
green algae. POSITION-RELA T ION SHIP 
39 
Further the chemical composition of bacteria is more like 
that of other fungous plants than of any of the forms classed 
as animals. 
Fig. 14.-Spherical bacteria. Sev- 
eral pairs are shown. 
Fig. 15.-Yeast cells. Some show 
typical budding. 
The food of bacteria is always taken up in solution by 
diffusion through the outer covering of the cell as it is in all 
plants. Plant cells never surround and engulf particles of 
solid food and digest them within the cell as many single- 
Fig. 16.-A portion of the mycelium of a mold. Note the large size and 
the branching. 
celled animals do, and as the leukocytes and similar ame- 
boid cells in practically all multicelled animals do.1 
One of the most marked differences between animals and 
plants is with respect to their energy relationships. Plants 
are characteristically storers of energy while animals are 
liberators of it. Some bacteria which have the power of 
1 Myxomycetes excepted, and they are probably to be regarded as animals 
-Mycetozoa. 40 
POSITION-RE LA TIONSHIP 
swimming in a liquid certainly liberate relatively large 
amounts of energy, and in the changes which bacteria bring 
about in the material which they use as food considerable 
heat is evolved ("heating" of manure, etc.). Nevertheless 
the evidence is good that the bacteria as a class store much 
more of the energy contained in the substances actually 
taken into the body cell as food than is liberated in any 
form. 
Bacteria do show some resemblance to the protozoa, or 
single-celled animal forms, in that the individuals of each 
group consist of one cell only and some bacteria have the 
power of independent motion from place to place in a liquid 
as most "infusoria" do, but here the resemblance ceases. 
Bacteria are among the smallest of organisms, so small 
that it requires the highest powers of the microscope for 
their successful study, and the use of a special unit for their 
measurement. This unit is the one-thousandth part of a 
millimeter and is called the micro-millimeter or micron. 
Its symbol is the Greek letter mu (p.). 
The size varies widely among different kinds but is fairly 
constant in the same kind. The smallest described form is 
said to be only 0.18P- long by 0.06^ thick and is just visible 
with the highest power of the microscope, though it is pos- 
sible and even probable that there are forms still smaller 
which cannot be seen. Some large rare forms may measure 
40u in length, but the vast majority are from 1^ to 4^ or 
long, and from one-third to one-half as wide. 
From the above description a bacterium might be said to 
be a microscopic, unicellular plant, without chlorophyl, which 
reproduces by dividing transversely. PART I. 
MORPHOLOGY 
CHAPTER II. 
CELL STRUCTURES. 
The essential structures which may by appropriate means 
be distinguished in the bacterial cell are cell wall and cell 
contents, technically termed protoplasm, cytoplasm. The 
cell wall is not so dense, relatively, as that of green plants, 
but is thicker than the outer covering of protozoa. It is 
very similar to the cell wall of other lower fungi. Diffusion 
takes place readily through it. with very little selective action 
on substances absorbed as judged by the comparative com- 
position of bacteria and their surrounding medium. 
Cytoplasm.-The cytoplasm according to Biitschli and 
others is somewhat different and slightly denser in its outer 
portion next to the cell wall. This layer is designated the 
ectoplasm, as distinguished from the remainder of the cell 
contents, the endoplasm. When bacteria are suddenly trans- 
ferred from a given medium into one of decidedly greater 
density, there sometimes results a contraction of the endo- 
plasm, due to the rapid diffusion of water. This phenomenon 
is designated plasmolysis (Fig. 17), and is similar to what 
occurs in the cells of higher plants when subjected to the 
same treatment. This is one of the methods which may be 
used to show the different parts of the cell just described. 
If bacteria are suddenly transferred from a relatively 
dense medium to one which is of decidedly less density, it 42 
CELL STRUCTURES 
occasionally happens that water diffuses into the cell and 
swells up the endoplasm so much more rapidly than the cell 
wall that the latter ruptures and some of the endoplasm 
exudes in the form of droplets on the surface of the cell 
wall. This phenomenon is called plasmoptysis. Students 
will seldom observe the distinction between cell wall and cell 
contents, except that in examining living bacteria the outer 
portion appears more highly refi active. This is chiefly due 
to the presence of a cell wall, but is not a proof of its exist- 
ence. 
Fig. 17.-Cells of bacteria show- 
ing plasmolysis. The cell sub- 
stance of three of the cells in the 
middle of the chain has shrunk 
until it appears as a round black 
mass. The cell wall shows as the 
lighter area. 
Fig. 18.-Vacuoles in the bac- 
terial cell. The lighter areas are 
vacuoles. 
Nucleus.-Douglas and Distaso1 summarize the various 
opinions with regard to the nucleus in bacteria as follows: 
1. Those who do not admit the presence of a nucleus or of 
anything equivalent to it. (Fischer, Migula, Massart). 
2. Those who consider that the entire bacterial cell is the 
equivalent of a nucleus and contains no protoplasm. (Ruz- 
icka). 
3. Those who admit the presence of nuclein but say that 
this is not morphologically differentiated from the proto- 
plasm as a nucleus. (Weigert). 
1 Centralblatt f. Bakteriologie, etc. LXIII. 1 Abt. Orig. 1912, 4, idemLXVI. 
1 Abt. Orig. 1912, 323. VACUOLES 
43 
4. Those who consider the bacterial protoplasm to con- 
sist of a central endoplasm throughout which the nuclein 
is diffused and an external layer of ectoplasm next to the 
cell wall. (Biitschli, Zettnow). 
5. Those who say that the bacterial cell contains a dis" 
tinct nucleus, at least in most instances. These authors 
base their claims on staining with a Giemsa stain. (Fein- 
berg, Ziemann, Neuvel, Dobell, Douglass and Distaso). 
That nucleoproteins are pi esent in the bacterial cell in 
relatively large amounts is well established. Also that 
there are other proteins and that the protoplasm is not all 
nuclein. 
Some workers as noted above have been able to demon- 
strate collections of nuclein by staining, especially in very 
young cells. In older cells this material is in most instances 
diffused throughout the protoplasm and can not be so 
differentiated. 
The following statement probably represents the generally 
accepted view at the present time: 
A nucleus as such is not present in bacterial cells, except in 
.a few large rare forms and in very young cells. Nuclein, the 
characteristic chemical substance in nuclei, which when 
aggregated forms the nucleus, is scattered throughout 
the cell contents and thus intimately mingled with the 
protoplasm, and cannot be differentiated by staining as in 
most cells. 
The close association of nuclein and protoplasm may ex- 
plain the rapid rate of division of bacteria (Chapter VIII, 
P-91). 
The chemical composition of the bacterial cell is discussed 
in Chapter VII. 
In addition to the essential parts just described the bac- 
terial cell may show some of the following accidental struc- 
tures: vacuoles, capsules, metachromatic granules, flagella, 
spores. 
Vacuoles.-Vacuoles appear as clear spaces in the proto- 
plasm when the organism is es amined in the living condition 
or when stained very slightly (Fig. 18). During life these 
.are filled with liquid or gaseous material which is sometimes 44 
CELL STRUCTURES 
waste, sometimes reserve food, sometimes digestive fluids. 
Students are apt to confuse vacuoles with spores (p. 47), 
Staining is the surest way to differentiate (Chapter XIX, 
p. 209). If vacuoles have any special function, it is an 
unimportant one. 
Capsule.--The capsule is a second covering outside the cell 
wall and probably developed from it (Fig. 19). It is usually 
gelatinous, so that bacteria which form capsules frequently 
stick together when growing in a fluid, so that the whole 
mass has a jelly-like consistency. The term zodgloea was 
formerly applied to such masses, but it is a poor term and 
misleading (zobn = an animal) and should be dropped. The 
masses of jelly-like material frequently found on decaying 
wood, especially in rainy weather, are in some cases masses 
Fig. 19.-Bacteria seen within 
capsules. 
Fig. 20.-Metachromatic gran- 
ules in bacteria. The dark round 
spots are the granules. The cells 
of the bacteria are scarcely visible. 
of capsule-forming bacteria, though a part of the jelly is a 
product of bacterial activity, a gum-like substance which 
lies among the capsulated organisms. When these masses 
dry out, they become tough and leathery, but it is not to be 
presumed that capsules are of this consistency. On the 
contrary, they are soft and delicate, though they certainly 
serve as an additional protection to the organism, doubtless 
more by selective absorption than mechanically. Certain 
bacteria which cause disease form capsules in the blood of 
those animals which they kill and not in the blood of those 
in which they have no effect (Bacterium anthracis in guinea FLAGELLUM 
45 
pig's blood and in rat's blood). The presence of capsules 
around an organism can be proved only by staining the 
capsule. Many bacteria when stained in albuminous fluids 
show a clear space around them which appears like a cap- 
sule. It is due to the contraction of the fluid away from the 
organism during drying. 
Metachromatic Granules.-The term "metachromatic" is 
applied to granules which in stained preparations take a 
color different from the protoplasm as a whole (Fig. 20). 
They vary widely in chemical composition. Some of them 
are glycogen, some fat droplets. Others are so-called "gran- 
ulose" closely related to starch but probably not true 
starch. Others are probably nuclein. Of many the chemi- 
cal composition is unknown. They are called "Babes- 
Ernst corpuscles" in certain bacteria (typhoid bacillus). 
Since they frequently occur in the ends of cells the term 
"polar granules" is also applied. Their presence is of 
value in the recognition of but few bacteria ("Neisser gran- 
ules" in diphtheria). 
Flagellum.-A flagellum is a very minute thread-like pro- 
cess growing out from the cell wall, probably filled with a 
strand of protoplasm. The vibrations of the flagella move 
the organism through the liquid medium. Bacteria which 
are thus capable of independent movement are spoken of 
as "motile bacteria." The actual rate of movement is very 
slight, though in proportion to the size of the organism it 
may be considered rapid. Thus Alfred Fischer determined 
that some organisms have a speed for short periods of about 
40 cm. per hour. This is equivalent to a man moving more 
than 200 miles in the same time. 
It is obvious that bacteria which can move about in a 
liquid have an advantage in obtaining food, since they do 
not need to wait for it to be brought to them. This advan- 
tage is probably slight. 
An organism may have only one flagellum at the end. It 
is then said to be monotrichic (Fig. 21) (puoyo<? = alone, single; 
Tpixoc = hair). This is most commonly at the front end, so 
that the bacterium is drawn through the liquid by its motion. 
Rarely it is at the rear end. Other bacteria may possess a 46 
CELL STRUCTURES 
bundle of flagella at one end and are called lophotrichic (Fig. 
22) (Xo4>oi; = tuft). Sometimes at approaching division the 
flagella may be at both ends and are then amphitrichic (Fig. 
23) (a^$i = both). It is probable that this condition does 
Fig. 21.-A bacterium showing a 
single flagellum at the end-mono- 
trichic. 
Fig. 22.-A bacterium showing 
a bundle of four flagella at the end 
-lophotrichic. 
not persist long, but represents the development of flagella 
at one end of each of a pair resulting from division of an 
organism which has flagella at one end only. In many 
bacteria the flagella arise from all parts of the surface of the 
Fig. 23.-A bacterium showing 
flagella at each end-amphitrichic. 
Fig. 24.-A bacterium showing 
flagella all around-peritrichic. 
cell. Such bacteria are peritrichic (Fig. 24) (zepi = around). 
The position and even the number of the flagella are very 
constant for each kind and are of decided value in identifi- 
cation. SPORES 
47 
Flagella are too fine and delicate to be seen on the living 
organism, or even on bacteria which have been colored by 
the ordinary stains. They are rendered visible only by 
certain methods which cause a precipitate on both bacteria 
and flagella which are thereby made thick enough to be 
seen (Chapter XIX, p. 210). The movement of liquid 
around a bacterium caused by vibrations of flagella can 
sometimes be observed with large forms and the use of 
"dark-field" illumination. 
Flagella are very delicate and easily broken off from the 
cell body. Slight changes in the density or reaction of the 
medium frequently cause this breaking off, so that prepara- 
tions made from actively motile bacteria frequently show 
no flagella. For this reason and also on account of their 
fineness the demonstration of flagella is not easy, and it is 
not safe to say that a non-motile bacterium has no flagella 
except after very careful study. 
The motion of bacteria is characteristic and a little prac- 
tice in observing will enable the student to recognize it and 
distinguish between motility and "Brownian" or molecular 
motion. Dead and non-motile bacteria show the latter. 
In fact, any finely divided particles suspended in a liquid 
which is not too viscous and in which the particles are not 
soluble show Brownian motion or "pedesis." This latter is 
a dancing motion of the particle within a very small area 
and without change of place, while motile bacteria move 
from place to place or even out of the field of the microscope 
with greater or less speed. There is a marked difference in 
the character of the motion of different kinds of bacteria. 
Some rotate around the long axis when moving, others 
vibrate from side to side. 
Among the higher thread bacteria there are some which 
show motility without possessing flagella. Just how they 
move is little understood. 
Spores.-Under certain conditions some bacterial cells 
undergo transformations which result in the formation of 
so-called spores. If the process is followed under the micro- 
scope, the changes observed are approximately these: A 
very minute point appears in the protoplasm which seems to 48 
CELL STRUCTURES 
act somewhat like the centrosome of higher cells as a "center 
of attraction" so that the protoplasm gradually collects 
around it. The spot disappears or is enclosed in the col- 
lected protoplasm. This has evidently become denser as it 
is more highly refractive than before. In time all or nearly 
all of the protoplasm is collected. A new cell wall is devel- 
oped around it which is thicker than the cell wall of the 
bacterium. This thickened cell wall is called the "spore 
capsule." Gradually the remnants of the former cell con- 
tents and the old cell wall disappear or dissolve and the 
spore becomes "free" (Fig. 25). 
Fig. 25.-The smaller oval bodies in the middle of the field are free spores 
If the spore is placed in favorable conditions the proto- 
plasm absorbs water, swells, the capsule bursts at some 
point, a cell wall is formed and the bacterium grows to 
normal size and divides, that is, it is an active growing cell 
again. This process is called "germination" of the spore. 
The point at which the spore capsule bursts to permit the 
new cell to emerge is characteristic for each kind of bac- 
terium. It may be at the end when the germination is said 
to be polar (Fig. 26). It may be from the middle of one 
side which gives equatorial germination (Fig. 27). Rarely it 
is diagonally from a point between the equator and the pole, 
which type may be styled oblique germination. In one or 
two instances the entire spore swells up, lengthens and 
becomes a rod without any special germination unless this 
type might be designated bi-polar. 
Spores are most commonly oval or elliptical in shape, 
though sometimes spherical. A spore may be formed in the SPORES 
49 
middle of the organism without (Fig. 28) or with (Fig. 29) 
a change in size of the cell around it. If the diameter 
through the cell is increased, then the cell with the con- 
Fig. 26.-Spores showing polar 
germination. The lighter part of 
the two organisms just below A 
and B is the developing bacterium. 
In the original slide the spore was 
stained red and the developing 
bacterium a faint blue. 
Fig. 27.-A spore showing equa- 
torial germination. The spore in 
the center of the field shows a rod 
growing out of it laterally. In the 
original slide the spore was stained 
red and the developing bacterium 
blue. 
tained spore becomes spindle-shaped. Such a cell is termed 
a " clostridium." Sometimes the spore develops in the end 
of the cell either without (Fig. 30) or with enlarging it 
(Fig. 31). In a few forms the spore is placed at the end of 
Fig. 28.-Spores in the middle of 
the rod without enlargement of the 
rod. The lighter areas in the rods 
are spores. 
Fig. 29.-Spores in the^middle 
of the rod with enlargement of the 
rod around them. The lighter 
areas in the rods are spores. 
the rod and shows a marked enlargement. This is spoken 
of as the "plectridium" or more commonly the "drumstick 
spore" (Fig. 32). The position and shape of the spore are 50 
CELL STRUCTURES 
constant for each kind of bacteria. In one or two instances 
only, two spores have been observed in a single organism. 
Fig. 30.-Spores in the end of the 
rod with no enlargement of the rod 
around them. The lighter areas 
in the rods are spores. 
Fig. 31.-Spores in the end of 
the rod with enlargement of the 
rod. A, A, A, A. 
The fact that the protoplasm is denser and the spore cap- 
sule thicker (the percentage of water in each is decidedly 
less than in the growing cell) gives the spore the property 
of much greater resistance to all destructive agencies than 
Fig. 32.-Drumstick spores at the end of the rod 
the active bacterium has. For example, all actively growing 
cells are destroyed by boiling in a very few minutes, while 
some spores require several hours' boiling. The same rela- 
tion holds with regard to drying, the action of chemicals, 
light, etc. That the coagulation temperature of a protein 
varies inversely with the amount of water, it contains, is 
shown by the following table from Frost and McCampbell, 
" General Bacteriology ": SPORES 
51 
Egg albumin plus 50 per cent, water coagulates at 
56° 
" " " 25 per cent. " " 
74-80° 
" " " 18 per cent. " " " 
88-90° 
" " " 6 per cent. " " 
145° 
" " dry " " " 
160-170° 
This resistance explains why it happens that food materials 
boiled and sealed in cans to prevent the entrance of organ- 
isms sometimes spoil. The spores have not been killed by 
the boiling. It explains also in part the persistence of some 
diseases like anthrax and black l^g in pastures for years. 
From the above description it follows that the spore is to 
be considered as a condensation of the bacterial protoplasm 
surrounded by an especially thick cell wall. Its function is 
the preservation of the organism under adverse conditions. It 
corresponds most closely to the encystment of certain pro- 
tozoa-the ameba for example. Possibly the spore repre- 
sents a very rudimentary beginning of a reproductive func- 
tion such as is gradually evolved in the higher thread bac- 
teria, the fission yeasts, the yeasts, the molds, etc. Its 
characteristics are so markedly different, however, that the 
function of preservation is certainly the main one. 
It must not be supposed that spores are formed under 
adverse conditions only, because bacteria showing vigorous 
growth frequently form spores rapidly. Special conditions 
are necessary for their formation just as they are for the 
growth and other functions of bacteria (Chapters VI and 
VII). CHAPTER III. 
CELL FORMS. 
Though there is apparently a wide variation in the shapes 
of different bacterial cells, these may all be reduced to 
three typical cell forms. These are: first and simplest, the 
round or spherical, typified by a ball and called the coccus 
form, or coccus, plural cocci1 (Fig. 33). The coccus may be 
large, that is, from 1.5[j. to 2p, in diameter. The term macro- 
coccus is cometimes applied to these large cocci. If the 
coccus is less than Ip. in diameter, it is sometimes spoken 
of as a micrococcus; in fact, this term is very commonly 
applied to any coccus. When cocci are growing together, 
many of the cells do not appear as true spheres but are more 
or less distorted from pressure of their neighbors or from 
failure to grow to full size after recent division. Most cocci 
divide into hemispheres and then each half grows to full 
size. A few cocci elongate before division and then appear 
oval or elliptical. 
The second cell form is that of a cylinder or rod typified 
by a section of a lead-pencil. The name bacillus, plural 
bacilli, is applied to this type (Fig. 34). The bacillus may 
be short (Fig. 35), or less in length, or long, up to 40[i 
in rare cases. Most bacilli are from 2^ to 5p. or long. 
The ends of the rod are usually rounded, occasionally square 
and very rarely pointed. It is evident that a very short 
rod with rounded ends approaches a coccus in form and it 
is not always easy to differentiate in such cases. Most 
bacilli are straight, but some are slightly curved (Fig. 36). 
1 The pronunciation of this word according to English standards is kok-si ; 
the continental pronunciation is kok-kee; the commonest American seems 
to be kok-ki. We prefer the latter since it is easier and more natural and 
should like to see it adopted. (Author.) CELL FORMS 
53 
The third cell form is the spiral, typified by a section of a 
cork-screw and named spirillum, plural spirilla (Fig. 37). A 
very short spiral consisting of only a portion of a turn is 
Fig. 33.-Cocci. 
Fig. 34.-Bacilli. 
sometimes called vibrio (Fig. 38). Vibrios when seen under 
the microscope look like short curved rods. The distinc- 
tion between the two can be made only by examining the 
organism alive and moving in a liquid. The vibrio shows a 
Fig. 35.-Short bacilli. 
Fig. 36.-Curved bacilli. Only 
the one in the center of the field is 
in focus. The others curve out of 
focus. 
characteristic spiral twisting motion. Very long, flexible 
spirals are usually named spirochetes (Fig. 39). The spir- 
ochetes are motile but flagella have not been shown to be 
present. 
Besides the three typical cell forms bacteria frequently 
show very great irregularities in shape. They may be 
pointed, bulged, club-shaped or even slightly branched. 54 
CELL FORMS 
These peculiar and bizarre forms practically always occur 
when some of the necessary conditions for normal growth, 
discussed in Chapters VI and VII, are not fulfilled. They 
Fig. 37.-Spirilla. 
Fig. 38.-Vibrio forms of spirilla 
Compare with Fig. 36. 
are best regarded as involution or degeneration forms for this 
reason (Fig. 40). In a very few cases it is not possible to 
obtain the organism without these forms (the diphtheria 
Fig. 39.-Spirochetes. 
Fig. 40.-Involution forms. The 
organisms are tapering and branch- 
ed at one end. 
group). It is probable that these cell forms are normal in 
such cases, or else conditions suitable for the normal growth 
have not been obtained. CHAPTER IV. 
CELL GROUPINGS. 
It has been stated that bacteria reproduce by trans- 
verse division, that is, division across the long axis. Fol- 
lowing repeated divisions the new cells may or may not 
remain attached. In the latter case the bacteria occur as 
separate isolated individuals. In the former, arrangements 
characteristic of the particular organism almost invariably 
result. These arrangements are best described as cell 
groupings or growth forms. 
Fig. 41.--Streptospirillum grouping. 
Fig. 42.-Diplobacillus grouping. 
In the case of spiral forms it is obvious that there is only 
one possible grouping, that is, in chains of two or more 
individuals adherent end to end. A chain of two spirilla 
might be called a diplospirillum (BixXo? = double); of 
three or more, a streptospirillum (aTpe~T6<; = necklace, chain) 
(Fig. 41). These terms are rarely used, since spirilla do 
not ordinarily remain attached. Likewise the bacillus can 
grow only in chains of two or more, and the terms diplo- 
bacillus (Fig. 42), bacilli in groups of two, and streptobacillus 
(Fig. 43), bacilli in chains are frequently used. Still the 56 
CELL GROUPINGS 
terms thread, filament, or chain are more common for strepto- 
bacillus. 
Since the coccus is spherical, transverse division may occur 
in any direction, though in three planes only at right angles 
to each other. Division might occur in one plane only as in 
spirilla and bacilli, or in two planes only or in all three planes. 
Fig. 43.-Steptobacillus grouping 
Fig. 44.-Typical diplococcus 
grouping. Note that the indivi- 
dual cocci are flattened on the 
apposing sides. 
As a matter of fact these three methods of division are 
found among the cocci, but only one method for each par- 
ticular kind of coccus. As a result there may be a variety 
of cell groupings among the cocci. When division occurs in 
Fig. 45.-Long streptococcus 
grouping. 
Fig. 46.-Short streptococcus 
grouping. 
one plane only, the possible groupings are the same as among 
the spirilla or bacilli. The cocci may occur in groups of two 
-diplococcus grouping (Fig. 44), or in chains -streptococcus 
grouping (Figs. 45 and 46). When the grouping is in diplo- CELL GROUPINGS 
57 
cocci, the individual cocci most commonly appear as hemi- 
spheres with the plane surfaces apposed (Fig. 44). Some- 
times they appear as spheres and occasionally are even 
somewhat elongated. The individuals in a streptococcus 
grouping are most commonly elongated, either in the same 
direction as the length of the chain, or at right angles to it. 
The latter appearance is probably due to failure to enlarge 
completely after division. Streptococci frequently appear 
as chains of diplococci, that is, the pair resulting from the 
division of a single coccus remain a little closer to each 
other than to neighboring cells, as a close inspection of Fig. 
45 will show. 
If division occurs in two planes only, there may result the 
above groupings and several others in addition. The four 
cocci which result from a single division may remain to- 
gether, giving the tetracoccus or tetrad grouping. Very 
rarely all the cocci divide evenly and the result is a regular 
rectangular flat mass of cells, the total number of which is a 
multiple of four. The term merismopedia (from a genus 
of algae which grows the same way) is applied to such a 
grouping. If the cells within a group after a few divisions 
do not reproduce so rapidly (lack of food), as usually happens, 
the number of cells becomes uneven or at least not neces- 
sarily a multiple of four and the resultant flat mass has an 
irregular, uneven outline. This grouping is termed staphylo- 
coccus (<jTa(J)uXo<; = a bunch of grapes) (Fig. 47). It is the 
most common grouping among the cocci. 
When division occurs in all three planes, there is in addi- 
tion to all the groupings possible to one- and two-plane divi- 
sion a third grouping in which the cells are in solid packets, 
multiples of eight. • The name sarcina is applied to this 
growth form (Fig. 48). The individual cells in a sarcina 
packet never show the typical coccus form so long as they 
remain together, but are always flattened on two or more 
sides. 
The above descriptions indicate how the method of divi- 
sion may be determined. If in examining a preparation the 
sarcina grouping appears, that shows three-plane division. 
If there are no sarcina, but tetrads or staphylococci (rarely CELL GROUPINGS 
58 
merismopedia), then the division is in two planes. If none 
of the foregoing is observed but only diplo- or streptococci, 
these indicate one-plane division only. Cocci show their 
characteristic groupings only when grown in a liquid medium, 
and such should always be used before deciding on the plane 
of division. 
Fig. 47.-Staphylococcus group- 
ing. The large flat masses are sta- 
phylococcus grouping. Diplococcus 
grouping, tetrads and short strep- 
tococci are also evident. 
Fig. 48.-Sarcina grouping. 
As the above description shows, these terms which are 
properly adjectives describing the cell grouping, are quite 
generally used as nouns. Thus the terms a diplococcus, a 
tetrad, a streptococcus, etc., are common, meaning a bac- 
terium of the cell form and cell grouping indicated. 
Cell Form. 
Cell Grouping. 
diplococcus-in 2's. 
streptoccus-in chains. 
tetracoccus, tetrads-in 4's. 
staphylococcus-irrgular flat masses 
sarcina-regular, solid packets, 
. multiples of 8. 
coccus-round or spherical. 
bacillus--rod-shaped or cylindri- 
cal. 
j diplobacillus-in 2's. 
I streptobacillus-in chains, 
diplospirillum-in 2's, little used. 
streptospirillum-in chains, little used. 
spirillum-spiral-shaped. CHAPTER V. 
CLASSIFICATION. 
The arrangement of living organisms in groups accord- 
ing to their resemblances and the adoption of fixed names 
is of the greatest advantage in their scientific study. For 
.animal forms and for the higher plants this classification is 
gradually becoming standardized through the International 
Congress of Zoologists and of Botanists respectively. Un- 
fortunately, the naming of the bacteria has not as yet been 
taken up by the latter body, though announced as one of 
the subjects for the Congress of 1916 (postponed on account 
of the war). Hence there is at present no system which 
■can be regarded as either fixed or official. 
Since Muller's first classification of "animalcules" in 1786 
numerous attempts have been made to solve the problem. 
Only those beginning with Ferdinand Cohn (1872-75) are 
•of any real value. As long as bacteria are regarded as 
plants it appears that the logical method is to follow the 
well-established botanical principles in any system for nam- 
ing them. Botanists depend on morphological features 
almost entirely in making their distinctions. The preceding 
chapters have shown that the minute plants which are 
discussed have very few such features. They are, to re- 
capitulate, cell wall, protoplasm, vacuoles, metachromatic 
granules, capsules, flagella, spores, cell forms and cell group- 
ings. Most bacteria show not more than three or four of 
these features, so that is is impossible by the aid of morphol- 
ogy alone to distinguish from each other the large number 
of different kinds which certainly exist. In the various 
systems which are conceded to be the best these charac- 
teristics do serve to classify them down to genera, leaving 
the "species" to be determined from their physiological 60 
CLASSIFICATIONS 
activities. One of these systems was adopted by the 
laboratory section of the American Public Health Associa- 
Fig. 49.-Illustrates the genus 
Streptococcus. Typical chains, no 
staphylococcus grouping, no sar- 
cina grouping, no flagella. 
Fig. 50.-Illustrates the genus 
Micrococcus. Diplococcus, tetrads 
short chains and staphylococcus; 
no sarcina, no flagella. 
tion and by the Society of American Bacteriologists and was 
practically the standard in this country until superseded by 
the Society's own classification. It is that of the German 
Fig. 51.--Illustrates the genus 
Sarcina. Sarcina grouping, no flag- 
ella. 
Fig. 52.-Illustrates the genus 
Bacillus. A bacillus with peritrichic 
flagella. (Student preparation.) 
Bacteriologist Migula and is given below for comparison. 
Since practically the entire discussion in this book is con- 
cerned with the first three families the generic characteristics CLASSIFICATIONS 
61 
Fig. 53.-Illustrates the genus 
Pseudomonas. A bacillus with 
flagella at the end only. 
Fig. 54.-Illustrates the genus 
Microspira. It is (though the 
photograph does not prove it) a 
short spiral with one flagellum at 
the end. 
Fig. 55.-Illustrates the genus 
Spirillum. Spiral bacteria with 
more than three, in this case four, 
flagella at the end. 
Fig. 56.-Illustrates the genus 
Spirochaeta. 
Fig. 57.-Illustrates the genus 
Chlamydothrix. Fine threads with 
a delicate sheath. 
Fig. 58.-Illustrates the genus 
Crenothrix. The thickness of the 
cell walls is due to deposits of iron 
hydroxide. (After Lafar.) 62 
CLASSIFICATIONS 
in these only will be given. The full classification as well 
as a thorough discussion of this subject is given in Lafar's 
Handbuch, whence the following is adopted: 
Fig. 59.-Illustrates the genus Beggiatoa. The filament A is so full of 
sulphur granules that the individual cells are not visible. B has fewer sul- 
phur granules. In C the granules are nearly absent and the separate cells 
of the filament are seen. (After Winogradsky, from Lafar.) 
ORDER I. Eubacteria. 
Cells without nuclei, free from sulphur granules and from 
bacteriopurpurin (p. 112); colorless, or slightly colored. 
1. Family: Coccace/b (Zopf) Migula, all cocci. 
Genus 1. Streptococcus Billroth: division in one plane only (Fig. 
49). 
I " 2. Micrococcus (Hallier) Cohn: division in two planes 
। only (Fig. 50). 
I " 3. Sarcina Goodsir: division in three planes only (Fig. 
51.) 
Non-flag 
ellated,- 
Non-mo- 
tile 
Flagellated, 
motile 
4. Pianococcus Migula: division in two planes only. 
5. Planosarcina Migula: division in three planes only. 
2. Family: Bacteriaceje Migula, all bacilli. 
Genus 1. Bacterium (Ehrenberg) Migula: no flagella; non- 
motile. 
" 2. Bacillus (Cohn)-Migula: flagella peritrichic (Fig. 52). 
" 3. Pseudomonas Migula: flagella at the end: monotrichic, 
lophotrichic, amphitrichic (Fig. 53). CLASSIFICATIONS 
63 
3. Family: Spibillace^e Migula, all spirilla. 
Cells stiff- 
Genus 1. Spirosoma Migula: non flagellated; non-motile. 
" 2. Microspira (Schrceter) Migula: flagella one to three 
at the end (Fig. 54). 
" 3. Spirillum (Ehrenberg) Migula: flagella more than three 
at the end (Fig. 55). 
Cell flexible 
" 4. Spirochada Ehrenberg: motile; no flagella (Fig. 56.) 
4. Family: Chlamtdobacteriace«. 
Cells cylindrical in long threads and surrounded by a 
sheath. Reproduction also by gonidia formed from an 
entire cell. 
Genus 1. Chlamydothrix Migula (Fig. 57). 
■ " 2. Crenothrix Cohn (Fig. 58). 
" 3. Pragmidiothrix Engler. 
" 4. Spherotilus (including Cladothrix). 
ORDER II. THIOBACTERIA: SULPHUR BACTERIA. 
Cells without a nucleus, but containing sulphur granules, 
may be colorless or contain bacteriopurpurin and be colored 
reddish or violet. 
1. Family Beggiatoace^e. 
Genus 1. Thiothrix Winogradsky. 
" 2. Beggiatoa Trevisan. Of interest since it is without a 
a sheath, is motile, but without flagella (Fig. 59). 
2. Family Rhodobacteriacea:. 
This has five subfamilies and twelve genera, most of which 
are due to the Russian bacteriologist Winogradsky who did 
more work than anyone else with the sulphur bacteria. 
THE CLASSIFICATION OF 
THE SOCIETY OF AMERICAN BACTERIOLOGISTS. 
The Committee on Classification of the Society of Ameri- 
can Bacteriologists at the meeting held in December, 1919, 
submitted its final report. This report has not been for- 
mally adopted as a whole, but in all probability will be sub- 64 
CLASSIFICATIONS 
stantially as outlined below. This outline does not attempt 
to give the detailed characterizations of the different groups 
as defined by the committee, but does show the names to be 
applied to the commoner organisms. These organisms are 
included in the 4th and 5th orders. Details of the first 
three orders have not been worked out. They are listed 
merely for completeness. 
CLASS. SCHIZOMYCETES. 
Unicellular, chlorophyl-free plants, reproducing by transverse div- 
ision (some forms by gonidia also). 
Orders: 
A. Myxobacteriales -Cells united during vegetative stage into a 
pseudo-plasmodium which passes over into a highly developed 
cyst-producing resting stage. 
B. Thiobacteriales -Sulphur bacteria. 
C. Chlamydobacteriales -Iron bacteria and other sheathed bac- 
teria. 
D. Actinomycetales -Actinomyces, tubercle and diphtheria ba- 
cilli. 
E. Eubacteriales -All the other common bacteria. 
Genera of Orders D and E. 
D. ACTINOMYCETALES- 
Family I. Actinomycetace.® Buchanan, 1918. 
Genus 1. Actinobacillus, Brampt, 1900. 
Type species, Actinobacillus lignieresi Brampt, 1900. 
Genus 2. Leptotrichia Trevisan, 1879. 
Type species, Leptotrichia buccalis (Robin, 1847) Trevisan. 
Genus 3. Actinomyces Harz, 1877. 
Type species, Actinomyces bovis Harz. 
Genus 4. Erysipelothrix Rosenbach, 1909. 
Type species, Erysipelothrix rhusiopathice (Kitt, 1893) 
Rosenbach, swine erysipelas. 
Family II. Mycobacteriace® Chester, 1897. 
Genus 1. Mycobacterium Lehmann and Neumann, 1896. 
Type species, Mycobacterium tuberculosis (Koch, 1882) L. 
and N. 
Genus 2. Corynebacterium Lehmann and Neumann, 1896. 
Type species, Corynebacterium diphtheriae (Loeffler, 1882) 
L.and N. CLASSIFICATIONS 
65 
Genus 3.. Fusiformis Hoelling, 1910. 
Type species, Fusiformis termitidis Hoelling. Vincent's 
angina. 
Genus 4. Pfeifferella Buchanan, 1918. 
Type species, Pfeifferella mallei (Loeffler, 1896) Buchanan. 
Glanders bacillus. 
E. EUBACTERIALES 
Family I-Nitrobacteriace^e-Proto- or autotrophic for N 
or C and sometimes for both (except Acetobacter). 
Tribe I-NiTROBACTERE^E-autotrophic for C. 
Genus 1. Hydrogenomonas Jensen, 1909. 
Type species, Hydrogenomonas pantotropha (Kaserer, 1906) 
Jensen; oxidizes free H. 
Genus 2. Methanomonas Jensen, 1909. 
Type species, Methanomonas methanica (Sohngen) Jensen; 
oxidizes CH4. 
Genus 3. Carboxydomonas Jensen, 1909. 
Type species, Carboxydomonas oligocarbophila (Beijerinck 
and Van Delden, 1903) Jensen; oxidizes CO. 
Genus 4. Acetobacter Fuhrman, 1905. 
Type species, Acetobacter aceti (Thompson, 1852) Fuhrman; 
oxidizes alcohol to acetic acid. 
Genus 5. Nitrosomonas Winogradsky, 1892. 
Type species, Nitrosomonas europoea Winogradsky; oxi- 
dizes ammonia or ammonium salts to nitrous acid, 
hence nitrites. 
Genus 6. Nitrobacter Winogradsky, 1892. 
Type species, Nitrobacter Winogradskyi Committee of 1917; 
oxidizes nitrous acid (nitrites) to nitric acid (nitrates). 
Tribe II-Azotobactere^e-prototrophic for N. 
Genus 7. Azotobacter Beijerinck, 1901; large, free-living, 
aerobic N absorbers. 
Type species, Azotobacter chroococcum Beijerinck. • 
Genus 8. Rhizobium Frank, 1889. 
Type species, Rhizobium leguminosarum Frank; root tub- 
ercle bacteria of legumes. 
Family II--Pseudomonadace^, Committee of 1917. 
Genus 1. Pseudomonas Migula, 1894. 
Type species, Pseudomonas violacea (Schroeter, 1872) 
Migula. 
Family III-Spirillace^e Migula, 1894-'all spiral bacteria. 
Genus 1. Vibrio Muller, 1786, emended by E. F. Smith, 
1905. 66 
CLASSIFICATIONS 
Type species, Vibrio choleras (Koch, 1884) Schroeter, 1886. 
Genus 2. Spirillum Ehrenberg, 1830, emended Migula, 1894. 
Type species, Spirillum undula (Muller, 1786) Ehrenberg. 
Family IV-Coccace^e Zopf, 1884, emended Migula, 1894- 
all cocci. 
Tribe I-Neissereas 
Genus 1. Neisseria Trevisan, 1885. 
Type species, Neisseria gonorrhoeae Trevisan. 
Tribe II-Streptococce^e Trevisan, 1889. 
Genus 2. Diplococcus Weichselbaum, 1886. 
Type species, Diplococcus pneumoniae Weichselbaum. 
Genus 3. Leuconostoc Van Tieghem, 1878. 
Type species, Leuconostoc mesenterioides (Cienkowski) Van 
Tieghem. 
Genus 4. Streptococcus Rosenbach, 1884; emended Winslow 
and Rogers, 1905. 
Type species, Streptococcus pyogenes Rosenbach. 
Tribe III-Micrococce® Trevisan, 1889. 
Genus 5. Staphylococcus Rosenbach, 1884; animal parasites. 
Type species, Staphylococcus aureus Rosenbach. 
Genus 6. Micrococcus Cohn, 1872, emended Winslow and 
Rogers, 1905. Facultative parasites or saprophytes. 
Type species. Micrococcus luteus (Schroeter, 1872) Cohn. 
Genus 7. Sarcina Goodsir, 1842, emended Winslow and 
Rogers, 1905. 
Type species, Sarcina ventriculi Goodsir. 
Genus 8. Rhodococcus Zopf, 1891, emended Winslow and 
Rogers, 1905; cocci with red pigment. 
Type species, Rhodococcus rhodochrous Zopf. 
Family V-Bacteriace^e Cohn, 1872, emended by Committee 
of 1917; bacilli without spores not above included. 
Tribe I-Chromobactere^ Committee of 1919; producing 
red or violet pigment, mainly water forms. 
Genus 1. Erythrobacillus Fortineau, 1905. 
Type species, Erythrobacillus prodigiosus (Ehrenberg, 1848) 
Fortineau. 
Genus 2. Chromobacterium Bergonzini, 1881. 
Type species, Chromobacterium violaceum Bergonzini. 
Tribe II-Erwine® Committee 1919; plant pathogens. 
Genus 3. Erwinia Committee 1917. 
Type species, Erwinia amylovora (Burrill, 1883) Committee 
1917. 
Tribe III-Zopfe® Committee of 1919; Gram +, no pigment, 
no n-carbohydrate-fermenting. CLASSIFICATIONS 
67 
Genus 4. Zopfius Wenner and Rettger, 1919. 
Type species, Zopfius zopfii (Kurth) Wenner and Rettger. 
Tribe IV-Bactere^e Committee of 1919; Gram -, carbo- 
hydrate fermenters. 
Genus 5. Proteus Hauser, 1885; liquefy gelatin. 
Type species, Proteus vulgaris Hauser. 
Genus 6. Bacterium Ehrenberg, 1828, emended Jensen, 1909; 
liquefy gelatin rarely. 
Type species, Bacterium coli. 
Tribe VI-Lactobacille.e Committee of 1919; Gram +, 
high acid, thermophils. 
Genus 7. Lactobacillus Beijerinck, 1901. 
Type species, Lactobacillus caucasicus (Kern ?) Beijerinck; 
Bulgarian bacillus. 
Tribe VI-Pasteurelle.® Committee of 1919; organisms of 
hemorrhagic septicemia. 
Genus 8. Pasteurella Trevisan, 1888. 
Type species, Pasteurella cholerae-gallinarum (Fliigge, 1886); 
Trevisan. 
Tribe VII-Hemophile^ Committee of 1917; require hemo- 
globin for growth. 
Genus 9. Hemophilus Committee of 1917. 
Type species, Hemophilus influenzae (Pfeiffer, 1893) 
Committee of 1917. 
Family VI-BacillacetE Fischer, 1895. Spore forming rods. 
Genus 1. Bacillus Cohn, 1872; aerobic, no change of form 
around the spore. 
Type species, Bacillus subtilis Cohn. 
Genus 2. Clostridium Prazmowski, 1880; anaerobic, frequent- 
ly enlarged around spore. 
Type species, Clostridium butyricum Prazmowski. 
As compared with Migula's classification it is to be noted 
that there are 38 genera listed by the Committee instead of 13 
in the same general groups. 
The following list of Genera conservanda submitted by the 
Committee was formally adopted by the Society and these 
are therefore its official names for the organisms included in 
these genera. 
Acetobacter Fuhrman 
Actinomyces Harz 
Bacillus Cohn 
Bacterium Ehrenberg 68 
CLASSIFICATIONS 
Chromobacterium Bergonzini 
Clostridium Prazmowski 
Erythrobacillus Fortineau 
Leptotrichia Trevisan 
Leuconostoc Van Tieghem 
Micrococcus Cohn 
Rhizobium Frank 
Sarcina Goodsir 
Spirillum Ehrenberg 
Staphylococcus Rosenbach 
Streptococcus Rosenbach 
Vibrio Muller 
It is greatly to be desired that the Society's Classification 
when finally completed shall become the standard in the United 
States at least. 
Such names as have been adopted by the Society are used 
throughout this work. 
The Committee also submitted the following artificial 
key for determining the genera in the two orders ACTINO- 
MYCETALES AND EUBAC TERI ALES: 
A-Typically filamentous forms... Actinomycetaceae 
B-Mycelium and conidia formed Actinomyces 
BB-No true mycelium 
C-Cells show branching 
D-Gram negative Actinobacillus 
DD-Gram positive Erysipelothrix 
CC-Cells never branch. Gram positive threads later fragment- 
ing into rods Leptotrichia 
AA-Typically unicellular forms (though chains of cells may occur) 
B-Cells spherical-COCCACEcE 
C-Parasitic forms (except Leuconostoc'), cells generally grouped 
in pairs or chains, never in packets, generally active 
fermenters. 
D-Cells in flattened coffee-bean-like pairs, gram -. 
Neisseria 
DD-Not as D 
E-Saprophytes in zobgloea masses in sugar solutions. 
Leuconostoc 
EE-Not as E. Gram + . 
F-Cells in lanceolate pairs or in chains. Growth on 
media not abundant. CLASSIFICATIONS 
69 
G-Cells in lanceolate pairs. Inulin generally fermented. 
Diplococcus 
GG-Cells in chains. Inulin not generally fermented. 
Streptococcus 
FF-Cells in irregular groups. Growth in media fairly 
vigorous. White or orange pigment. 
Staphylococcus 
CC--Saprophytic forms. Cells in irregular groups or packets, 
not in chains. Fermentative powers low. 
D-Packets Sarcina 
DD-No packets. 
E-Yellow pigment Micrococcus 
EE-Red pigment Rhodococcus 
BB-Rods: 
C. Spiral rods 
D-Short, comma-like rods. One to three flagella. 
Vibrio 
DD-Long spirals. Five to twenty flagella. 
Spirillum 
CC. Straight rods. 
D. No endospores. 
E-Rods of irregular shape or showing branched or filament- 
ous involution forms. 
F-Cells irregular in shape.. Staining unevenly. Animal 
parasites. 
G-Acid fast... Mycobacterium 
GG-Not acid fast. 
H-Cells elongated, fusiform Fusiformis 
HH-Cells not elongated, sometimes branching. 
I-Gram positive. Slender, sometimes club-shaped. 
C orynebacterium 
II-Gram negative. Rods sometimes form threads. 
Characteristic honey-like growth on potato. 
Pfeifferella 
FF-Cells staining unevenly but with branched or fila- 
mentous forms at certain stages. Never acid fast- 
Not animal parasites. 
G-Metabolism simple, growth processes involving oxi- 
dation of alcohol or fixation of free N (latter in sym- 
biosis with green plants). 
H.-Cells minute. Symbiotic in roots of legumes.. 
Rhizobium 
HH-Oxidizing alcohol. Branching forms common.. 
Acetobacter 70 
CLASSIFICATIONS 
GG-Not as G. Proteus-like colonies. 
H.-'Not attacking carbohydrates. . Zopfius 
HH. Fermenting glucose and sucrose at least. 
Proteus 
EE-Regularly formed rods. 
F-Metabolism simple, growth processes involving oxida- 
dation of C, H, or their simple compounds or the fixa- 
tion of free N.-NITROBACTERIACEAE. 
G-Fixing N or oxidizing its simple compounds. 
H-Fixing N, cells large, free in soil. 
Azotobacter 
HH-Oxidizing N compounds. 
I-Oxidizing NH4 compounds. .Nitrosomonas 
II--Oxidizing nitrites Nitrobacter 
GG-Not as G. 
H-Oxidizing free H Hydrogenomonas 
HH-Oxidizing simple C compounds, not free H. 
I-Oxidizing CO Carboxydomonas 
II-Oxidizing CH4 Methanomonas 
FF-Not as F. 
G-Flagella usually present, polar-PSEUDOMON AD- 
ACE AE..................... Pseudomonas 
GG-Flagella when present peritrichic-BACTERIA- 
CEAE 
H-Parasitic forms showing bi-polar staining. 
Pasteurella 
HH-Not as H. 
I-Strict parasites growing only in presence of hemo- 
globin Hemophilus 
II-Not as I. 
J-Water forms producing red or violet pigment. 
K-Pigment red Erythrobacillus 
KK-Pigment violet Chromobacterium 
JJ-Not as J. 
K-Plant pathogens Erwinia 
KK-Not plant pathogens. 
L-Gram +, forming large amount of acid 
from carbohydrates, sometimes CO 2, 
never H Lactobacillus 
LL-Gram -, forming H as well as CO 2 if 
gas is produced Bacterium 
DD-Endospores present-BACILLACEAE 
E-Aerobes, rods not swollen at sporulation. 
Bacillus 
EE-Anaerobes, rods swollen at sporulation. 
Clostridium PART II. 
PHYSIOLOGY. 
CHAPTER VI. 
GENERAL CONDITIONS FOR GROWTH. 
OCCURRENCE. 
Bacteria are probably the most widely distributed of 
living organisms. They are found practically everywhere 
on the surface of the earth. Likewise in all surface waters, 
in streams, lakes and the sea. They occur in the air imme- 
diately above the surface, since they are carried up mechan- 
ically by air currents. They cannot fly of themselves. 
There is no reason to believe that any increase in numbers 
occurs to an appreciable extent in the air. The upper air, 
for example, on high mountains, is nearly free from them. 
So also is the air over midocean, and in high latitudes. As 
a rule, the greater the amount of dust in the air, the more 
numerous are the bacteria. Hence they are found more 
abundantly in the air in cities and towns than in the open 
country. The soil is especially rich in numbers in the upper 
few feet, but they diminish rapidly below and almost disap- 
pear at depths of about six feet unless the soil is very porous 
and open, when they may be carried farther down. Hence 
the waters from deep wells and springs are usually devoid 
of these organisms. In the sea they occur at all levels and 
have been found in bottom ooze dredged from depths of 
several miles. It is perhaps needless to add that they are 
found on the bodies and in the alimentary tract of human 72 
GENERAL CONDITIONS FOR GROWTH 
beings and animals; on clothing, utensils; in dwellings, 
stables, outhouses, etc. From one-fourth to one-half of the 
dry weight of the feces of animals and men is due to the 
bacteria present. The urine is practically free from them 
in health. 
While bacteria are thus found nearly everywhere, it is an 
entirely mistaken idea to suppose that all are injurious to 
man. As a matter of fact, those which are dangerous are 
relatively few and are for the most part found only in close 
association with man. Most bacteria are harmless and the 
vast majority are beneficial or even essential to man's 
existence on the earth. These facts must be constantly 
borne in mind, and it is hoped that the pages which follow 
will make them clear. 
In order that any organism may thrive there are a number 
of general environmental conditions which must be fulfilled. 
These conditions vary more or less for each kind of organ- 
ism. Bacteria are no exception to this general rule. These 
conditions may be conveniently considered under the gen- 
eral heads of moisture; temperahire; light; oxygen supply; 
osmotic pressure; action of electricity; of Rontgen and radium 
rays; pressure; mechanical vibration; and chemical environ- 
ment, including the reaction of the medium, the effect of in- 
jurious chemicals, and especially the food requirements of 
bacteria. For each of these conditions there is a maximum, 
meaning the greatest amount of the given condition which 
the organism can withstand, a minimum, or the least amount, 
and an optimum or that amount which is most favorable for 
development. Further, there might be distinguished a maxi- 
mum for mere existence and a lower maximum for develop- 
ment; also a minimum for mere existence and a higher mini- 
mum for development. These maxima, minima, and optima, 
for bacteria have been determined with exactness for only 
a very few of the general conditions and for comparatively 
few kinds. MOISTURE 
73 
MOISTURE. 
The maximum moisture is absolutely pure water, and no 
organism can thrive in' this alone owing to the factor of too 
low osmotic pressure and to the further factor of absence of 
food material. There are many bacteria which thrive in 
water containing only traces of mineral salts and a large 
class whose natural habitat is surface water. These "water 
bacteria" are of great benefit in the purification of streams. 
They are as a class harmless to men and animals. Some 
of the disease-producing bacteria like Bacterium typhosum 
(of typhoid fever) and Vibrio choleras (of Asiatic cholera) 
were undoubtedly originally water bacteria, and it is rather 
striking that in these diseases conditions are induced in 
the intestine (diarrheas) which simulate the original watery 
environment. The minimum moisture condition is abso- 
lute dryness, and no organism can even exist, not to say 
develop, in such a condition since water is an essential con- 
stituent of living matter. Some bacteria and especially 
most spores may live when dried in the air or by artificial 
means for months and even years, while some are destroyed 
in a few hours or days when dried (typhoid, cholera, etc.). 
The optimum amount of moisture has not been determined 
with any great accuracy and certainly a rather wide range 
in percentage of water is permissable with many, though 
a liquid medium is usually most favorable for artificial 
growth. The "water bacteria" have been mentioned. In 
the soil a water content of 5 to 15 per cent, seems to be 
most suitable for many of the organisms which aid in plant 
growth. In animals and man the organisms infecting the 
intestinal tract prefer a high percentage of moisture as a 
rule, especially those causing disease here. Those found on 
the surface of the body (pus cocci) need a less amount of 
water, while those invading the tissues (tuberculosis, black- 
leg, etc.) seem to be intermediate in this respect. In arti- 
ficial culture media a water content of less than 30 per cent, 
inhibits the growth of most bacteria. 
As a general rule those bacteria which require the largest 
percentage of water are most susceptible to its loss and are 74 
GENERAL CONDITIONS FOR GROWTH 
most readily killed by drying. The typhoid and cholera 
organisms die in a few hours when dried, while pus cocci 
and tubercle bacilli live much longer. 
TEMPERATURE. 
The temperature conditions for bacterial existence and 
growth have been determined more accurately than any of 
the other general conditions. The maximum for existence 
must be placed at or near 100° since it is known that all 
bacteria including spores may be killed by boiling in time. 
Nevertheless, certain forms have been reported as thriving 
in hot springs where the water temperature was 93°. This 
is the highest known temperature for development. The 
minimum for existence lies at or near the absolute zero 
(-273°) since certain organisms have been subjected to the 
temperature produced by the sudden evaporation of liquid 
hydrogen (-256° to -265°) and have remained alive. 
Whether they could withstand such temperatures indefinitely 
is not known. The minimum for development is near the 
freezing-point of water, since reproduction by division has 
been observed in the water from melting sea-ice at a tem- 
perature of -1.5°. Thus bacteria as a class have a range 
for existence of about 373° (- 273° to + 100°) and for de- 
velopment of 94.5° (-1.5° to + 93°) certainly much wider 
ranges than any other group of organisms.1 
The optimum temperature for development varies within 
rather wide limits for different organisms. In general it 
may be stated that the optimum temperature is approxi- 
mately that of the natural habitat of the organism, though 
there are exceptions. The optimum of the "hot spring" 
bacteria just mentioned is apparently that of the springs 
(93° in this case). Many soil organisms are known whose 
optimum is near 70° (a temperature rarely, if ever, attained 
in the soil), but only when grown in air or oxygen; but is 
very much lower when grown in the absence of oxygen. 
1With the possible exception of blue green algae which have been found 
with bacteria in the above-mentioned hot springs. Seeds of many plants 
have been subjected to as low temperatures as those above-mentioned with- 
out apparent injury. LIGHT 
75 
Many other soil organisms exhibit very little difference in 
rate or amount of growth when grown at temperatures 
which may vary as much as 10° or 15°, apparently an 
adaptation to their normal environment. The disease-pro- 
ducing organisms show much narrower limits for growth, 
especially those which are difficult to cultivate outside the 
body. For example, the bacterium of tuberculosis in man 
scarcely develops beyond the limits of 2° or 3° from the 
normal body temperature of man (37°), while the bacterium 
of tuberculosis in birds grows best at 41° to 45°, the normal 
for birds, and the bacterium of so-called tuberculosis of cold- 
blooded animals at 14° to 18°. 
Those bacteria whose optimum temperature is above 40° 
are sometimes spoken of as the " thermophil" bacteria. The 
fixing of the "thermal death-point" that is, the minimum 
temperature at which the bacteria are killed is a matter of 
great practical importance in many ways and numerous 
determinations of this have been made with a great many 
organisms and by different observers. The factors which 
enter into such determinations are so many and so varied 
that unless all the conditions of the experiment are given 
together with the time of application, the mere statements 
are worthless. It may be stated that all young, actively 
growing (non-spore-containing) disease-producing bacteria, 
when exposed in watery liquids and in small quantities are 
killed at a temperature of 60° within half an hour. It is evi- 
dent that this fact has very little practical application, 
since the conditions stated are rarely, if ever, fulfilled except 
in laboratory experiments. (See Sterilization and Pasteur- 
ization, Chapter XIII.) 
LIGHT. 
Speaking generally, it can be said that light is destructive 
to bacteria. Many growing forms are killed in a few hours 
when properly exposed to direct sunlight and die out in 
several days in the diffuse daylight of a well-lighted room. 
Even spores are destroyed in a similar manner, though the 
exposure must be considerably longer. Certain bacteria 76 
GENERAL CONDITIONS FOR GROWTH 
which produce colors may grow in the light, since the pig- 
ments protect them. Some few kinds, like the sulphur bac- 
teria, which contain a purplish-red pigment that serves 
them to break up H2S, need light for their growth. Since 
disease-producing bacteria are all injuriously affected by 
1'ght, the advantage of well-lighted habitations both for 
men and animals is obvious. 
OXYGEN SUPPLY. 
Oxygen is one of the constituents of protoplasm and is 
therefore necessary for all organisms. This does not mean 
that all organisms must obtain their supply from free oxygen, 
however, as animals and plants generally do. This fact 
is well illustrated by the differences among bacteria in this 
respect. Some bacteria require free oxygen for their growth 
and are therefore called aerobic bacteria or aerobes (sometimes 
strict aerobes, though the adjective is unnecessary). Others 
cannot grow in the presence of free oxygen and are therefore 
named anaerobic bacteria or anaerobes (strict is unnecessary). 
There are still other kinds which may grow either in the 
presence of free oxygen or in its absence, hence the term 
facultative anaerobes (usually) is applied to them. The dis- 
tinction between facultative aerobe and facultative anaerobe 
might be made. The former means those which grow best 
in the absence of free oxygen, though capable of growing in 
its presence, while the latter term means those which grow 
best in the presence of free oxygen, but are capable of grow- 
ing in its absence. The amount of oxygen in the atmos- 
phere in which an organism grows may be conveniently 
expressed in terms of the oxygen pressure, i. e., in milli- 
meters of mercury. It is evident that the maximum, mini- 
mum and optimum oxygen pressures for anaerobic bacteria 
are the same, namely, 0 mm. Hg. This is true only for nat- 
ural conditions, since a number of anaerobic organisms have 
been gradually accustomed to increasing amounts of O, so 
that by this process of training they finally grew in ordinary 
air, that is, at an oxygen pressure of about 150 mm. Hg. 
(Normal air pressure is 760 mm. Hg. and oxygen makes up OXYGEN SUPPLY 
77 
one-fifth of the air.) The minimum O pressure for faculta- 
tive anaerobes is also 0 mm. Hg. Some experiments have 
been made to determine the limits for aerobes, but on a few 
organisms only, so that no general conclusions can be drawn 
from them. To illustrate: Bacillus subtilis (a common 
"hay bacillus") will grow at 10 mm. Hg. pressure but 
not at 5 mm. Hg. It will also grow in compressed oxygen 
at a pressure of three atmospheres (2280 mm. Hg.), but not 
at four atmospheres (3040 mm. Hg.), though it is not de- 
stroyed. 
Parodko has determined the oxygen limits for five com- 
mon organisms as follows: 
Minimum 
Maximum. 
Vol. 
Mm. 
In atmospheres. 
Mm. Hg. 
per cent. 
Hg. 
Bacterium fluorescens.. 
... .1.94 to 2.51 
1474 to 1908 
0.00016 = 
0.0012 
Sarcina lutea 
.. . .2.51 to 3.18 
1908 to 2417 
0.00015 = 
0.0011 
Proteus vulgaris 
. . .3.63 to 4.35 
2749 to 3306 
0 
0 
Bacterium coli 
.. .4.09 to 4.84 
3108 to 3478 
0 
0 
Erythrobacillus prodigiosus 5.45 to 6.32 
3152 to 4800 
0 
0 
These few instances do not disclose any general principles 
which may be applied either for the growth or for the dis- 
tinction of aerobes or facultative anaerobes. 
It has been shown that compressed oxygen will kill some 
bacteria but this method of destroying them has little or 
no practical value. Oxygen in the form of ozone, O3, is 
rapidly destructive to bacteria, and this fact is applied prac- 
tically in the purification of water supplies for certain cities 
■where the ozone is generated by electricity obtained cheaply 
from water power. The same is true of oxygen in the 
"nascent state" as illustrated by the use of hypochlorites 
for the same purpose. 
It was stated (p. 74) that certain thermophil bacteria in 
the soil have an optimum temperature for growth in the air 
which is much higher than is ever reached in their natural 
habitat and that they grow at a moderate temperature under 
anaerobic conditions. It has been shown that if these organ- 
isms are grown with aerobes or facultative anaerobes they 
thrive at ordinary room temperature. These latter Organ- 78 
GENERAL CONDITIONS FOR GROWTH 
isms by using up the oxygen apparently keep the tension 
low, and this explains how such organisms grow in the soil.1 
OSMOTIC PRESSURE. 
Like all living cells bacteria are very susceptible to changes 
in the density of the surrounding medium. If placed in a 
medium less concentrated than their own protoplasm water 
is absorbed and they "swell up"; while if placed in a denser 
medium, water is given off and they shrink (plasmoptysis 
or plasmolysis). Should these differences be marked or 
the transition be sudden, the cell walls may even burst and 
the organisms be destroyed. If the differences are not 
too great or if the transition is made gradually, the organ- 
isms may not be destroyed, but will either cease to grow and 
slowly die out, or will show very much retarded growth, or 
will produce abnormal cell forms. This is illustrated in the 
laboratory in attempting to grow bacteria on food material 
which has dried out. A practical application of osmotic 
effects is in the use of a high percentage of sugar in preserv- 
ing fruits, etc., and in the salting of meats. Neither the 
cane-sugar nor the common salt themselves injure the bac- 
teria chemically, but by the high concentration prevent their 
development. In drying material in order to preserve it 
there are two factors involved: first, the loss of water neces- 
sary for growth and second, the increased osmotic pressure. 
In a medium of greater density diffusion of water is 
outward from the cell and this will continue until an equili- 
brium is established between cell contents and medium. 
Food for the organism must be in solution and enter the cell 
by diffusion. Therefore, growth ceases in a medium too 
dense, since water to carry food in solution does not enter 
the cell. 
JIt is popularly supposed that in canning fruit, vegetables, meats, etc., 
all the air must be removed, since the organisms which cause "spoiling" 
cannot grow in a vacuum. The existence of anaerobic and facultative anae- 
robic bacteria shows the fallacy of such beliefs. PRESSURE 
79 
Careful experimenters have shown that the electric cur- 
rent, either direct or alternating, has no direct destructive 
effect on bacteria. In a liquid medium the organisms may 
be attracted to or repelled from one or the other pole or 
may arrange themselves in definite ways between the poles 
(galvanotaxis), but are not injured. However, electricity 
through the secondary effects produced, may be used to 
destroy bacteria. If the passage of the electric current 
increases the temperature of the medium sufficiently, the bac- 
teria will be killed, or if injurious chemical substances are 
formed (ozone, chlorine, acids, bases, etc.), the same result 
will follow (see Ozone, pp. 77 and 157). 
ELECTRICITY. 
RADIATIONS. 
Rontgen or ar-rays and radium emanations when prop- 
erly applied to bacteria will destroy them. The practical 
use of these agents for the direct destruction of bacteria in 
diseases of man or animals is restricted to those cases where 
they may be applied directly to the diseased area, since they 
are just as injurious to the animal cell as they are to the 
bacteria, and even more so. Their skilful use as stimuli to 
the body cells to enable them to resist and overcome bacteria 
and other injurious organisms or cell growths is an entirely 
different function and will not be considered here. 
PRESSURE. 
Hydrostatic pressure up to about 10,000 pounds per 
square inch is without appreciable effect on bacteria as has 
been shown by several experimenters and also by finding 
living bacteria in the ooze dredged from the bottom of the 
ocean at depths of several miles. 
Pressures from 10,000 to 100,000 pounds show variable 
effects. Some bacteria are readily killed and others, even 
non-spore formers, are only slightly affected. The time 
factor is important in this connection. The presence of 
acids, even CO2, or organic acids, results in the destruction 
of most non-spore formers. 80 
GENERAL CONDITIONS FOR GROWTH 
Vibrations transmitted to bacteria in a liquid may be 
injurious to them under certain circumstances. Some of 
the larger forms like Bacillus subtilis may be completely 
destroyed by shaking in a rapidly moving shaking machine 
in a few hours. Bacteria in liquids placed on portions of 
machinery where only a slight trembling is felt, have been 
found to be killed after several days. Reinke has shown 
that the passing of strong sound waves through bacterial 
growths markedly inhibits their development. 
MECHANICAL VIBRATION. CHAPTER VII. 
CHEMICAL ENVIRONMENT. 
REACTION OF MEDIUM. 
Most bacteria are very susceptible to changes in the 
degree of acidity or alkalinity of the medium in which they 
grow. Some kinds prefer a slightly acid reaction, some a 
slightly alkaline, and some a neutral (with reference to 
litmus as indicator). The organism which is the commonest 
cause of the souring of milk thrives so well in the acid 
medium it produces that it crowds out practically all other 
kinds, though its own growth is eventually stopped by too 
much acid. Acid soils are usually low in numbers of bac- 
teria and as a consequence produce poor crops. The disease- 
producing bacteria as a class grow best in a medium which 
is slightly alkaline. 
Accurate determination of limits have been made on but 
few organisms. The reaction is a most important factor in 
growing bacteria on artificial media (see Making of Media, 
Chapter XVI). 
INJURIOUS CHEMICAL SUBSTANCES. 
(See Disinfection and Disinfectants, Chapter XIII.) 
CHEMICAL COMPOSITION. 
The chemical composition is subject to wide variation 
chiefly for two reasons: First, the cell wall in most instances 
seems to exert only a slight selective action in the absorption 
of mineral salts so that their concentration within the cell 
is very nearly that of the surrounding medium. Second, 
the chief organic constitutents vary remarkably with the 
kind and amount of food material available-a rich protein 82 
CH EM IC A L EN VI RON MEN T 
pabulum increases the protein, a plentiful supply of carbo- 
hydrates or of fat results in the storing of more fat, especially 
and vice versa. These facts must be borne in mind in con- 
sidering the chemistry of bacteria. 
Of the chemical elements known, only the following 
seem to be essential in the structure of bacteria: carbon, 
hydrogen, oxygen, nitrogen, sulphur, phosphorus, chlorine, 
potassium, calcium, magnesium, iron, manganese. Other 
elements, as sodium, iodine, silicon, aluminum, lithium, 
copper, etc., have been reported by different analysts, but 
none of them can be regarded as essential, except possibly 
in isolated instances. 
These elements exist in the bacterial cell in a great variety 
of combinations of which the most abundant is water. The 
amount of water varies in different species from 75 to 90 
per cent, of the total weight in growing cells, and is less in 
spores. The amount of ash has been shown by different 
observers tq vary from less than 2 per cent, to as much as 
30 per cent, of the dry weight. The following table com- 
piled from various sources will give an idea of the relative 
abundance of the different elements in the ash. 
S as SO3 7.64 per cent, (much more in sulphur bacteria) 
P as P2O5 18.14 " to 73.94'per cent. 
Cl 2.29 
K as K20 11.1 " to 25.59 " 
Ca as CaO 12.64 " to 14.0 
Mg as MgO 0.7 " to 11.55 " 
Fe as Fe2O3 1.0 " to 8.15 " (iron bacteria) 
Mn traces 
As to the form in which the last six elements in the table 
exist in the cell, little is known. The sulphur and phos- 
phorus are essential constituents of various proteins. The 
high percentage of phosphorus points to nuclein compounds 
as its probable source. 
The carbon and nitrogen, together with most of the 
hydrogen and oxygen not united as water, make up the 
great variety of organic compounds which compose the main 
substances in the bacterial cell. 
It has already been stated that the essential structures in 
the bacterial cell are cell wTall and protoplasm, including. CHEMICAL COMPOSITION 
83 
the nuclein. These differ markedly in chemical composi- 
tion. It is well known that the cell walls of green plants 
consist largely of cellulose and closely related substances.1 
True cellulose has been recognized in but very few bacteria. 
(Sarcina ventriculi, Migula; Mycobacterium tuberculosis, Ham- 
merschlag, Dreyfuss, Nishimura; Bacillus subtilis, Drey- 
fuss; Acetobacter xylinum, Brown; Acetobacter acidi oxalici, 
Banning; and a few others.) It is certainly not an import- 
ant constituent of the cell wall in many. On the other hand, 
hemicellulose and gum-like substances have been identified 
in numerous organisms of this class as important constit- 
uents of the cell wall and of the capsule which is probably 
an outgrowth from the latter. Practically always associ- 
ated with these substances are compounds containing nitro- 
gen. One of these has been certainly identified as chitin or 
a closely similar substance. Chitin is the nitrogenous sub- 
stance which enters largely into the composition of the hard 
parts of insects, spiders and crustaceans. It is an interesting 
fact to find this substance characteristic of these animals in 
bacteria, as well as other fungi. 
Though it is extremely difficult to separate the cell wall 
of bacteria from the cell contents, in the light of our present 
knowledge it can be stated that the cell walls are composed 
of a carbohydrate body closely related to cellulose, though 
not true cellulose, probably in close combination with chitin. 
Of the organic constituents of the cell contents the most, 
abundant are various proteins which ordinarily make up- 
about one-half of the dry weight of the entire cell. The 
"Mycoproteid" of Nencki, 1879, and other earlier workers, 
is deserving of little more than historical interest, since these 
substances were certainly very impure and probably con- 
sisted of mixtures of several "proteins" in the more recent, 
sense. 
111 By cellulose is understood a carbohydrate of the general formula CeHioOj, 
not soluble in water, alcohol, ether, or dilute acids but soluble in an ammo- 
niacal solution of copper oxide. It gives with iodine and sulphuric acid a 
blue color and with iodine zinc chloride a violet and yields dextrose on hydrol- 
ysis."-H. Fischer. 84 
CHEMICAL ENVIRONMENT 
From later studies it seems probable that substances 
resembling the albumin of higher forms do not occur in 
bacteria, at least in appreciable quantities. Globulin has 
been reported by Hellmich in an undetermined bacterium, 
but is certainly not commonly found. The larger portion 
of the protein is of a comparatively simple type, in fact, 
consists of protamins most of which are in combination 
with nucleic acid as nucleoprotamins. Practically all re- 
cent workers find a high percentage of nuclein, both actually 
isolated and as indicated by the amounts of purin bases- 
xanthin, guanin, adenin-obtained, as well as by the abun- 
dance of phosphorus in the ash, already mentioned. Some 
of these nucleins have been shown to have poisonous prop- 
erties. 
Closely related to but not identical with the proteins are 
the enzymes and toxins which are formed in the cell and 
exist there as endo-enzymes or endo-toxins respectively. 
These substances will be discussed later under the heading 
"Physiological Activities of Bacteria" (Chapter XII). 
Carbohydrates are not commonly present in the cell con- 
tents, though glycogen has been observed in a few and a 
substance staining blue with iodine in one or two others. 
This latter substance was at first considered to be starch 
"granulose," but is probably more closely related to gly- 
cogen. 
Fats seem to be very generally present. The commoner 
fats-tri-olein, tri-palmitin, tri-stearin have been found by 
many analysts. The "acid-fast bacteria" are particularly 
rich in fatty substances, especially the higher wax-like fats. 
Lecithins (phosphorized fats) and cholesterins (not fats but 
alcohols) have been repeatedly observed and probably occur 
in all bacteria as products of katabolism. 
Organic acids and esters occur as cell constituents but 
will be discussed in connection with their more character- 
istic occurrences as products of bacterial activity, as will 
also pigments which may likewise be intracellular in some 
instances. 
The following analysis of tubercle bacilli, from de 
Schweinitz and Dorset, while not intended as typical for CHEMICAL COMPOSITION 
85 
all bacteria, still illustrates the high percentage of protein 
compounds which undoubtedly occurs' in most, as well as 
showing the large amount of fatty substance in a typical 
"acid-fast" organism: 
' 8.5 per cent, tuberculinic acid 
24.5 " nucleoprotamin 
'23.0 " neucleoprotein 
। 8.3 " proteinoid 
26.5 " fat and wax 
9.2 " ash 
In the dried 
organisms 
55.8 per cent, protein. CHAPTER VIII. 
CHEMICAL ENVIRONMENT (Continued). 
GENERAL FOOD RELATIONSHIPS. METABOLISM. 
The foregoing brief review of the chemical composition 
of the bacterial cell illustrates the variety of compounds 
which necessarily occurs, but affords no definite clue as to 
the source of the elements which enter into these compounds. 
These elements come from the material which the organism 
uses as food. Under this term are included elements or 
compounds which serve as building material, either for new 
cell substance or to repair waste, or as sources of energy. 
An organism which is capable of making use of an element 
in the free state is said to be prototrophic for that particular 
element. Thus aerobes and facultative anaerobes are proto- 
trophic for O. The "root-tubercle bacteria" of leguminous 
and other plants and certain free living soil organisms are 
prototrophic for N.1 
On the other hand, if the element must be secured from 
compounds, then the organism is metatrophic in respect to 
the element in question. Should the compound be inor- 
ganic, the term autotrophic is applied to the organism and 
heterotrophic if the compound is organic. It is very probable 
that anaerobes, exclusive of a few nitrogen absorbers, are 
metatrophic for all the elements they utilize. With the 
exception of the anaerobes it seems that all bacteria are mixo- 
trophic, that is, prototrophic for one or two elements and 
auto- or heterotrophic for the others.2 
' The sulphur bacteria are partially prototrophic for S; probably the iron 
bacteria also for Fe. Some few soil bacteria have been shown to be capable 
of utilizing free H, and it seems certain that the bacteria associated with 
the spontaneous heating of coal may oxidize free C. So far as known no 
elements other than these six are directly available to bacteria. 
2 Only a few lands of bacteria so far as known are pi oto-autotrophic. The 
nitrous and nitric organisms of Winogradsky which are so essential in the 
soil, and which might have been the first of all organisms so far as their food 
is concerned, and some of the sulphur bacteria are examples. METABOLISM 
87 
Those bacteria whose food consists of dead material are 
spoken of as saprophytes, while those whose natural habitat, 
without reference to their food, is in or on other living 
organisms are called parasites. The host is the organism in 
or on which the parasite lives. Parasites may be of several 
kinds. Those which neither do injury nor are of benefit to 
the host are called non-pathogenic parasites or commensals; 
many of the bacteria in the intestines of man and other ani- 
mals are of this class. Those which do injury to the host 
are called pathogenic or disease-producing, as the organisms 
causing the transmissible diseases of animals and plants.1 
Finally, we have those parasites which are of benefit to and 
receive benefit from the host. These are called symbionts 
or symbiotic parasites and the mutual relationship symbiosis. 
Certain of the intestinal bacteria in man and especially in 
herbivorous animals are undoubted symbionts, as are also 
the "root-tubercle bacteria" already mentioned. 
It is evident that all parasites that may be cultivated 
outside the body are for the time saprophytic, hence the terms 
strict parasites and facultative parasites, which should re- 
quire no further explanation. 
The changes which the above-mentioned types of food 
material undergo in the various anabolic and katabolic pio- 
cesses within the cell are as yet but very slightly known. 
Nevertheless there are a number of reactions brought about 
by bacteria acting on various food materials, partly within 
but largely without the cell which are usually described as 
"physiological activities "or "biochemicalreactions." Some 
of these changes are to be ascribed to the utilization of 
certain of the elements and compounds in these materials 
as tissue builders, some as energy-yielding reactions and still 
others as giving rise to substances that are of direct benefit 
to the organism concerned in its competition with other 
organisms. 
Though all of the twelve elements already mentioned are 
essential for the growth of every bacterium, twm of them 
1 The term pathogenic is also applied to certain non-parasitic saprophytic 
bacteria whose products cause disease conditions, as one of the organisms 
causing a type of food poisoning in man (Clostridium botulinum'), which also 
probably causes "forage poisoning" in domestic animals. 88 
CHEMICAL EN VI RON MEN T 
are of especial importance for the reason that most of the 
"physiological activities" to be described in the next chap- 
ters are centered around their acquisition and utilization. 
These elements are carbon and nitrogen. Some few of the 
special activities of certain groups have to do with one or 
the other of the remaining nine, as will be shown later. 
But generally speaking when a bacterium under natural con- 
ditions secures an adequate supply of cai bon and nitrogen, the 
other elements are readily available in sufficient amount. 
Carbon is necessary not only because it is an essential 
constituent of protoplasm but because its oxidation is the 
chief source of the energy necessary for the internal life of 
the cell, though nitrogen and sulphur replace it in this 
function with a few forms. This latter use of carbon con- 
stitutes what may be called its respiratory function. Bac- 
teria like other organisms in their respiration utilize oxygen 
and give off carbon dioxide. The amount of the latter given 
off from the cell in this way is very small as compared with 
that which is frequently produced as an accompaniment of 
other reactions (see Fermentation, next chapter). But 
there is no doubt of its formation and it has been determined 
by a few investigators. On account of this use of carbon, 
bacteria require relatively large amounts of this element. 
One group of bacteria concerned in the spontaneous heating 
of coal seems to be able to use free carbon from this material. 
Another gioup is said to be able to oxidize marsh gas, CH4, 
and use this as its source of carbon. The nitrite, nitrate 
and sulphur bacteria mentioned later utilize carbon dioxide 
and carbonates as their carbon supply, and one kind has 
been described which uses carbon monoxide. With these 
few exceptions bacteria are dependent on organic compounds 
for their carbon and cannot use CO2 as green plants do. 
The oxygen requirement is high partly for the same 
reason that carbon is, i. e., respiration. Oxygen is one of 
the constituents of protoplasm, and combined with hydro- 
gen forms water which makes up such a large part of the 
living cell. Anaerobic bacteria are dependent on so-called 
"molecular respiration" for their energy. That is, through 
a shifting or rearrangement of the atoms in the compounds METABOLISM 
89 
used as food the oxidation of carbon is brought about. 
Enzymes are probably responsible for this action. Car- 
bon dioxide is produced by anaerobes as well as by aerobes, 
and frequently in amounts readily collected. A carbohy- 
drate is usually though not always essential for the growth 
of anaerobes and serves them as the best source of energy. 
Nitrogen is the characteristic element of living material. 
Protoplasm is a chemical substance in unstable equilibrium 
and nitrogen is responsible for this instability. No other 
of the commoner elements is brought into combination with 
such difficulty, nor is so readily liberated when combined 
(all commercial explosives are nitrogen compounds). Bac- 
teria, like other forms of protoplasm, require nitrogen. More 
marked peculiarities are shown by bacteria with reference 
to the sources from which they derive their nitrogen than 
for carbon. Some can even combine the free nitrogen of the 
air and furnish the only natural means of any importance 
for this reaction. Some few forms (the nitrite and nitrate 
formers, Chapter XI) obtain their energy from the oxida- 
tion of inorganic nitrogen compounds, ammonia and nitrites 
respectively, and not from carbon. These latter bacteria 
use carbon from carbon dioxide and carbonates. A great 
many bacteria can secure their nitrogen from nitrates but 
some are restricted to organic nitrogen. Many bacteria 
obtain their carbon from the same organic compounds from 
which their nitrogen is derived. 
Sulphur serves mainly as a constituent of protein com- 
pounds in the protoplasmic structure. In some of the sul- 
phur bacteria it is a source of energy, since either free sul- 
phur or H2S is oxidized by them. Some of these bacteria 
can obtain their carbon from CO2 or carbonates, and their 
nitrogen from nitrates or ammonium salts. 
Whether the iron bacteria, belonging to the genus Creno- 
thrix of the higher, thread bacteria, use this element or its 
compounds as sources of energy is still a disputed question. 
The evidence is largely in favor of this view. 
Free hydrogen has been shown to be oxidized by some 
forms which obtain their energy in this way. 
Whether there is a special class of phosphorus bacteria 
remains to be discovered. That phosphorus is oxidized dur- 90 
CHEMICAL ENVIRONMENT 
ing the activity of many bacteria is undoubted, but whether 
this represents a source of energy or is the accidental by- 
product of other activities is undetermined. 
Practically nothing is known about the metabolism of the 
other elements as such. 
From the preceding brief review of the relation of certain 
bacteria to some of the elements in the free state and from 
the further fact that there is scarcely a known natural 
organic compound which cannot be utilized by some kind 
of bacterium, it is evident that this class of organisms has 
a far wider range of adaptability than any other class, and 
this adaptability helps to explain their seemingly universal 
distribution. 
As to the metabolism within the cell, no more is known than 
is the case with other cells, nor even as much. The mater- 
ials used for growth and as sources of energy are taken into 
the cell, built up into various compounds some of which 
have been enumerated and in part broken down again. 
Carbon dioxide and water are formed in the latter process. 
What other katabolic products occur it is not easy to deter- 
mine. Certainly some of the substances mentioned in the 
next chapters are such products but it is not always possible 
to separate those formed inside the cell from those formed 
outside. Perhaps most of the latter should be considered 
true metabolic products. It would seem that on account 
of the simplicity of structure of the bacterial cell and of the 
compounds which they may use as food they would serve 
as excellent objects for the study of the fundamental prob- 
lems of cell metabolism. Their minuteness and the nearly 
impossible task of separating them completely from the 
medium in or on which they are grown makes the solution 
of these problems one of great difficulty. 
When all of the environmental conditions necessary for 
the best development of a given bacterium are fulfilled, it 
will then develop to the limit of its capacity. This develop- 
ment is characterized essentially by its reproduction, which 
occurs by transverse division. The rate of this division 
varies much with the kind even under good conditions. 
The most rapid rate so far observed is a division in eighteen ME TAB0LISM 
91 
minutes. A great many reproduce every half-hour and this 
may be taken as a good average rate. If such division 
could proceed without interruption, a little calculation will 
show that in about sixty-five hours a mass as large as the 
earth would be produced. 
Starting with 1 coccus, 1 [1 in diameter, 
its volume = 0.0000000000005 cc. 
y. hour = 2 
1 hour = 4 
2 hours = 16 
4 hours = 256 
5 hours = 1024 = 103 + 
15 hours = 1,000,000,000 = 109 = 0.5 cc. 
35 hours = 1021 + = 500.0 cu.m. 
About 65 hours= 2x IO42 + = 5 x 1020 cu.m. = a mass as 
large as the earth. 
Such a rate of increase evidently cannot be kept up long 
on account of many limiting factors, chief of which is the 
food supply. 
The foregoing calculation is based on the assumption that 
the organism divides in one plane only. If it divides in 2 or 
3 planes, the rate is much faster, as is shown by the follow- 
ing formulae, which indicate the theoretical rate of division: 
S = number of bacteria after a given number of divisions, 
a = number at the beginning, and n = number of divisions. 
1 plane division S = 2"a 
2 " " S = 22«a 
3 " " 8 = 23«a 
With two-plane or three-plane division, assuming that 
each organism attains full size, as was assumed in the first 
calculation, the "mass as large as the earth" would be 
attained in about thirty-two and twenty-two hours respect- 
ively. 
This extraordinary rate of increase explains in large 
measure why bacteria are able to bring about such great 
chemical changes in so short a time as is seen in the rapid 
"spoiling" of food materials, especially liquids. The reac- 
tions brought about by bacteria on substances which are 
soluble and diffusible are essentially "surface reactions." 
The material diffuses into the cell over its entire surface 
with little hindrance. The bacteria are usually distributed 
throughout the medium, so that there is very intimate con- 92 
CHEMICAL EN VI RON MEN T 
tact in all parts of the mass which favors rapid chemical 
action. The following calculation illustrates this: 
The volume of a coccus in diameter is 0.5236 x 10-13 cc. 
The surface of a coccus in diameter is It x 10-8 sq. cm. 
It is not uncommon to find in milk on the point of sour- 
ing 1,000,000,000 bacteria per cc. 
Assuming these to be cocci of Ijx diameter the volume of 
these bacteria in a liter is only 0.05 cc. or in the liter there 
would be 19999 parts of milk and only 1 part bacteria. The 
surface area of these bacteria is 3141.6 sq. cm. With this 
large surface exposed, it is not strange that the change from 
"on the point of souring" to "sour" occurs within an hour 
or less. 
Although large numbers of bacteria can and do cause 
great chemical changes the amount of material actually 
utilized for maintenance of the cell is very slight, infinites- 
imal almost, and yet is fairly comparable to that required 
for man, as is illustrated by the following computations: 
E. Kohn has shown that certain water bacteria grew well 
in water to which there was added per liter 0.000002 mg. 
dextrose, 0.00000007 mg. (NH^SO* and 0.0000000007 mg. 
(NH^HPCK The bacteria numbered about 1000 per cc. 
Taking the specific gravity at 1 (a little too low) the mass 
of the bacteria in the liter was about 0.001 mg. Hence the 
bacteria used 0.002 of their weight of carbohydrate and 
0.00007 of ammonium sulphate. A 150-pound (75-kilo) man 
can live on 375 g. of sugar (0.005 of his weight) and 52.5 g. of 
protein (0.0007 of his weight). From these figures it can 
be calculated that the man utilizes about two and a half 
times as much carbohydrate and about seven times as much 
nitrogen as the bacterium, relatively speaking. CHAPTER IX. 
PHYSIOLOGICAL ACTIVITIES. 
The physiological activities of motion, reproduction and 
metabolism within the cell have been discussed in previous 
chapters. The objects in view in the discussion of the 
"physiological activities" (sometimes spoken of as "bio- 
chemical" activities) of bacteria in this and subsequent 
chapters are to familiarize the student to some extent with 
the great range of chemical changes brought about by these 
minute organisms, to show their usefulness, even their 
necessity, and to impress the fact that it is chiefly by a care- 
ful study of these "activities" that individual kinds of 
bacteria are identified. It should always be borne in mind 
that the bacteria, in bringing about these changes which are 
so characteristic in many instances, are simply engaged in 
their own life struggle, n securing the elements which they 
need for growth, in liberating energy for vital processes, or 
occasionally in providing conditions which favor their own 
development and hinder that of their competitors. 
FERMENTATION. OF CARBOHYDRATES. 
By this is meant the changes which different carbohy- 
drates undergo when subjected to bacterial action.1 
These changes are marked chiefly by the production of 
gas or acid. The former is called "gaseous fermentation" 
the latter "acid fermentation." The gases commonly pro- 
'The term ' ' fermentation ' ' was originally used to denote the process which 
goes on in fruit juices or grain extracts when alcohol and gas are formed. 
Later it was extended to apply to the decomposition of almost any organic 
substance. In recent years the attempt has been made to give a chemical 
definition to the word by restricting its use to those changes in which by 
virtue of a "wandering" or rearrangement of the carbon atoms "new sub- 
stances are formed which are not constitutents of the original molecule." 
It may be doubted whether this restriction is justified or necessary. A 
definition is at present scarcely possible except when the qualifying adjective 
is included as "alcoholic fermentation, " "ammoniacal fermentation," "lactic 
acid fermentation," etc. PHYSIOLOGICAL ACTIVITIES 
94 
duced are carbon dioxide (CO2) hydrogen and marsh gas 
(CH 4). Other gases of the paraffin series may also be formed 
as ethane (C2H6), acetylene (C2H2), etc. CO2 and H are the 
ones usually formed from sugars by the few gas-forming 
Fig. 60.-Cylinder to show the 
formation of gas by bacteria. 
The gauge shows 265 pounds. 
It went beyond 500 pounds. 
Fig. 61.--A burning natural gas 
well at night. From a photograph 
colored. FERMENTATION OF CARBOHYDRATES 
95 
bacteria which produce disease, though even here some CH4 
is present. The common Bacterium coli forms all three, 
though the CH4 is in smallest quantity. 
In the fermentation of the polysaccharids-starch and 
especially cellulose and woody material-large amounts of 
CH 4 occur, particularly when the changes are due to anae- 
robic bacteria. This phenomenon may be readily observed 
in sluggish streams, ponds and swamps where vegetable 
matter accumulates on the bottom. The bubbles of gas 
which arise when the mass is disturbed explode if a lighted 
match is applied to them. 
The author has conducted a number of experiments to 
demonstrate this action as follows: Material taken from the 
botton of a pond in the fall after vegetation had died out 
was packed into a cylinder five feet long and six inches in 
diameter, water was added to within about 2 inches of the 
top. After leaving them open for a few days to permit all 
the dissolved oxygen to be used up by the aerobes, the cylin- 
ders were tightly capped and allowed to stand undisturbed. 
Pressure gauges reading to 500 lbs. were attached (Fig. 60). 
At the end of six months the gauge showed a pressure beyond 
the limits of the readings on it. Most of the gas was col- 
lected and measured 146 liters. An analysis of portions 
collected when about one-half had been allowed to escape 
showed the following composition, according to Prof. D. J. 
Demorest of the Department of Metallurgy: 
CO2 18.6 per cent. 
CH4 ■ 76.1 
H 1.0 
N 4.3 
In the author's opinion natural gas and petroleum have 
been formed in this way1 (Figs. 61 and 62). 
One of the very few practical uses of the gaseous fermen- 
tation of carbohydrates is in making "salt rising" bread. 
The "rising" of the material is due not to yeasts but to the 
formation of gas by certain bacteria which are present on 
the corn meal or flour used in the process (Fig. 63). 
1 See "Oil and Gas in Ohio," Bownocker: Geological Survey of Ohio, 
Fourth Series, Bull. I, pp. 313-314. 96 
PHYSIOLOGICAL AC TIC I TIES 
Another is in the formation of the "holes" or "eyes" so 
characteristic of Swiss and other types of cheese (Fig. 64). 
A great many organic acids are formed during the "acid 
fermentation4 ' of carbohydrates by bacteria. Each kind of 
Fig. 62.-A "flowing" oil well. 
bacterium, as a rule, forms several different acids as well as 
other substances, though usually one is produced in much 
larger amounts, and the kind of fermentation is named from 
this acid. One of the commonest of these acids is lactic. FERMEN TA TION OF CA RBOH YD RA TES 
97 
The "lactic acid bacteria" form a very large and important 
group and are indispensable in many commercial processes. 
Fig. 63.-A loaf of "salt rising" bread. The porous structure is due to the 
gas formed by bacilli and not by yeasts. 
In the making of butter the cream is first "ripened," as is 
the milk from which many kinds of cheese are made (Fig. 65). 
Fig. 64.-Ohio Swiss cheese. The "eyes" are due to gas formed by bacteria 
during the ripening of the cheese. 
The chief feature of this "ripening" is the formation of lac- 
tic acid from the milk-sugar by the action of bacteria. A 98 
PH Y BIOLOGICAL ACT IVI TIES 
similar change occurs in the popular "Bulgarian fermented 
milk." The reaction is usually represented by the equa- 
tion : 
Milk-sugar. Lactic acid. 
C12H22O11 -J- H2O -p (bacteria) = 4C3H6O3 
It is not probable that the change occurs quantitatively as 
indicated, because a number of other substances are also 
formed. Some of these are acetic and succinic acids and 
alcohol. Another industrial use of this acid fermentation 
Fig. 65.-A cream ripener. In this apparatus cream is "ripened," i. e 
undergoes lactic acid fermentation, preparatory to making it into butter. 
is in the preparation of "saner kraut." These bacteria are 
chiefly anaerobic and grow best in a relatively high salt 
concentration. They occur naturally on the cabbage leaves. 
In the formation of ensilage (Fig. 66) the lactic acid bac- 
teria play a very important part, as they do also in "sour 
mash" distilling, and in many kinds of natural "pickling." 
In fact, whenever green vegetable material "sours" spon- 
taneously, lactic acid bacteria are always present and 
account for a large part of the acid. This property of lactic FERMENTATION OF CARBOHYDRATES 
99 
acid formation is also taken advantage of in the preparation 
of lactic acid on a commercial scale in at least one plant in 
this country. 
Acetic acid is another common product of acid fermenta- 
tion. However, in vinegar making the acetic acid is not 
formed directly from the sugar in the fruit juice by bac- 
teria. The sugar is first converted into alcohol by yeasts, 
Fig. 66.-Filling a silo on the University farm. 
then the alcohol is oxidized to acid by the bacteria (Fig.67). 
The reaction may be represented as follows: 
Dextrose. Ethyl alcohol. Acetic acid. 
C6Hi2O6 = 2C2H5OH + 2CO2 
C2H5OH + O2 + (bacteria) = CH3COOH + H2O 
Butyric acid is generally produced where fermentation of 
carbohydrates occurs under anaerobic conditions. Some of 
the "strong" odor of certain kinds of cheese is due to this 
acid which is formed partly from the milk-sugar remaining 
in the cheese. Most of it under these conditions comes from 
the proteins of the cheese and especially from the fat (see 
page 101). 100 
PH YSIOLO GIGA L A C TIVI TIES 
As has been indicated alcohol is a common accompani- 
ment of most acid fermentations, as are the esters of acids 
other than the chief product. Bacteria are not used in 
a commercial way to produce alcohol, however, as the 
yield is too small. There are some few bacteria in which 
the amount of alcohol is prominent enough to call the pro- 
Fig. 67.-A vinegar ripen er. The tank shown opened at the side is filled 
with a special type of beech shavings which thus provide a very large sin-face. 
The apple juice which has been previously fermented with yeast, which 
converts the sugar into alcohol, is allowed to trickle through the openings 
at the top over the shavings. The acetic acid bacteria on the shavings 
rapidly oxidize the alcohol to acetic acid. The vinegar is drawn off below. 
cess an "alcoholic fermentation" rather than an acid one. 
In brewing and distilling industries, yeasts are used to make 
the alcohol, though molds replace them in some countries 
("sake" and "arrak" from rice). 
Under ordinary conditions the carbohydrate is never com- 
pletely fermented, since the accumulation of the product- SPLITTING OF FATS 
101 
acid-stops the reaction. If the acid is neutralized by the 
addition of an alkali-calcium or magnesium carbonate is 
best-then the sugar may all be split up. Where such fer- 
mentation occurs under natural conditions, the products 
are further split up, partly by molds and partly by acid- 
destroying bacteria into simpler acids and eventually to 
carbon dioxide and water, so that the end-products of the 
complete fermentation of carbohydrate material in nature 
are carbon dioxide, hydrogen, marsh gas, and water. 
In all of these fermentations the bacteria are utilizing the 
carbon both as building material and for oxidation and the 
fermentations are incidental to this use. As a rule, the acid- 
forming bacteria can withstand a higher concentration of 
acid than the other bacteria that would utilize the same 
material, and in a short time crowd out their competitors 
or inhibit their growth, and thus' have better conditions for 
their own existence, though finally their growth is also 
checked by the acid. 
SPLITTING OF FATS. 
The splitting of fats into glycerin and the particular acid 
or acids involved may be brought about by bacteria. An 
illustration is the development of rancidity in butter at 
times and the "strong" odor of animal fats on long keeping 
and of many kinds of cheese-"limburger"-in this country. 
Generally speaking, however, fats are not vigorously at- 
tacked, as is illustrated by the difficulties due to accumula- 
tion of fats in certain types of sewage-disposal works. The. 
chemical change is represented by the equation: 
Fat. Glycerin. Fatty acid. 
C3H6 (CnHsn-! O2)3 + 3 H20 = C3H6 (0H)3 + 3 (CnH2n 02). CHAPTER X. 
PHYSIOLOGICAL ACTIVITIES (Continued). 
PUTREFACTION OF PROTEINS. 
The word "putrefaction" is now restricted to the action of 
bacteria on the complex nitro gen-containing substances, pro- 
teins, and their immediate derivatives. The process is 
usually accompanied by the development of foul odors. 
Bacteria make use of proteins chiefly as a source of nitro- 
gen, but also as a source of carbon and other elements. 
Proteins contain nitrogen, carbon, hydrogen, oxygen, sul- 
phur and frequently phosphorus. Some of the metals- 
potassium, sodium, calcium, magnesium, iron and manganese 
and the non-metal chlorine-are nearly always associated 
with them more or less intimately. Since these bodies 
are the most complex of natural chemical substances it 
follows that the breaking up of the molecule to secure a 
part of the nitrogen gives rise to a great variety of products. 
There are marked differences among bacteria in their 
ability to attack this class of compounds. Some can break 
up the most complex natural proteins such as albumins, 
globulins, glyco-, chromo-, and nucleoproteins, nucleins and 
albuminoid derivatives like gelatin. The term saprogenic 
( <raTCpo<;= rotten) is sometimes applied to bacteria which 
have this power. These proteins are large-moleculed and 
not diffusable, so that the first splitting up that they under- 
go must occur outside the bacterial cell. The products of 
this first splitting may diffuse into the cell and be utilized 
there. The bacteria of this class attack not only these pro- 
teins in the natural state or in solution, but also in the coag- 
ulated state. The coagulum becomes softened and finally 
changed into a liquid condition. The process when applied 
to the casein of milk is usually called "digestion," also when PUTREFACTION OF PROTEINS 
103 
coagulated blood serum is acted on. In the latter case the 
serum is more commonly said to be "liquefied" as is the 
case when gelatin is the substance changed. Most of these 
bacteria have also the property of coagulating or curdling 
milk in an alkaline medium, and then digesting the curd. 
A second class of bacteria has no effect on the complex 
proteins just mentioned but readily attacks the products of 
their first splitting, i. e., the proteoses, peptones, polypep- 
tids and amino-acids. They are sometimes called sapro- 
phitic bacteria. 
Other bacteria derive their nitrogen from some of the 
products of the first two groups, and still further break down 
the complex protein molecule. Under normal conditions 
these various kinds of bacteria all occur together and thus 
mutually assist one another in what is equivalent to a sym- 
biosis or rather a metabiosis, a "successive existence," one 
set living on the products of the other. The result is the 
complete splitting up of the complete protein molecule. A 
part of the nitrogen is built up into the bodies of the bacteria 
which are using it as food. A part is finally liberated as 
free nitrogen or as ammonia after having undergone a series 
of transformations many of which are still undetermined. 
One class of compounds formed received at one time 
much attention because they were supposed to be respon- 
sible for a great deal of illness. These are the "ptomaines," 
basic nitrogen compounds of definite composition-amines 
-some few' of which are poisonous, most of them not. The 
basic character of ptomaines may be understood if they be 
regarded as made up of one or more molecules of ammonia 
in which the hydrogen has been replaced by alkyl or other 
radicals. Thus ammonia (NH3) may be represented as 
/H /CH 3 
N-H. The simplest ptomaine is n-H, in which one H is 
\H \H 
replaced by methyl, methylamine, a gaseous ptomaine. 
/CH3 
With two hydrogens replaced by methyl, n-CH3, dimethyl- 
\H 
amine, also a gas at ordinary temperature, is formed. Tri- 104 
PH YSIOWGICAL ACTIV1 TIES 
/CH 3 
methylamine, n-CH3, a liquid, results when three hydro- 
\CH3 
gens are similarly replaced. All three of these occur in her- 
ring brine and are responsible for the characteristic odor 
of this material. Putrescin and cadaverin-tetramethyl- 
ene-diamine, and pentamethylenediamine respectively- 
occur generally in decomposing flesh, hence the names. 
They are only slightly poisonous. One of the highly poi- 
sonous ptomaines is neurin C5Hi3NO or C>H3N(CH3)3OH 
= trimethyl-vinyl ammonium hydroxide. This is a stronger 
base than ammonia, liberating it from its salts. Numerous 
other ptomaines have been isolated and described. These 
bodies were considered for a long time to be the cause of 
various kinds of "meat posrsoning," "ice cream poison- 
ing," "cheese poisoning," etc. It is true that they may 
sometimes cause these conditions, but they are very much 
rarer than the laity generally believe. Most of the "meat 
poisonings" in America are due, not to ptomaines, but to 
infections with certain bacilli of the Bacterium enteritidis 
group. Occasionally a case of poisoning by the true toxin 
(see Chapter XII) of Clostridium botulinum occurs, and in 
recent years has become entirely too common due to in- 
sufficient heating of canned goods. The boiling of such 
material will destroy this toxin. The safest ride to follow is 
not to eat any canned material that shows any departure from 
the normal in flavor, taste or consistency. 
As ptomaines result from the putrefaction of proteins, so 
they are still further decomposed by bacteria and event- 
ually the nitrogen is liberated either as free nitrogen or 
as ammonia. 
Another series of products are the so-called aromatic 
compounds-phenol (carbolic acid), various cresols, also 
indol and skatol or methyl indol (these two are largely 
responsible for the characteristic odor of human feces). 
All of these nitrogen compounds are attacked by bacteria and 
the nitrogen is eventually liberated, so far as it is not locked 
up in the bodies of the bacteria, as free nitrogen or as am- 
monia. PUTREFACTION OF PROTEINS 
105 
The carbon which occurs in proteins accompanies the 
nitrogen in many of the above products, but also appears in 
nitrogen-free organic acids, aldehydes and alcohols which 
are all eventually split up, so that the carbon is changed to 
carbon dioxide or in the absence of oxygen partly to marsh 
gas. 
The intermediate changes which the sulphur in proteins 
undergoes are not known, but it is liberated as sulphuretted 
hydrogen (H2S) or as various mercaptans (all foul-smelling), 
or is partially oxidized to sulphuric acid Some of the H2S 
and the sulphur of the mercaptans are oxidized by the 
sulphur bacteria to free sulphur and finally to sulphuric 
acid. 
Phosphorus is present especially in the nucleoproteins 
and nucleins. Just what the intermediate stages are, on 
whether there are any, so far as the phosphorus is concerned, 
in the splitting up of nucleic acid by bacterial action is not 
determined. The phosphorus may occur as phosphoric acid 
in such decompositions, or when the conditions are 
anaerobic, as phosphine (PH3), which burns spontaneously 
in the air to phosphorus pentoxide (P2O5), and water.1 
The hydrogen in proteins appears in the forms above 
indicated: H4C, H3N, H3P, H2S, H2O and as freeH. The 
oxygen as CO2 and H2O. 
In the breaking down of the complex protein molecule 
even by a single kind of bacterium there is not a perfect 
descending scale of complexity as might be supposed from 
the statement that there result proteoses, peptones, poly- 
peptids, amino-acids. These substances do result, but at 
the time of their formation simpler ones are formed also, 
even CO2, NH3 and H2S. It appears that the entire mole- 
cule is shattered in such a way that less complex proteins 
are formed from the major part, while a minor portion breaks 
up completely to the simplest combinations possible. A 
more complete knowledge of these decompositions will aid 
Ut is probable that this is the way "Jack o'lanterns" or "Will o' the wisps" 
are ignited. Marsh gas is produced as above outlined from the vegetable 
and animal matter decomposing in swampy places under anaerobic condi- 
tions and likewise phosphine. These escape into the air and the "spontan- 
eous combustion" of the phosphine ignites the marsh gas. 106 
PHYSIOLOGICAL ACTIVITIES 
in the further unravelling of the structure of proteins. The 
presence or absence of free oxygen makes a difference in the 
end-products, as has been indicated. There are bacteria 
which oxidize the ammonia to nitric acid and the H2S to 
sulphuric acid. (See Oxidation, Chapter XI.) Bacteria 
which directly oxidize phosphorus compounds to phosphoric 
acid have not been described. It does not seem that such 
are necessary since this is either split off from nucleic acid 
or results from the spontaneous oxidation of phosphine when 
this is formed under anaerobic conditions. 
Not only are proteins decomposed as above outlined, but 
also their waste products, that is, the form in which their 
nitrogen leaves the animal body. This is largely urea in 
mammals, with much hippuric acid in herbivorous animals 
and uric acid in birds and reptiles. These substances yield 
NH3, CO 2 and H2O with a variety of organic acids as inter- 
mediate products in some cases. The strong odor of am- 
monia in stables and about manure piles is the every-day 
evidence of this decomposition. 
Where the putrefaction of proteins occurs in the soil with 
moderate amounts of moisture and free access of air a large 
part of the products is retained in the soil. Thus the am- 
monia and carbon dioxide in the presence of water form 
ammonium carbonate; the nitric, sulphuric and phosphoric 
acids unite with some of the metals which are always present 
to form salts. Some of the gases do escape and most where 
the oxygen supply is least, since they are not oxidized. 
The protein-splitting reactions afford valuable tests in 
aiding in the recognition of bacteria. In the study of patho- 
genic bacteria the coagulation and digestion of milk, the 
digestion or liquefaction of blood serum, the liquefaction of 
gelatin and the production of indol and H2S are those usually 
tested for. In dairy bacteriology the coagulation of milk 
and the digestion of the casein are common phenomena. 
Most bacteria which liquefy gelatin also digest blood serum 
and coagulate and digest milk, though there are exceptions. 
In soil bacteriology the whole range of protein changes is of 
the greatest importance. PUTREFACTION OF PROTEINS 
107 
Nuclein oFanimal 
Atolls 
Animals s' 
Nuclein of plant 
'cells 
Green plants 
Decorhposition 
^■fbactb^ia 
Unknown * 
A oxidizes 
compoindssiiont"neou^ 
Phosphoric 
acid 
Phosphates in the soil 
Fig. 68.-Diagram to illustrate the circulation of phosphorus through the 
agency of bacteria. 
Fatyfmd 
various C/compounds 
Animals 
DecQmposi tion 
/I \bacteria 
Various 
cl 
compounds 
eventually 
to I 
Animal X 
\res^iiVation 
Carbohydrates^ 
fatSand'' \ 
other C campounds\ 
Green ^Plant respiration / 
in' 
the air 
Fig. 69.-Diagram to illustrate the circulation of carbon through the agency 
of bacteria. 
Dead anintidprotein 
Decomposition bacteria 
Anin/als --"^re^t^^' vjX ^Free N taken 
Animals urea,et^ NH^ompounds up by free living 
I Dead plant protein \ N absorbers and 
Plait protein ^NifritTl^^ tubercle bacteria 
\ Nitrites 
Gre^n plants Nitrate bacteria 
Nitrates in soil 
Fig. 70.-Diagram to illustrate the circulation of nitrogen through the agency 
of bacteria. 108 
PH YSIOLOGICAL ACTIVI TIES 
The three physiological activities already discussed ex- 
plain how bacteria break down the chief complex, energy- 
rich substances-carbohydrates, fats and proteins which 
constitute the bulk of the organic material in the bodies of 
plants and animals, as well as the waste products of the 
latter-into energy-free compounds like carbon dioxide, 
water, ammonia, nitric, sulphuric and phosphoric acids- 
mineralize them, as is frequently said. By so doing the 
bacteria act as the great scavengers of nature removing the 
dead animal and vegetable matter of all kinds which but 
for this action would accumulate to such an extent that all 
life, both on land and in the water, must cease. It is fur- 
ther to be noted that not only is all this dead organic matter 
removed, but it is converted into forms which are again 
Dead animal protein 
Decompo^mon bacteria 
Animals 
Plant protein 
Greenxplants 
Dead plcCht 
^pr^tein 
1 
Sulphur bacteria 
r 
Fre;e S 
Sulphur bacteria 
Sulphates in the soil 
Fig. 71.-Diagram to illustrate the circulation of sulphur through the agency 
of bacteria. 
available for plant growth. Carbon dioxide forms the source 
of the carbon in all green plants, hence in all animals; the 
sulphates and phosphates are likewise taken up by green 
plants and built up again into protein compounds; the 
ammonia is not directly available to green plants to any 
large extent but is converted by the nitrifying bacteria 
(Chapter XI) into nitrates which is the form in which nitro- 
gen is assimilated by these higher types. Even the free 
nitrogen of the air is taken up by several kinds of bacteria, 
the symbiotic "root-tubercle bacteria" of leguminous and 
other plants, and some free-living forms, and made avail- PUTREFACTION OF PROTEINS 
109 
able. Hence bacteria are indispensable in nature, especially 
in keeping up the circulation of nitrogen. They are also 
of great service in the circulation of carbon, sulphur and 
phosphorus. Though some few kinds cause disease in man 
and animals, if it were not for the saprophytic bacteria 
above outlined, there could be no animals and higher plants 
to acquire these diseases. CHAPTER XI. 
PHYSIOLOGICAL ACTIVITIES (Continued). 
PRODUCTION OF ACIDS. 
The production of organic acids has been sufficiently dis- 
cussed in preceding chapters. It should be noted that not 
only these in great variety are produced by bacteria but that 
under certain conditions mineral acids, such as nitric, sul- 
phuric and phosphoric may be formed (see Oxidation, p. 114). 
Acid production is of great value in the identification of 
bacteria in dairy and soil work and in connection with 
certain types of pathogenic bacteria. 
GAS PRODUCTION. 
It will be sufficient merely to enumerate collectively the 
various gases mentioned in preceding paragraphs and to 
state that those commonly observed in the study of patho- 
genic bacteria are the first six mentioned. Most of them 
come in in dairy work either in the study of bacteria causing 
milk and cheese "failures" or as affecting the flavors of 
butter or cheese. In the study of soil organisms, any or all 
of them are liable to be of importance. The gases are: 
CO2, H, CIL, N, NH3, H2S, gaseous mercaptans, gaseous 
ptomaines, volatile fatty acids, etheral salts or esters and 
others, both of pleasant and of foul odor, but of unknown 
composition. 
PRODUCTION OF ESTERS. 
The production of esters, as mentioned in Chapters IX 
and X, of various alcohols and aldehydes are activities 
which are sometimes of value in the study of bacteria, but 
need not be further discussed. PHOSPHORESCENCE OR PHOTOGENESIS 
111 
PRODUCTION OF "AROMATIC" COMPOUNDS. 
These have been mentioned in discussing the putrefac- 
tion of proteins, as indol, skatol, phenol and various cresols. 
Of these only the first is ordinarily tested for in the study 
of bacteria, though others of the group become of value in 
certain special cases. 
Fig. 72.-Culture of phosphorescent bacteria in an Ehrlenmeyer flask 
photographed by their own light. Time of exposure twelve hours. (Molisch, 
from Lafar.) 
PHOSPHORESCENCE OR PHOTOGENESIS. 
This is a most interesting phenomenon associated with 
the growth of some bacteria. The "fox fire" frequently 
seen on decaying wood which is covered with a slimy deposit 
is most commonly due to bacteria, though also to other fungi. 
Phosphorescent bacteria are very common in sea water, 
hence they are frequently found on various sea foods, espe- 112 
PHYSIOLOGICAL ACTIVITIES 
cially when these are allowed to decompose, such as fish, 
oysters, clams, etc. The light is due to the conversion of 
the energy of unknown easily oxidizable compounds directly 
into visible radiant energy through oxidation without appre- 
ciable quantities of heat. The light produced may be suffi- 
cient to tell the time on a watch in absolute darkness, and 
also, to photograph the growths with their own light, but 
only after several hours' exposure (Fig. 72). None of the 
phosphorescent bacteria so far discovered produce disease 
in the higher animals or man. 
PRODUCTION OF PIGMENT OR CHROMOGENESIS. 
One of the most striking results of bacterial activity is 
this phenomenon. The particular color which results may 
be almost any one throughout the range of the spectrum, 
though shades of yellow and of red are of more frequent 
occurrence. 
In the red sulphur bacteria the "bacteriopurpurin" which 
they contain appears to serve as a true respiratory pigment 
in a manner similar to the chlorophyl in green plants, except 
that these bacteria oxidize H2S in the light as a source of 
energy instead of splitting up CO2. The red pigment pro- 
duced by certain bacteria has been shown to have a capacity 
for combining with 0 resembling that of hemoglobin, and 
some investigators have believed that such bacteria do 
store O in this way for use when the supply is diminished. 
With these few exceptions the pigments seem to be merely 
by-products of cell activity which are colored and have no 
known function. 
The red sulphur bacteria above mentioned and one or 
two other kinds retain the pigments formed within the cell. 
Such bacteria are called chromophoric as distinguished from 
the chromoparic bacteria whose pigment lies outside the 
cell. 
The chemical composition of no bacterial pigment has 
been determined up to the present. Some are soluble in 
water, as shown by the discoloration of the substances on 
which they grow. Others are not soluble in water but are REDUCING ACTIONS 
113 
in alcohol, or in some of the fat solvents as ether, chloro- 
form, benzol, etc. These latter are probably closely related 
to the lipochromes or "fat colors" of higher plants and 
animals. Attempts have been made to render the produc- 
tion of pigments a still more reliable means of identification 
of species of bacteria through a careful examination of the 
spectra of their solutions, but such study has not as yet led 
to any valuable practical results. 
The production of pigment depends on the same general 
factors which determine the growth of the organism but does 
not necessarily run parallel with these. It is especially in- 
fluenced by the oxygen supply (only a very few organisms 
are known which produce pigment anaerobically-Spirillum 
rubrum is one); by the presence of certain food substances 
(starch, as in potato, for many bacteria producing yellow and 
red colors; certain mineral salts, as phosphates and sul- 
phates, for others); by the temperature (many bacteria 
cease to produce color at all if grown at body temperature, 
37°-Erythrobacillus prodigiosus-or if grown for a longer 
time at temperatures a few degrees higher). 
Reduction of nitrates to nitrites or to ammonia or even 
to free nitrogen is brought about by a great many different 
kinds of bacteria. In many instances this phenomenon is 
due to a lack of free oxygen, which is obtained by the bac- 
teria from these easily reducible salts. In other cases a 
portion of the nitrogen is removed to be used as food material 
in the building up of new protein in the bacterial cell. This 
latter use of the nitrogen of nitrates by bacteria might 
theoretically result in considerable loss of " available nitro- 
gen" in the soil as has actually been shown in a few experi- 
ments The reduction of nitrates as above mentioned 
would also diminish this supply, but probably neither of 
these results has any very great practical effect on soil fer- 
tility. The building up of protein from these mineral salts 
by bacteria in the intestines of herbivorous animals has been 
suggested by Armsby as a considerable source of nitrogenous 
food, and this suggestion appears possible. 
REDUCING ACTIONS. 114 
PHYSIOLOGICAL ACTIVITIES 
The liberation of nitrogen from nitrates or nitrites, either 
as free nitrogen or as ammonia, is spoken of as "dentrifica- 
tion, " though this term was formerly applied to such libera- 
tions, from compounds of nitrogen generally even from pro- 
teins. 
Certain bacteria may also reduce sulphates and other 
sulphur compounds to H2S, a phenomenon frequently ob- 
served in sewage and likewise of importance in the soil. 
It is possible that phosphates may be similarly reduced.1 
Further and more careful study of the reducing actions of 
bacteria is needed. 
As has been stated in discussing the respiration of bac- 
teria (Chapter VIII) most of these organisms gain their 
energy through the oxidation of carbon in various forms, 
chiefly organic, so that CO2 is a product of the activity of 
nearly all bacteria. Some few oxidize CO to CO2, others 
CH4 and other paraffins to CO2 for this purpose. One class 
of bacteria even oxidizes H in small amounts for its energy 
and uses the carbon dioxide of the air or traces of organic 
carbon in the air as a source of carbon for "building" pur- 
poses. 
One of the familiar oxidations of organic carbon is that 
of the acetic acid bacteria in the making of vinegar. These 
oxidize the alcohol which results from the action of yeast 
to acetic acid according to the formula CH3CH2OH + O2= 
CH3COOH-f-H2O (see Fig. 67). 
Of the various phenomena of oxidation due to bacteria, 
the formation of nitrites and nitrates has the greatest prac- 
tical importance, since it is by this means that the ammonia 
which results from the decomposition of animal and vege- 
table tissue and waste products is again rendered available 
to green plants as food in the form of nitrates. Practically 
all the nitrates found in nature, sometimes in large quanti- 
ties, are formed in this way. There are two distinct kinds 
of bacteria involved. One, the nitrous bacteria, oxidizes 
the ammonia to nitrous acid which forms nitrites with bases, 
and the other, the nitric bacteria, oxidizes the nitrous to 
OXIDATION. 
1 Dr. H. H. Green, of Pretoria, South Africa, has isolated from "cattle 
dips" a bacterium that reduces arsenates to arsenites. OXIDATION 
115 
nitric acid, giving nitrates with bases. A striking peculiar- 
ity of these two classes of organisms is that they may live 
entirely on inorganic food materials, are proto-autotrophic, 
prototrophic for oxygen (aerobic) and autotrophic for the 
other elements. Their carbon is derived from CO2 or car- 
bonates. The importance of such organisms in keeping up 
the supply of nitrates in the soil can scarcely be overesti- 
mated. 
Fig. 73.-Sprinkling filters of the Columbus sewage-disposal plant-de- 
vices which provide a good supply of oxygen for the bacteria that oxidize 
the organic matter in the sewage. 
The oxidation of the H2S, which is formed in the putre- 
faction of proteins, to free S by the sulphur bacteria and the 
further oxidation of this free S to sulphuric acid, and of the 
phosphorus, so characteristic of the nucleins, to phosphoric 
acid have been referred to. These activities of bacteria are 
of great value in the soil. Doubtless the commercial "phos- 
phate rock" owes its origin to similar bacterial action in 
ages past. 116 
PHYSIOLOGICAL ACTIVITIES 
The oxidation of H2S to free S may be an explanation of 
the origin of the great deposits of sulphur which are found 
in Louisiana and along the Gulf coast. These deposits occur 
in the same general regions as natural gas and oil. The 
sulphur might have been derived from the same organic 
material carried down by the Mississippi which yielded the 
oil and gas.1 
A purposeful utilization of the oxidizing power of bacteria 
is in "contact beds," "sprinkling filters" and "aerated 
sludge tanks" in sewage disposal works. In these instances 
the sewage is thoroughly mixed with air and brought in 
contact with large amounts of porous material so as to ex- 
pose an extensive surface for oxidation (Fig. 73). 
Fig. 74.-One of the University hot beds. 
PRODUCTION OF HEAT. 
A direct result of the oxidizing action of bacteria is the 
production of heat. Under most conditions of bacterial 
growth this heat is not appreciable. It may become well 
marked. The "heating" of manure is one of the commonest 
illustrations. The temperature in such cases may reach 
70°. The heating of hay and other green materials is due 
chiefly to bacterial action. This heating may lead to "spon- 
taneous combustion." The high temperatures (60° to 70°) 
favor the growth of thermophil bacteria which cause a still 
further rise. The heat dries out the material, portions of 
which are in a state of very fine division due to the disin- 
1 Dr. Green (1. c.) has also isolated an organism which causes some deteri- 
oration of cattle dips by oxidizing arsenites to arsenates. ABSORPTION OF FREE NITROGEN 
117 
tegrating action of the organisms. The hot, dry, finely 
divided material oxidizes so rapidly on contact with the air 
that it ignites. 
A practical use of heat production by bacteria is in the 
making of "hot beds" for forcing vegetables (Fig. 74). 
This is likewise one of the most important practical 
activities of certain types of bacteria present in the soil. 
ABSORPTION OF FREE NITROGEN. 
Fig. 75.-Root tubercles on soy bean, x %• 
The ability of plants of the legume family to enrich the 
soil has been known and taken advantage of for centuries, 
but it is only about thirty years since it was demonstrated 
that this property is due to bacteria. These plants, and 
several other kinds as well, have on their roots larger or 
smaller nodules (Fig. 75) spoken of as "root tubercles" 
which are at certain stages filled with bacteria. When 
conditions are favorable, these bacteria live in symbiotic 
relationship with the plant tissues, receiving carbonaceous 
and other food material from them and in return furnishing 118 
PH YS10LOGICA L A C TIVI TIES 
nitrogenous compounds to the plant. This nitrogenous 
material is built up from free nitrogen absorbed from the 
air by the bacteria. The utilization of this peculiar prop- 
erty through the proper cultivation of clover, alfalfa, soy 
beans and other legumes is one of the best ways of building 
up and maintaining soil fertility in so far as the nitrogen is 
concerned. The technical name of these bacteria is Rhizobium 
leguminosarum. 
There are also types of "free-living," as distinguished 
from these symbiotic, bacteria which absorb the free nitro- 
gen of the air and aid materially in keeping up this supply 
Fig. 76.-Free-living nitrogen absorbing bacteria "Azotobacter." Note 
their large size as compared with other bacteria shown in this book. 
under natural conditions. One of the most important of 
these types is the aerobic "Azotobacter" (Fig. 76), while 
another is the anaerobic Clostridium pasteurianum. The 
nitrogen which is absorbed is built up into the protein 
material of the cell body and this latter must in all proba- 
bility be "worked over" by various types of decomposition 
bacteria and by the nitrous and nitric organisms and be 
converted into utilizable nitrates just as other protein 
material is, as has been discussed in Chapter X. At any 
rate there is as yet no definite knowledge of any other 
method of transformation. Up to the present no intentional 
practical utilization of this valuable property of these free- 
living forms has been made. 
Nitrogen Nutrition of Green Plants.-It is the belief of 
botanists that green plants obtain their nitrogen chiefly 
in the form of nitrates, though ammonium salts may be ABSORPTION OF FREE NITROGEN 
119 
utilized to some extent by certain plants at least. Excep- 
tions to this general rule are those plants provided with 
root tubercles (and the bog plants and others which have 
mycorrhiza?). These plants obtain their nitrogen in the 
form of organic compounds made for them by the bacteria 
growing in the tubercles. That nitrogen circulates through- 
out the structure of plants in organic combination is certain. 
There does not appear to be any reason why similar com- 
pounds which are soluble and diffusible (amino-acids?) 
should not be taken up through the roots of plants and 
utilized as such. It seems to the author that this is very prob- 
ably the case. Arguments in favor of this view are: (1) The 
nitrogen nutrition of leguminous and other plants with root 
nodules. (2) The close symbiosis between " Azotobacter " 
and similar nitrogen-absorbing bacteria and many species 
of algae in sea water at least. (3) The vigorous growth of 
plants in soils very rich in organic matter, which inhibits 
the production of nitrates by the nitrous-nitric bacteria 
when grown in culture, and possibly (?) in the soil, so that 
nitrates may not account for the vigorous growth. (4) The 
effect of nitrate fertilizers is to add an amount of nitro- 
gen to the crop much in excess of the amount added as 
nitrate. (5) The most fertile soils contain the largest num- 
bers of bacteria. The doctrine that nitrates furnish the 
only nitrogen to plants was established before the activities- 
of bacteria in the soil were suspected, and, so far as the 
author is aware, has not been supported by experiments 
under conditions rigidly controlled as to sterility. 
It would seem that one of the chief functions of soil bac- 
teria is to prepare soluble organic compounds of nitrogen 
for the use of green plants and thus to make a "short cut" 
in the nitrogen cycle (p. 107), as now believed in, direct 
from the "decomposition bacteria" to green plants. 
Experiments have been made by different observers in 
growing seedling plants of various kinds in water culture 
with one or in some cases several of the amino-acids as 
sources of nitrogen. Most of these experiments were disap- 
pointing. Plant proteins are not so different from animal 
proteins, or plant protoplasm (apart from the chlorophyl 120 
PHYBIOLOGICAL ACTIVI TIES 
portions of plants) from animal protoplasm as to lead one to 
suppose that it could be built up from one or two amino- 
acids any more than animal protoplasm can. The author 
is strongly convinced that this subject should be thoroughly 
investigated. It will require careful experimentation and 
perhaps rather large funds to provide the amounts of amino- 
acids that would probably be needed, but might result in a 
decided change in our ideas of soil fertility, and especially 
in the use of nitrogen fertilizers. CHAPTER XII. 
PHYSIOLOGICAL ACTIVITIES (Continued). 
PRODUCTION OF ENZYMES. 
Most of the physiological activities of bacteria which 
have been discussed are due to the action of these peculiar 
substances, so that a knowledge of their properties is essen- 
tial. This knowledge cannot as yet be exact because no 
enzyme has, up to the present, been obtained in a "pure 
state, " though it must be admitted that there are no certain 
criteria which will enable this "pure state" to be recognized. 
It was formerly thought that they were protein in nature, 
but very "pure" and active enzymes have been prepared 
which did not give the characteristic protein reactions, so 
this idea must be abandoned. That they are large mole- 
culed colloidal substances closely related to the proteins in 
many respects must still be maintained. There are certain 
characteristics which belong to enzymes, though no one of 
them exclusively. These may be enumerated as follows: 
1. Enzymes are dead organic chemical substances. 
Dead is used in the sense of non-living, never having 
lived, not in the sense of "ceased to be alive." 
2. They are always produced by living cells: 
Sometimes as active enzymes, sometimes as pro-enzymes 
or zymogens which are converted into enzymes outside the 
cell by acids, other inorganic substances or other enzymes. 
3. They produce very great chemical changes without 
themselves being appreciably affected. 
Enzymes will not continue to act indefinitely, but are 
used up in the process (combination with products?). The 
amount of change is so great in proportion to the amount of 
enzyme that the above statement is justified in the rela- 
tive sense. Thus a milk-curdling enzyme has been prepared 122 
PHYSIOLOGICAL ACTIVITIES 
that would precipitate 100,000,000 times its own weight of 
caseinogen. 
4. Their action is specific in that each enzyme acts on 
one kind of chemical substance only, and the products are 
always the same. 
The substance may be combined with a variety of other 
chemical substances so that the action appears to be on 
several, but in reality it is on a definite group of molecules 
in each instance. For example, emulsin attacks several 
different glucosides but always sets free dextrose from them. 
5. The action is inhibited and eventually stopped, and in 
some cases the enzyme is destroyed by an accumulation of 
the products of the action. If the products are removed, the 
action will continue, if the enzyme is not destroyed. This 
effect is explained partly because the enzyme probably 
■combines with some of the products, since it does not act 
indefinitely, and partly because of the reversibility of the 
reaction. 
6. Like many chemical reactions those of enzymes are 
reversible, that is, the substance broken up may be reformed 
by it from the products produced in many instances. Thus: 
maltose-|-maltase glucose+glucose + maltase, 
fat+lipase glycerin+fatty acid + lipase. 
7. The presence of certain mineral salts seems to be 
essential for their action. These and other substances which 
are necessary are sometimes called co-enzymes. A salt of 
calcium is most favorable for a great many. 
8. They may be adsorbed like other colloids by "shaking 
out" with finely divided suspensions like charcoal or kaolin, 
or by other colloids like aluminum hydroxide or proteins. 
9. When properly introduced into the tissues or blood of 
an animal, they cause the body cells to form anti-enzymes 
which will prevent the action of the enzyme (see Chapter 
XXVII). 
10. Though inert, they show many of the characteristics 
of living organisms, that is 
(a) Each enzyme has an optimum, a maximum and a 
minimum temperature for its action. PRODUCTION OF ENZYMES 
123 
All chemical reactions have such temperature limits, the 
distinction is that for enzymes as for living substance the 
range is relatively narrow. 
(6) High temperatures destroy enzymes. All in water 
are destroyed by boiling in time and most at temperatures 
considerably below the boiling-point. When dry, many will 
withstand a higher degree of heat than 100° before they are 
destroyed. 
(c) Temperatures below the minimum stop their action, 
though they are not destroyed by cold. 
(d) Many poisons and chemical disinfectants (Chapter 
XIV) which kill living organisms will also stop the action of 
enzymes, though generally more of the substance is required, 
so that it is possible to destroy the living cells by such means 
and yet the action of the enzyme will continue. 
(e) Most enzymes have an optimum reaction of medium 
either acid, alkaline or neutral, depending on the particular 
enzyme, though some few seem to act equally well within a 
considerable range on either side of the neutral point. 
The final test for an enzyme is the chemical change it brings 
about in the specific substance acted on. 
The most prominent characteristic of enzymes is that 
they bring about very great chemical changes without them- 
selves being appreciably affected. This property is also 
shown by many inorganic substances which are spoken of 
as "catalytic agents" or catalyzers" so that enzymes are 
sometimes called "organic catalyzers." The function of 
catalytic agents seems to be to hasten the rate of a reaction 
which would occur spontaneously, though in a great many 
cases with extreme slowness. 
Just how enzymes act is not certain and probably will not 
be until their composition and constitution are known. 
Most probably they form a combination with the substance 
acted on (the substrate) as a result of which there is a rear- 
rangement of the atoms in such a way that new compounds 
are formed, nearly always at least two, and the enzyme is 
at the same time set free. It is rather remarkable that 
chiefly optically active substances are split up by enzymes 
and where two modifications exist it is usually the dextro- 124 
PHYSIOLOGICAL ACTIVITIES 
rotatory one which is attacked. No single enzyme attacks 
both. This probably means that the structure of the 
enzyme corresponds to that of the substrate, "fits it as a 
key fits a lock," as Emil Fischer says. 
The production of enzymes is by no means restricted to 
bacteria since all kinds of living cells that have been inves- 
tigated have been shown to produce them and presumably 
all living cells do. Hence the number of different kinds of 
enzymes and of substances acted upon is practically unlim- 
ited. Nevertheless they may be grouped into a compara- 
tively few classes based on the general character of the 
change brought about by them. 
I. Class I is the so-called "splitting" enzymes whose 
action is for the most part hydrolytic, that is, the substance 
takes up water and then splits into compounds that were ap- 
parently constituents of the original molecule. As examples 
may be mentioned diastase, the enzyme first discovered, 
which changes starch into a malt-sugar, hence is more com- 
monly called amylase1 (starch-splitting enzyme); invertase,1 
which splits cane-sugar into dextrose and levulose: C12H22O11 
+ H2O = CbH^Ob + CeH^Os. Lipase1 or a fat-splitting 
enzyme, which decomposes fat into glycerin and fatty acid: 
CaHsCOCnH^-xOs + 3H2O = C3H8(OH)3 + 3CnH2nO2. 
Fat Glycerin Fatty acid 
Proteases, which split up proteins into proteoses and pep- 
tones. 
Other classes of "splitting enzymes" break up the prod- 
ucts of complex protein decomposition, such as proteoses, 
peptones and amino-acids. A variety of the "splitting 
enzymes" is the group of 
"Coagulases" or coagulating enzymes as the rennet (lab, 
chymosin) which curdles milk; fibrin ferment (thrombin, 
thrombase) which causes the coagulation of blood. These 
apparently act by splitting up a substance in the fluids 
mentioned, after which splitting one of the new products 
formed combines with other compounds present (usually a 
1 It will be noted that the names of enzymes (except some of those first 
discovered) terminate in ase which is usually added to the stem of the name 
of the substance acted on, though sometimes to a word which indicates the 
substance formed by the action, as lactacidase, alcoholase. PRODUCTION OF ENZYMES 
125 
mineral salt, and in the cases mentioned a calcium salt) to 
form an insoluble compound, the curd or coagulum. 
Another variety is the "activating" enzymes or "kinases" 
such as the enterokinase of the intestine. The action here 
is a splitting of the zymogen or mother substance or form in 
which the enzyme is built up by the cell so as to liberate the 
active enzyme. 
Of a character quite distinct from the splitting enzymes 
are 
II. The zymases. Their action seems to be to cause a 
"shifting on rearrangement of the carbon atoms" so that new 
compounds are formed which are not assumed to have 
been constituents of the original molecule. Most commonly 
there is a closer combination of the carbon and oxygen 
atoms, frequently even the formation of CO2 so that con- 
siderable energy is thus liberated. Examples are the zymase 
or alcoholase of yeast which converts sugar into alcohol and 
carbon dioxide; CeHnOs = 2C2H6O + 2CO2: also urease, 
which causes the change of urea into ammonia and carbon 
dioxide. Another common zymase is the lactacidase in 
lactic acid fermentation. 
III. Oxidizing enzymes also play an important part in 
many of the activities of higher plants and animals. Among 
the bacteria this action is illustrated by the formation of 
nitrites, nitrates and sulphates and the oxidation of alcohol 
to acetic acid as already described. 
IV. Reducing enzymes occur in many of the dentrifying 
bacteria and in those which liberate H2S from sulphates. 
A very widely distributed reducing enzyme is "catalase" 
which decomposes hydrogen peroxide. 
As previously stated, most of the physiological activities 
of bacteria are due to the enzymes that they produce. It is 
evident that for action to occur on substances which do 
not diffuse into the bacterial cell-starches, cellulose, com- 
plex proteins, gelatin-the enzymes must pass out of the 
bacterium and consequently may be found in the surround- 
ing medium. . Substances like sugars, peptones, alcohol, 
which are readily diffusible, may be acted on by enzymes 
retained within the cell bodv. In the former case the en- 126 
PHYSIOLOGICAL ACTIVITIES 
zymes are spoken of as extra-cellular or "exo-enzymes, " 
and in the latter as intra-cellular or " endo-enzymes." The 
endo-enzymes and doubtless also the exo-enzymes may after 
the death of the cell digest the contents to a greater or less 
extent and thus furnish substances that are not otherwise 
obtainable. This process of "self-digestion" is known tech- 
nically as " autolysis. 
A distinction was formerly made between "organized" 
and "unorganized ferments." The former term was ap- 
plied to the minute living organisms, bacteria, yeasts, molds, 
etc., which bring about characteristic fermentative changes, 
while the latter term was restricted to enzymes as just 
described. Since investigation has shown that the changes 
ascribed to the "organized ferments" are really due to their 
enzymes, and that, enzymes are probably formed by all liv- 
ing cells, the distinction is scarcely necessary at present. 
PRODUCTION OF TOXINS. 
The injurious effects of pathogenic bacteria are due in 
large part to the action of these substances, which in many 
respects bear a close relationship to enzymes. The chemical 
composition is unknown since no toxin has been prepared 
"pure" as yet. It was formerly thought that they were 
protein in character, but very pure toxins have been pre- 
pared which failed to show the characteristic protein reac- 
tions. It is well established that they are complex sub- 
stances, of rather large molecule and are precipitated by 
many of the reagents which precipitate proteins. Toxins 
will be further discussed in Chapter XXVII. It will be 
sufficient at this point to enumerate their chief peculiarities 
in order to show their marked resemblance to enzymes. 
1. Toxins are dead organic chemical substances. 
2. They are always produced by living cells. 
3. They are active poisons in very small quantities.1 
4. Their action is specific in that each toxin acts on a 
particular kind of cell. The fact that a so-called toxin acts 
1 Tetanus toxin is about 120 times as poisonous as strychnin, both of which 
act on the same kind of nerve cells. PRODUCTION OF TOXINS 
127 
on several different kinds of cells, possibly indicates a mix- 
ture of several toxins, or action on the same substance in the 
cells. 
5. Toxins are very sensitive to the action of injurious 
agencies such as heat, light, etc., and in about the same 
measure that enzymes are, though as a rule they are some- 
what more sensitive or "labile." 
6. Toxins apparently have maxima, optima, and minima 
of temperature for their action, as shown by the destructive 
effect of heat and by the fact that a frog injected with te- 
tanus toxin and kept at 20° shows no indication of poison, 
but if the temperature is raised to 37°, symptoms of poison- 
ing are soon apparent. Cold, however, does not destroy a 
toxin. 
7. When properly introduced into the tissues of animals 
they cause the body cells to form antitoxins (Chapter 
XXVII) which are capable of preventing the action of the 
toxin in question. 
8. The determining test for a toxin is its action on a living 
cell. 
It is true that enzymes are toxic, as are also various 
foreign proteins, when injected into an animal, but in much 
large doses than are toxins. 
A marked difference between enzymes and toxins is that 
the former may bring about a very great chemical change 
and still may be recovered from the mixture of substances 
acted on and produced, while the toxin seems to be perma- 
nently used up in its toxic action and cannot be so recovered. 
Toxins seem very much like enzymes whose action is restricted 
to living cells. 
Just as enzymes are probably produced by all kinds of 
cells and not by bacteria alone, so toxins are produced 
by other organisms. Among toxins which have been care- 
fully studied are ricin, the poison of the castor oil plant 
(Ric inns communis'); abrin of the jequirity bean (Abrus pre- 
catorius'); robin of the common locust (Robinia pseudacacia'); 
poisons of spiders, scorpions, bees, fish, snakes and sala- 
manders. 
It has been stated that some enzymes are thrown out 
from the cell and others are retained within the cell. The 128 
DISINFECTIONS TERI LIZ A TION 
same is true of toxins, hence we speak of exo-toxins or toxins 
excreted from, and endo-toxins or toxins retained within the 
cell. Among the pathogenic bacteria there are very few 
which secrete toxins when growing outside the body. Clostri- 
dium tetani or lockjaw bacillus, Corynebacterium diphtherias or 
the diphtheria bacillus, Clostridium botulinum or a bacillus 
causing a type of food poisoning, Pseudomonas pyocyanea or 
the blue pus bacillus are the most important. Other patho- 
genic bacteria do not secrete their toxins under the above 
conditions, but only give them up when the cell is disinte- 
grated either within or outside the body. For the reason 
that endotoxins are therefore difficult to obtain, their char- 
acteristics have not.been much studied. The description of 
toxins as above given is intended.to apply to the exo-toxins 
of bacteria, sometimes spoken of as true toxins, and to the 
vegetable toxins (phytotoxins) which resemble them. 
The snake venoms and probably most of the animal toxins 
(zootoxins) are very different substances. (See Chapter 
XXIX.) 
CAUSATION OF DISEASE. 
This subject belongs properly in special pathogenic bac- 
teriology. It will be sufficient to indicate that bacteria may 
cause disease in one or more of the following ways: (a) 
blocking circulatory vessels, either blood or lymph, directly 
or indirectly; (b) destruction of tissue; (c) production of 
non-specific poisons (ptomaines, bases, nitrites, acids, gases, 
etc.); (d) production of specific poisons (toxins). 
ANTIBODY FORMATION. 
Bacteria cause the formation of specific " antibodies" when 
properly introduced into animals. This must be considered 
as a physiological activity since it is by means of substances 
produced within the bacterial cell that the body cells of 
animals are stimulated to form antibodies. (See Chapters 
XXVI-XXIX.) STAINING 
129 
STAINING. 
The reaction of bacteria to various stains is dependent on 
their physico-chemical structure and hence is a result of 
physiological processes, but is best discussed separately 
(Chapter XIX). 
CULTURAL CHARACTERISTICS. 
The same is true of the appearance and growth on differ- 
ent culture media. (Chapter XX.) CHAPTER XIII. 
DISINFECTION-STERILIZATION- 
DISINFECTANTS. 
The discussion of the physiology of bacteria in the pre- 
ceding chapters has shown that a number of environmental 
factors must be properly correlated in order that a given 
organism may thrive. Conversely, it can be stated that any 
one of these environmental factors may be so varied that 
the organism -will be more or less injured, may even be 
destroyed by such variation. It has been the thorough study 
of the above-mentioned relationships which has led to prac- 
tical methods for destroying bacteria, for removing them or 
preventing their growth when such procedures become 
necessary. 
The process of killing all the living organisms or of remov- 
ing them completely is spoken of as disinfection or as sterili- 
zation, according to circumstances. Thus the latter term is 
applied largely in the laboratory, while the former more 
generally in practice outside the laboratory. So also disin- 
fection is most commonly done with chemical agents and 
sterilization by physical means, though exceptions are 
numerous. The original idea of disinfection was the de- 
struction of " infective " organisms, that is, organisms produc- 
ing disease in man or animals. A wider knowledge of bac- 
teriology has led to the application of the term to the destruc- 
tion of other organisms as well. Thus the cheese-maker 
" disinfects " his curing rooms to prevent abnormal ripening of 
cheese, and the dairy-worker "disinfects" his premises to 
avoid bad flavors, abnormal changes in the butter or milk. 
Sterilization is more commonly applied to relatively small 
objects and disinfection to larger ones. Thus in the laboratory, 
instruments, glassware, apparatus, etc., are "sterilized" 
while desks, walls and floors are "disinfected." The sur- PHYSICAL AGENTS 
131 
geon "sterilizes" his instruments, but " disinfects " his 
operating table and room. The dairy-workers mentioned 
above sterilize their apparatus, pails, milk bottles, etc. 
Evidently the object of the two processes is the same, remov- 
ing or destroying living organisms, the name to be applied 
is largely a question of usage and circumstances. Any agent 
which is used to destroy microorganisms is called a "disin- 
fectant." Material freed from living organisms is "ster- 
ile." 
The process of preventing the growth of organisms without 
reference to whether they are killed or removed is spoken 
of as "antisepsis," and the agent as an antiseptic. Hence a 
mildly applied "disinfectant" becomes an "antiseptic," 
though it does not necessarily follow that an "antiseptic" 
may become a disfectant when used abundantly. Thus 
strong sugar solutions prevent the development of many 
organisms, though they do not necessarily kill them. 
Asepsis is a term which is restricted almost entirely to 
surgical operations and implies the taking of such precau- 
tions that foreign organisms are kept out of the field of opera- 
tion. Such an operation is an aseptic one, or performed 
aseptically. 
A "deodorant or deodorizer" is used to destroy or remove 
an odor and does not necessarily have either antiseptic or 
disinfectant properties. 
The agents which are used for the above-described pro- 
cesses may be conveniently divided into physical agents and 
chemical agents. 
PHYSICAL AGENTS. 
1. Drying.-This is doubtless the oldest method for pre- 
venting the growth of organisms, and the one which is used 
on the greatest amount of material at the present time. A 
very large percentage of commercial products is preserved 
and transported intact because the substances are kept free 
from moisture. In the laboratory many materials which 
are used as food for bacteria (see Chapter XVI) "keep" 
because they are dry. Nevertheless, drying should be con- 132 
DI SI NF EC TION-S TERI LIZ A TION 
sidered as an antiseptic rather than as a disinfectant process. 
While it is true that the complete removal of water would 
result in the death of all organisms this necessitates a high 
temperature, in itself destructive, and does not occur in 
practice. Further, though many pathogenic bacteria arc 
killed by drying, many more, including the spore formers, 
are not. Hence drying alone is not a practical method of 
disinfecting. 
2. Heat.-The use of heat in some form is one of the 
very best means for destroying bacteria. It may be made 
use of by combustion, or burning, as direct exposure to the 
open flame, as dry heat (hot air), or as moist heat (boiling 
water or steam). Very frequently in veterinary practice, 
Fig. 77.-A small laboratory hot-air sterilizer. 
especially in the country, occasionally under other condi- 
tions, the infected material is best burned. This method is 
thoroughly effective and frequently the cheapest in the end. 
Wherever there are no valid objections it should be used. 
Exposure to the open flame is largely a laboratory procedure 
to sterilize small metallic instruments and even small pieces 
of glassware. It is an excellent procedure in postmortem 
examinations to burn off the surface of the body or of an PHYSICAL AGENTS 
133 
organ when it is desired to obtain bacteria from the interior 
free from contamination with surface organisms. 
Dry Heat.-Dry heat is not nearly so effective as moist 
heat as a sterilizing agent. The temperature must be higher 
and continued longer to accomplish the same result. Thus 
a dry heat of 150° for thirty minutes is no more efficient than 
steam under pressure at 115° for fifteen minutes. Various 
forms of hot-air sterilizers are made for laboratory purposes 
(Fig. 77). On account of the greater length of time required 
for sterilization their use is more and more restricted to 
objects which must be used dry, as in blood and serum 
work, for example. In practice the use of hot air in disin- 
fecting plants is now largely restricted to objects which 
might be injured by steam, as leather goods, furs, and cer- 
tain articles of furniture, but even here chemical agents are 
more frequently used. 
Moist Heat.-Moist heat may be applied either by boil- 
ing in water or by the use of steam at air pressure, or, for 
rapid work and on substances that would not be injured, 
by steam under pressure. Boiling is perhaps the best house- 
hold method for disinfecting all material which can be so 
treated. The method is simple, can always be made use of, 
and is universally understood. It must be remembered that 
all pathogenic organisms, even their spores, are destroyed 
by a few minutes' boiling. The process may be applied to 
more resistant organisms, such as are met with in canning 
vegetables, though the boiling must be continued for several 
hours, or what is better, repeated on several different days. 
This latter process, known as "discontinuous sterilization," 
or "tyndallization" must also be applied to substances, 
which would be injured or changed in composition by too 
long-continued heating, such as gelatin, milk, and certain 
sugars. In the laboratory such materials are boiled or sub- 
jected to steaming steam for half an hour on each of three 
successive days. In canning vegetables the boiling should 
be from one to two hours each day. The principle involved 
is that the first boiling destroys the growing cells, but not 
all spores. Some of the latter germinate by the next day 
and are then killed by the second boiling and the remainder 134 
DI SI NF EC TION-S TERI LIZ A TION 
develop and are killed on the third day. Occasionally a 
fourth boiling is necessary. It is also true that repeated 
heating and cooling is more destructive to bacteria than 
continuous heating for the same length of time, but the 
development of the spores is the more important factor. 
Discontinuous heating may also be used at temperatures 
below the boiling-point for the sterilization of fluids like 
blood serum which would be coagulated by boiling. In 
this case the material is heated at 55° to 56° for one hour, 
but on each of seven to ten successive days. The inter- 
mittent heating and cooling is of the same importance as 
the development of the spores in this case. (Better results 
are secured with such substances by collecting them asep- 
tically in the first place.) 
Fig. 78.-The Arnold steam sterilizer for laboratory use 
Steam.-Steam is one of the most commonly employed 
agents for sterilization and disinfection. It is used either 
as "streaming steam" at air pressure or confined under 
pressure so that the temperature is raised. For almost all 
purposes where boiling is applicable streaming steam may PHYSICAL AGENTS 
135 
be substituted. It is just as efficient and frequently more 
easily applied. The principle of the numerous forms of 
"steam sterilizers" (Fig. 78) is essentially the same. There 
Fig. 79.-Vertical gas-heated lab- 
oratory autoclave. 
Fig. 80.-Horizontal gas-heated 
laboratory autoclave. 136 
DISINFECTIONS TERI LIZ A TION 
is a receptacle for a relatively small quantity of water and 
means for conducting the steam generated by boiling this 
water to the objects to be treated, which are usually placed 
immediately above the water. Surgical instruments may 
be most conveniently sterilized by boiling or by steaming in 
especially constructed instrument sterilizers. If boiled, the 
addition of carbonate of soda, about 1 per cent., usually 
prevents injury. 
Fig. 81.-A battery of two horizontal autoclaves in one of the author's 
student laboratories. Steam is furnished direct from the University central 
heating plant. 
Steam under pressure affords a much more rapid and cer- 
tain method of destroying organisms. Fifteen to twenty 
pounds pressure corresponding to temperatures of 121° to 
125° is commonly used. Variations depend on the bulk and 
nature of the material. Apparatus for this purpose may now PHYSICAL AGENTS 
137 
be obtained from sizes as small as one or two gallons up to 
huge structures which will take one or two truckloads of 
material (Figs. 79-91). The latter type is in common use 
in canning factories, dairy plants, hospitals, public institu- 
tions, municipal and governmental disinfecting stations. 
Very frequently there is an apparatus attached for pro- 
ducing a vacuum both to exhaust the air before sterilizing, 
so that the steam penetrates much more quickly and thor- 
oughly and for removing the vapor after sterilizing, thus 
hastening the drying out of the material disinfected. 
Fig. 82.-A "process kettle" (steam-pressure sterilizer) used in canning. 
Diameter, 40 inches; height, 72 inches. 
The smaller types of pressure sterilizers are called "auto- 
claves" and have become indispensable in laboratory work. 
Fifteen pounds pressure maintained for fifteen minutes is 
commonly sufficient for a few small objects. For larger 
masses much longer time is needed. The author found that 138 
DI SI NF EC TION-S TERI LIZ A TION 
in an autoclave of the type shown in Fig. 81 it required ten 
minutes for 500 cc. of water at 15 pounds pressure to reach 
Fig. 83.-Horizontal steam chest used in canning. Height, 32 inches; width, 
28 inches; length, 10 feet. 
a temperature of 100°, starting at room temperatures, 20° to 
25°. Autoclaves may be used as simple steam sterilizers by 
leaving the escape valves open so that the steam is not 
confined, hence they have largely replaced the latter.1 
1 In the author's laboratory in the past ten years all sterilization except 
those few objects in blood and serum work which must be dry, has been done 
in autoclaves of the type shown in Fig. 81 which are supplied with steam 
from the University central heating plant. A very great saving of time is 
thus secured. PHYSICAL AGENTS 
139 
A process closely akin to sterilization by heat is 'pasteur- 
ization. This means the heating of material at a tempera- 
Fig. 84.-A battery of horizontal rectangular steam chests in actual use in a canning factory. 
ture and for a time which will destroy the actively growing 
bacteria but not the spores. The methods for doing this 140 
DISINFECTIONS TERI LIZ A TION 
vary but are essentially two in principle. 1. The material 
in small quantities in suitable containers (bottles) is placed 
Fig. 85.-A battery of cylindrical process kettles in actual use in a canning factory. 
in the apparatus; the temperature is raised to 60° to 65° 
and maintained for twenty to thirty minutes and then the PHYSICAL AGENTS 
141 
whole is cooled (beer, wine, grape juice, bottled milk) (Figs. 
92, 93 and 94). 
Fig. 86.-A steam chamber used in government disinfection work. Size, 
4 feet 4 inches x 5 feet 4 inches x 9 feet. 
Fig. 87.-Circular steam chamber used, in government disinfection work. 
54 inches in diameter. 
2. Pasteurizing machines are used and the fluid flows 
through continuously. In one type the temperature is 142 
DISINFEC TION-S TERI LIZ A TION 
Fig. 88.-Portable steam chamber used in government disinfection work 
Fig. 89.-Steam chambers on deck of the U. S. quaratine station barge 
"Defender." PHYSICAL AGENTS 
143 
raised to 60° and by "retarders" is kept at this temperature 
for twenty to thirty minutes (Figs. 95 to 98). In another 
Fig. 90'-Steam chambers in hold of U. S. quarantine station barge "Pro- 
tector. ' ' Disinfected space. 
Fig. 91.-Municipal disinfecting station, Washington, D. C. Fig. 92.-A pasteurizer for milk in bottles. 
Fig. 93.-A pasteurizer for grape juice, cider, etc., in bottles. PHYSICAL AGENTS 
145 
type the temperature is raised to as high as 85° for a few 
seconds only, "flash process" (Fig. 99), and then the 
Fig. 94.-A pasteurizer for beer in bottles. 
Fig. 95.-A continuous milk pastuerizer. 146 
DI SI NF EC TION-S TERI LIZ A TION 
material is rapidly cooled. It is certain that all pathogenic 
microorganisms, except the very few spore formers in that 
Fig. 9S.-A pasteurizer for cream to be used in making ice-cream. 
stage, are killed by proper pasteurization. The process is 
largely employed in the fermentation and dairy industries. Fig. 97.-A continuous milk pasteurizer with holder; capacity 1500 
pounds per hour. A, pasteurizer-the milk flows in tubes inside of a jacket 
of water heated to the proper temperature; B, holder; C, water cooler; D, 
brine cooler. 
Fig. 98.-A continuous pasteurizing plant in operation. Similar to Fig. 
97 but larger. Capacity, 12,000 pounds per hour. A, pasteurizer; B, seven 
compartment holder; C, D, coolers. 148 
DISINFECTIONS TERI LIZ A TION 
3. Cold.-That cold is an excellent antiseptic is illustrated 
by the general use of refrigerators and "cold storage." 
Numerous experiments have shown that although many 
pathogenic organisms of a given kind are killed by tempera- 
tures below freezing, not all of the same kind are, and many 
kinds are only slightly affected. Hence cold cannot be con- 
sidered a practical means for disinfection. 
Fig. 99.-A "flash process" pasteurizing outfit, with holder. A, flash 
pasteurizer; B, holder; C, cooler. 
4. Light.-It has been stated (p. 75) that light is destruc- 
tive to bacteria, and the advisability of having well-lighted 
habitations for men and animals has been mentioned. The 
practice of "sunning" bedclothing, hangings and other large 
articles which can scarcely be disinfected in a more con- 
venient way is the usual method of employing this agent. 
Drying and the action of the oxygen of the air assist the 
process to some extent. Undoubtedly large numbers of 
pathogenic organisms are destroyed under natural condi- 
tions by the combined effects of drying, direct sunlight and PHYSICAL AGENTS 
149 
oxidation, but it should not be forgotten that a very slight 
protection will prevent the action of light (Figs. 100 and 101). 
5. Osmotic Pressure.-Increase in the concentration of 
substances in solution is in practical use as an antiseptic 
procedure. Various kinds of "sugar preserves," salt meats 
and condensed milk are illustrations. It must be remem- 
bered that a similar increase in concentration occurs when 
Fig. 100.-Effect of light on bacteria. X ho- The plate was inoculated 
in the usual way. A letter H of black paper was pasted on the bottom. 
The plate was then exposed for four hours to the sun in January outside the 
window and then incubated. The black paper protected the bacteria. Out- 
side of it they were killed except where they happened to be in large masses. 
Hence the letter shows distinctly. (Student preparation.) 
many substances are dried, and is probably as valuable in 
the preservative action as the loss of water. That the pro- 
cess cannot be depended on to kill even pathogenic organ- 
isms is shown by finding living tubercle bacilli in condensed 
milk. The placing of bacteria in water or in salt solution 
in order to have them die and disintegrate (greatly aided 
by vigorous shaking in a shaking machine) ("autolysis," 150 
DI SIN FEC TI0N-S TERI LIZ A TI0N 
p. 1?6) is a laboratory procedure to obtain cell constituents. 
It is not a practical method of disinfection, however. 
6. Electricity.-Electricity, though not in itself injurious 
to bacteria, is used as an indirect means for destroying bac- 
teria in a practical way. This is done by electrical produc- 
tion of some substance which is destructive to bacteria as 
in ozone water purification (Petrograd, Florence, and else- 
Fig. 101.-Effect of light on bacteria. X Mo- This plate was treated 
exactly as the plate in Fig. 100, except that the letter is L, and that it was 
exposed inside the window and wire screen. The window was plate glass. 
It is evident that few of the bacteria were killed, since the letter L is barely 
outlined. The exposure was at the same time as the plate in Fig. 100. (Stu- 
dent preparation.) 
where), or the use of ultra-violet rays for the same purpose 
(Marseilles, Paris) and for treatment of certain disease con- 
ditions. Electricity might be used as a source of heat for 
disinfecting purposes should its cheapness justify it. It has 
also been used in the preservation of meats to hasten the 
'penetration of the salt and thus reduce the time of pickling. 
Electrolyzed sea water has been tried as a means of flushing PHYSICAL AC ENTS 
151 
Fig. 102.-An electric milk purifier (pasteurizer). The milk flowing from 
cup to cup completes the circuit when the current is on. The effect is cer- 
tainly a heat effect. Sparking occurs at the lips of the cups. 
Fig. 103.-One of the ten filter beds of the Columbus water filtration plant 
with the filtering material removed. Sand is the filtering material. All 
of the beds together have a capacity of 30,000,000 gallons daily. 152 
DISINFECTIONS TERI LIZ A TION 
and disinfecting streets, but it is very doubtful if the added 
expense is justified by any increased benefit. A number of 
electric devices have been put forth for various sterilizing 
and disinfecting purposes and doubtless will continue to be, 
but everyone should be carefully tested before money is 
invested in it.1 
Fig. 104.-Suction filtration. A, Berkefeld filter in glass cylinder con- 
taining the liquid to be filtered; B, sterile flask to receive the filtrate as it is 
drawn through; C, water pump; D, manometer, convenient for detecting 
leaks as well as showing pressure; E, bottle for reflux water. 
7. Filtration.-Filtration is a process for rendering fluids 
sterile by passing them through some material which will 
'The author has tested an "electric milk purifier" (Fig. 102) which was 
as efficient as a first-class pasteurizer and left the milk in excellent condi- 
tion both chemically and as far as "cream line" was concerned. The cost 
of operation as compared with steam will depend on the price of electricity. PHYSICAL AGENTS 
153 
hold back the bacteria. It is used on a large scale in the 
purification of water for sanitary or manufacturing reasons 
(Fig. 103). Air is also rendered "germ free" in some surg- 
ical operating rooms, "serum laboratories" and breweries by 
filtration. In the laboratory it is a very common method of 
sterilizing liquids which would be injured by any other 
process. The apparatus consists of a porous cylinder with 
Fig. 105.-Pressure filtration. A, cylinder which contains the filter candle; 
B, cylinder for the liquid to be filtered; C, sterile flask to receive the filtrate; 
D, air pump to furnish pressure. 
proper devices for causing the liquid to pass through either 
by suction (Fig. 104) where the pressure will be only one 
atmosphere (approximately 15 pounds per square inch), 
or by the use of compressed air at any desired pressure (Fig. 
105). The two main types of porous cylinders ("filter 154 
DISINFEC TION-S TERI LIZ A TION 
candles," "bougies") are the Pasteur-Chamberland (Fig. 
106) and the Berkefeld. The former are made of unglazed 
porcelain of different degrees of fineness, the latter of dia- 
tomaceous earth (Fig. 107) The Mandler filter of this same 
material is now manufactured in the United States and is 
equal if not superior to the Berkefeld. The designs of com- 
plete apparatus are numerous. 
8. Burying.-This is a time-honored method of disposing 
of infected material of all kinds and at first thought might 
not be considered a means of disinfection. As a matter of 
fact, under favorable conditions it is an excellent method. 
Fig. 106.-Pasteur-Chamberland filter candles about one-half natural size. 
The infected material is removed. Pathogenic organisms 
tend to die out in the soil owing to an unfavorable environ- 
ment as to temperature and food supply, competition with 
natural soil organisms for what food there is, and the injuri- 
ous effects of the products of these organisms. Care must 
be taken that the burial is done in such a wav that the sur- PHYSICAL AGENTS 
155 
face soil is not contaminated either directly or by material 
brought up from below by digging or burrowing animals, 
insects, worms, or movement of ground water to the sur- 
face. Also that the underground water supply which is 
■drawn upon for use by men or animals is not contaminated. 
Frequently infected material, carcasses of animals, etc., are 
Fig. 107.-Berkefeld filter candles about one-half natural size. 
treated in some way so as to aid the natural process of 
destruction of the organisms present, especially by the use 
of certain chemical agents, as quicklime (see p. 158). CHAPTER XIV. 
DISINFECTION AND STERILIZATION (Continued). 
CHEMICAL AGENTS. 
A very large number of chemical substances might be 
used for destroying bacteria or preventing their growth 
either through direct injurious action or by the effect of 
concentration. Those which are practically useful are rela- 
tively few, though this is one of the commonest methods of 
disinfecting and the word "disinfectant" is frequently 
wrongly restricted to chemical agents. 
Chemical agents act on bacteria in a variety of ways. 
Most commonly there is direct union of the chemical with 
the protoplasm of the cell and consequent injury. Some 
times the chemical is first precipitated on the surface of the 
cell without penetrating at once. If removed soon enough, 
the organism is not destroyed. This is true of bichloride of 
mercury and formaldehyde. If bacteria treated with these 
agents in injurious strength be washed with ammonia or 
ammonium sulphate, even after a time which would other- 
wise result in their failure to grow, they will develop. Some 
chemicals change the reaction of the material in a direction 
unfavorable to growth, and if the change is enough, may 
even kill the bacteria. Some agents remove a chemical 
substance necessary to the growth of the organism and 
hence inhibit it. Such actions are mainly preventive (anti- 
septic) and become disinfectant only after a long time. 
Oxygen.-Oxygen as it occurs in the air is probably not 
injurious to living bacteria but aids them with the excep- 
tion of the anaerobes. In the nascent state especially as 
ELEMENTS. ELEMENTS 
157 
liberated from ozone (O3) hydrogen peroxide (H2O2) and 
hypochlorites (Ca(ClO)2) it is strongly bactericidal. 
Chlorine.-Chlorine is actively disinfectant and is coming 
into use for sterilizing water on a large scale in municipal 
plants (Fig. 108). 
Fig. 108.-Apparatus for sterilizing water with liquid chlorine. 
Iodine finds extended use in aseptic surgical operations 
and antiseptic dressings. Bromine, mercury, silver, gold, 
nickel, zinc and- copper are markedly germicidal in the 
elemental state but are not practical. 158 
DISINFECTION AND STERILIZATION 
COMPOUNDS. 
Calcium Oxide.-Calcium oxide (CaO), quick lime, is an, 
excellent disinfectant for stables, yards, outhouses, etc, 
where it is used in the freshly slaked condition as "white 
wash;" also to disinfect carcasses to be buried. It is very 
efficient against the typhoid bacillus in water, where it is 
much used to assist in the softening. 
Chloride of Lime.-Chloride of lime, bleaching powder, 
which consists of calcium hypochlorite, the active agent, 
and chloride and some unchanged quicklime is one of the 
most useful disinfectants. It is employed to sterilize water 
for drinking purposes on a large scale and to disinfect sewage 
plant effluents. A 5 per cent, solution is the proper strength 
for ordinary disinfection. Only a supply which is fresh or 
has been kept in air-tight containers should be used, as it 
rapidly loses strength on exposure to the air. The active 
agent is nascent oxygen liberated from the decomposition of 
the hypochlorite. 
Sodium Hypochlorite.-Sodium hypochlorite prepared by 
the electrolysis of common salt has been used to some extent. 
Bichloride of Mercury.-Bichloride of mercury, mercuric- 
chloride, corrosive sublimate (HgCL), is the strongest of all 
disinfectants under proper conditions. It is also extremely 
poisonous to men and animals and great care is necessary 
in its use. It is precipitated by albuminous substances and 
attacks metallic objects, hence should not be used in the 
presence of these classes of substances. 
It is used in a strength of one part HgCL to 1000 of water 
for general disinfection. Ammonium chloride or sodium 
chloride, common salt, in quantities equal to the bichloride,, 
or citric acid in one-half of the amount should be added in 
making large quantities of solution or for use with albumin- 
ous fluids to prevent precipitation of the mercury (Fig. 109). 
None of the other metallic salts are of value as practical 
disinfectants aside from their use in surgical practice. In 
this latter class come boric acid, silver nitrate, potassium 
permanganate. The strong mineral acids and alkalies are, 
of course, destructive to bacteria, but their corrosive effect ORGANIC COMPOUNDS 
159 
excludes them from practical use, except that "lye washes" 
are of value in cleaning floors and rough wood-work, but 
even here better disinfection can be done more easily and 
safelv. 
Fig. 109.-Tanks for bichloride of mercury, government quarantine dis- 
infecting plant. 
ORGANIC COMPOUNDS. 
Carbolic Acid or Phenol.-Carbolic acid or phenol (C6H5 
OH) is one of the commonest agents in this class. It is used 
mostly in 5 per cent, solution as a disinfectant and in 0.5 
per cent, solution as an antiseptic. For use in large quanti- 
ties the crude is much cheaper and, according to some experi- 
menters, even more active than the pure acid, owing to the 
cresols it contains. The crude acid is commonly mixed with 
an equal volume of commercial sulphuric acid and the mix- 
ture is added to enough water to make a 5 per cent, dilution, 
which is stronger than either of the ingredients alone in 5 
per cent, solution. 
Cresols.-The cresols (C6H4CH3OH, ortho, meta and 
para), coal-tar derivatives, as phenol, are apparently more 
powerful disinfectants. A great number of preparations 160 
DISINFECTION AND STERILIZATION 
containing them have been put on the market. Creolin is 
one which is very much used in veterinary practice and 
forms a milky fluid with water, while lysol forms a clear 
frothy liquid owing to the presence of soap. Both of these 
appear to be more active than carbolic acid and are less 
poisonous and more agreeable to use. They are used in 2 
to 5 per cent, solution. 
Alcohol.-Ordinary (ethyl) alcohol (C2H5OH) is largely 
used as a preservative, also as a disinfectant for the body 
surface, hands, and arms. Experiments show that alcohol 
of 70 per cent, strength is most strongly bactericidal and 
that absolute alcohol is very slightly so. 
Soap.-Experimenters have obtained many conflicting 
results with soaps when tested on different organisms, as 
is to be expected from the great variations in this article. 
Miss Vera McCoy in the author's laboratory carried out 
experiments with nine commercial soaps-Ivory, Naphtha, 
Packer's Tar, Grandpa's Tar, Balsam Peru, A. D. S. Car- 
bolic, German Green, Dutch Cleanser, Sapolio-and ob- 
tained abundant growth from spores otBacillusanthracis,tvom 
Bacterium coli and from Staphylococcus pyogenes aureus in 
all cases even when the organisms had been exposed twenty- 
four hours in 5 per cent, solutions. From these results and 
from the wide variations reported in the literature it is clear 
that soap solutions alone cannot be depended on as disinfec- 
tants. Medicated soaps do not appear to offer any advan- 
tages in this respect. The amount of the disinfectant which 
goes into solution when the soap is dissolved is too small to 
have any effect. 
Formaldehyde.-Formaldehyde (HCHO) is perhaps the 
most largely used chemical disinfectant at the present time. 
The substance is a gas but occurs most commonly in com- 
merce as a watery solution containing approximately 40 
per cent, of the gas. This solution is variously known as 
formalin, formol, and formaldehyde solution. The first two 
names are patented and the substance under these names 
usually costs more. It is used in the gaseous form for disin- 
fecting closed spaces of all kinds to the exclusion of most 
other means today. A great many types of formalin gen- ORGANIC COMPOUNDS 
161 
erators have been devised. The gas has little power of 
penetration and all material to be reached should be exposed 
as much as possible. The dry gas is almost ineffective, so 
that the objects must be moistened or vapor generated 
along with the gas. A common method in use is to avoid 
expensive generators by pouring the formaldehyde solution 
on permanganate of potash crystals placed in a vessel 
removed from inflammable objects on account of the heat 
developed which occasionally sets the gas on fire. The 
formalin is used in amounts varying from 20 to 32 ounces 
to 8^ to 13 ounces of permanganate to each 1000 cubic feet 
of space. This method is expensive since one pint (16 
ounces) of formalin is sufficient for each 1000 cubic feet, and 
since the permanganate is an added expense. Dr. Dixon, 
Commissioner of Health of Pennsylvania, recommends the 
following mixture to replace the permanganate, claiming 
that it works more rapidly and is less expensive and just 
as efficient: 
1. Sodium bichromate, ten ounces. 
2. Saturated solution of formaldehyde, sixteen ounces. 
3. Common sulphuric acid, one and a half ounces. 
Two and three are mixed together and when cool are 
poured on the bichromate which is placed in an earthen- 
ware jar of a volume about ten times the quantity of fluid 
used. The quantities given are for each 1000 cubic feet of 
space. 
A very simple method is to cause the formalin, diluted 
about twice with water to furnish moisture enough, to drop 
by means of a regulated "separator funnel" on a heated 
iron plate. The dropping should be so regulated that each 
drop is vaporized as it falls. The plate must have raised 
edges, pan-shaped, to prevent the drops rolling off when they 
first strike the plate. Formaldehyde has no corrosive (except 
on iron) or bleaching action, and is the most nearly ideal 
closed space disinfectant today. In disinfecting stations it 
is made use of in closed sterilizers such as were described 
under steam disinfection particularly in connection with 
vacuum apparatus. It is also used in solution as a pre- 
servative and as a disinfectant. The commonest strength is 162 
DISINFECTION AND STERILIZATION 
2 or 3 per cent, of formalin or 0.8 to 1.2 per cent, of the 
formaldehyde gas. As an antiseptic it is efficient in dilu- 
tions as high as 1 to 2000 of the gas. It is very irritant to 
mucous membranes of most individuals. 
Anilin Dyes.-Some of the anilin dyes show remarkable 
selective disinfectant and antiseptic action on certain kinds 
of bacteria with little effect on others. This has been well 
shown by Churchman in his work on Gentian Violet. This 
dye inhibits the growth of Gram positive organisms up to a 
dilution of one part in 300,000 while for Gram negative or- 
ganisms it is without effect even in saturated solution. 
This is nicely shown in the accompanying illustration. 
Fig. 110.-The lower half of the plate is plain agar medium, the upper 
half the same medium plus gentian violet to make one part in 300,000. The 
Gram positive organism is on the right and the Gram negative on the left. 
Streak inoculations were made across both media. 
This inhibiting effect of anilin dyes is taken advantage of in 
several methods of isolating bacteria (Chapter XVIII). 
In addition to the above-discussed disinfectants a large 
number of substances, particularly organic, are used in 
medicine, surgery, dentistry, etc., as more or less strong 
antiseptics, and the lisl is a constantly lengthening one. 
In the laboratory chloroform, H2O2, ether and other vola- 
tile or easily decomposable substances have been used to 
sterilize liquids which could not be treated by heat or by 
filtration. The agent is removed either by slow evapora- ORGANIC COMPOUNDS 
163 
tion or by exhausting the fluid with an air pump. The 
method is not very satisfactory, nor is absolute steriliza- 
tion easily accomplished. It is much better to secure such 
liquids aseptically where possible. CHAPTER XV. 
DISINFECTION AND STERILIZATION (Continued). 
CHOICE OF AGENT. 
The choice of the above-described agents depends on the 
conditions. Evidently a barn is not to be disinfected in the 
same way that a test-tube in the laboratory is sterilized. 
Among the factors to be considered in making a choice are 
the thing to be disinfected or sterilized, its size and nature, 
that is, whether it will be injured by the process proposed, 
cost of the agent, expecially when a large amount of material 
is to be treated. Among the conditions which affect the 
action of all agents the following should be borne in mind 
particularly when testing the disinfecting power of chemical 
agents: 
1. The kind of bacterium to be destroyed, since some are 
more readily killed by a given disinfectant than others, even 
though no spores are present. 
2. The age of the culture. Young bacteria less than twenty- 
four hours old are usually more readily killed than older 
ones since the cell wall is more delicate and more easily 
penetrated, though old growths may be weakened by the 
accumulation of their products and be more easily destroyed. 
3. Presence of spores, since they are much more resistant 
than the growing cells. 
4. Whether the organism is a"good" or "bad" growth,i.e., 
whether it has grown in a favorable environment and hence 
is vigorous, or under unfavorable conditions and hence is 
weak. 
5. The number of bacteria present, since with chemical 
agents the action is one of relative masses. 
6. Nature of the substance in which the bacteria are. Me- 
tallic salts, especially bichloride of mercury, are precipi- STANDARDIZATION OF DISINFECTANTS 
165 
tated by albuminous substances and if employed at all must 
be used in several times the ordinary strength. Solids 
require relatively more of a given solution than liquids. 
7. State of the disinfectant, whether solid, liquid or gas, 
and whether it is ionized or not. Solutions penetrate best 
and are therefore more quickly active and more efficient. 
8. The solvent. Water is the best solvent to use. Strong 
alcohol (90 per cent.+ ) diminishes the effect of carbolic 
acid, formaldehyde and bichloride of mercury. Oil has a 
similar effect. The action is probably to prevent the pene- 
tration of the disinfectant. 
9. Strength of solution. The stronger the solution, the 
more rapid and more certain the action, for the same reason 
as mentioned under 5. In fact, every disinfectant has a 
strength below the lethal at which it stimulates bacterial 
growth. 
10. Addition of salts. Common salt favors the action of 
bichloride of mercury and also of carbolic acid. Other salts 
may hinder by precipitating the disinfectant. 
11. Temperature. Chemical disinfectants, as a rule, fol- 
low' the general law that chemical action increases with the 
temperature, up to the point where the heat of itself is 
sufficient to kill. 
12. Time of action. It is scarcely necessary to point out 
that a certain length of time is necessary for any disinfec- 
tant to act. One may touch a red hot stove and not be 
burned. All the above-mentioned conditions are influenced 
by the time of action. 
STANDARDIZATION OF DISINFECTANTS-"PHENOL 
COEFFICIENT." 
Many attempts have been made to devise standard 
methods for testing the relative strengths of disinfectants.. 
The one most widely used in the United States is the so- 
called "Hygienic Laboratory" method of determining the- 
"phenol coefficient" of the given substance and is a modifi- 
cation of the method originally proposed by Rideal, and 
Walker in England. In this method as proposed by Ander- 166 
DISINFECTI0N AND STERILIZATION 
son and McClintic, formerly of the above laboratory, the 
strengths of the dilution of the disinfectant to be tested 
which kills a culture of Bacterium typhosum in 2^ minutes 
is divided by the strength of the dilution of carbolic acid 
which does the same; and the dilution which kills in 15 
minutes is likewise divided by the corresponding dilution 
of carbolic acid. The two ratios thus obtained are aver- 
aged and the result is the "phenol coefficient." For ex- 
ample 
Phenol 1:80 killed in 2 minutes 
Disinfectant "A" 1:375 " " " " 
Phenol 1:110 " " 15 " 
Disinfectant "A" 1:650 " " " " 
375~ 80=4.69 
6504-110=5.91 
2)10.60 
Average= 5.30= "phenol coefficient." 
Standard conditions of temperature, age of culture, medium, 
reaction, etc., and of making the dilutions and transfers are 
insisted on. Details may be found in the Journal of Infec- 
tious Diseases, 1911, 8, p. 1. 
This is probably as good a method as any for arriving at 
the relative strengths of disinfectants and in the hands of 
any given worker concordant results in comparative tests 
can usually be attained. Experience has shown that the 
results obtained by different workers with the same disin- 
fectant may be decidedly at variance. This is to be expected 
from a knowledge of the factors affecting the action of 
disinfectants above stated and from the known specific 
action of certain disinfectants on certain organisms (com- 
pare anilin dyes, p. 162). 
It seems that the only sure way to test the action of such 
a substance is to try it out in the way it is to be used. It is 
scarcely wise to adopt the "phenol coefficient" method as a 
legal standard method as some states have done. 
PRACTICAL STERILIZATION AND DISINFECTION. 
The methods for sterilizing in the laboratory have been 
discussed and will be referred to again in the next chapter. DISINFECTION AND STERILIZATION 
167 
In practical disinfection it is a good plan always to pro- 
ceed as though spores were present even if the organism is 
known. Hence use an abundance of the agent and apply it 
as long as practicable. Also it is best to secure the chemical 
substances used as such and not depend on patented mixtures 
purporting to contain them. As a rule the latter are more 
expensive in proportion to the results secured. 
Surgical instruments may be sterilized by boiling in water 
for fifteen minutes, provided they are clean, as they should 
be. If dried blood, pus, mucus, etc., are adherent, which 
should never be the case, they should be boiled one-half 
hour. The addition of sodium carbonate (0.5 to 1 per cent.) 
prevents rusting. Surgeons' sterilizers are to be had at 
reasonable prices and are very convenient. Whether the 
instruments are boiled or subjected to streaming steam 
depends on whether the supporting tray is covered with 
water or not. The author finds it a good plan to keep the 
needles of hypodermic syringes in a small wire basket in 
an oil bath. The oil may be heated to 150° to 200° and the 
needles sterilized in a very few minutes. The oil also pre- 
vents rusting. 
Rooms, offices and all spaces which may be readily made 
practically gas-tight are best disinfected by means of for- 
maldehvde bv any of the methods above described (Figs. 
Ill and 112). 
Stables and Barnyards (Mohler): " A preliminary cleaning 
up of all litter is advisable together with the scraping of the 
floor, mangers and walls of the stable with hoes and the 
removal of all dust and filth. All this material should be 
burned since it probably contains the infective agent. Heat 
may be applied to the surfaces, including barnyard, by means 
of a 'cyclone oil burner.' When such burning is impracti- 
cable, the walls may be disinfected with one of the following: 
1. Whitewash 1 gallon + chloride of lime 6 ounces. 
2. Whitewash 1 gallon + crude carbolic acid 7 ounces. 
3. Whitewash 1 gallon + formalin 4 ounces. 
The same may be applied with brushes or, more rapidly, 
sprayed on with a pump; the surface soil of the yard and 
surroundings should be removed to a depth of 5 or 6 inches, 168 
DISINFECTION AND STERILIZATION 
placed in a heap and thoroughly mixed with quicklime. 
The fresh surface of soil thus exposed may be sprinkled with 
a solution of a chemical disinfectant as above described. 
"Portions of walls and ceiling not readily accessible may 
be disinfected by chlorine gas liberated from chloride of 
Fig. 111.-Formaldehyde gen- 
erator used in city work for room 
disinfection. 
Fig. 112.-Government formal- 
dehyde generator. 
lime by crude carbolic acid. This is accomplished by mak- 
ing a cone of 5 or 6 pounds of chloride of lime in the top of 
which a deep crater is made for the placement of from 1 DISINFECTION AND STERILIZATION 
169 
to 2 pints of crude carbolic acid. The edge of the crater is 
thereupon pushed into the fluid, when a lively reaction fol- 
lows. Owing to the heat generated, it is advisable to place 
the chloride of lime in an iron crucible (pot), and to have 
nothing inflammable within a radius of two feet. The num- 
ber and location of these cones of chloride of lime depend 
on the size and structure of the building to be disinfected. 
As a rule it may be stated that chlorine gas liberated from 
the above sized cone will be sufficient for disinfecting 5200 
cubic feet of air space." 
Liquid manure, leachings, etc., where collected are thor- 
oughly disinfected by chloride of lime applied in the pro- 
portion of 2 parts to 1000 of fluid. 
Fig. 113.-Chamber used in government work for formaldehyde disinfec- 
tion. The small cylinder at the side is the generator. 
Vehicles may be thoroughly washed with 2 per cent, for- 
malin solution, or if closed space is available, subjected to 
formaldehyde gas disinfection, after cushions, hangings, etc., 
have been removed and washed with the disinfectant. 
Harness, brushes, combs should be washed with a solution 
of formalin, carbolic acid, or creolin as given under these 
topics. 
Washable articles should be boiled, dropped into disinfec- 
tant solutions as soon as soiled, and then boiled or steamed. 
Unwashable articles-burn all possible. Use formalde- 
hyde gas method in a closed receptacle (Fig. 113). 170 
DISINFECTION AND STERILIZATION 
Stock cars-the method described for stables is applicable 
here. 
Animals, large and small, may have the coat and surface 
of the body disinfected by washing with 1 to 1000 bichloride 
or strong hot soapsuds to which carbolic acid has been added 
to make a 5 per cent, solution; they should then be given a 
good warm bath. 
Frequently time and money are saved by a combination 
of steam and formaldehyde disinfection. This is a regular 
practice in municipal and quarantine disinfection (Fig. 114). 
Fig. 114.-Chamber in actual use at government quarantine station for 
disinfecting baggage and dunnage with steam or formaldehyde or both. 
The small cylinder at the side is the steam formaldehyde generator. 
Persons engaged in disinfection work should wear rubber 
boots, coats and caps which should be washed in a disinfec- 
tant solution and the change to ordinary clothing made in 
a special room so that no infective material will be taken 
away. PART III. 
IHE STUDY OF BACTERIA. 
CHAPTER XVI. 
CULTURE MEDIA. 
The study of bacteria may be taken up for the disciplin- 
ary and pedagogic value of the study of a science; with 
the idea of extending the limits of knowledge; or for the 
purpose of learning their beneficial or injurious actions with 
the object of taking advantage of the former and combating 
■or preventing the latter. 
Since bacteria are classed as plants, their successful study 
implies their cultivation on a suitable soil. A growth of 
bacteria is called a "cuZ/we" and the "soil" or material 
on which they are grown is called a "culture medium." 
In so far as the culture medium is made up in the laboratory 
it is an "artificial culture medium" as distinguished from a 
natural medium. A culture consisting of one kind of bac- 
teria only is spoken of as a "pure culture," and accurate 
knowledge of bacteria depends on obtaining them in "pure 
■culture. " After getting a " pure culture " the special charac- 
teristics of the organism must be ascertained in order to dis- 
tinguish it from others. The discussion of the morphology 
of bacteria in Chapters II, III, and IV shows that the 
morphological structures are too few to separate individual 
kinds. They serve at best to enable groups of similarly 
appearing forms to be arranged. Hence any further differ- 
,entiation must be based on a study of the physiology of the 172 
CULTURE MEDIA 
organism as discussed in the chapters on Physiological 
Activities of Bacteria. 
The thorough study of a bacterium involves, therefore: 
1. Its isolation in pure culture. 
2. Its study with the microscope to determine morpho- 
logical features and staining reactions. 
3. Growth on culture media for determining its physio- 
logical activities as well as morphological characteristics of 
the growths themselves. 
4. Animal inoculations in certain instances. 
5. Special serum reactions in some cases. 
Since isolation in pure culture requires material for grow- 
ing the organism, the first subject to be considered is 
culture media. 
A culture medium for a given bacterium should show the 
following essentials: 
1. It must contain all the elements necessary for the 
growth of the organism except those that may be obtained 
from the surrounding atmosphere. 
2. These elements must be in a form available to the 
organism. 
3. The medium must not be too dry, in order to furnish 
sufficient moisture, for growth and to prevent too great a 
concentration of the different ingredients. 
4. The reaction must be adjusted to suit the particular 
organism dealt with. 
5. There must be no injurious substances present in 
concentration sufficient to inhibit the growth of the organ- 
ism or to kill it. 
Ordinarily, more attention must be paid to the sources of 
the two elements N and C than to the others, for in. general 
the substances used to furnish these two and the water 
contain the other elements in sufficient amount. For very 
exact work on the products of bacteria, synthetic media 
containing definite amounts of chemicals of known composi- 
tion have been prepared, but for most of the work with 
bacteria pathogenic to animals such media are not needed. 
Culture media may be either liquid or solid, or for certain 
purposes may be liquid at higher temperatures and solid at MEAT BROTH 
173 
lower, as indicated later. Liquid media are of value for 
obtaining bacteria for the study of morphology and cell 
groupings and for ascertaining many of the physiological 
activities of the organisms. Solid media are useful for 
studying some few of the physiological activities and espe- 
cially for determining characteristic appearances of the 
isolated growths of bacteria. These isolated growths of 
bacteria on solid media are technically spoken of as "colo- 
nies, " whether they are microscopic in size or visible to the 
unaided eye. 
It is clear that the kinds of culture media used for the 
study of bacteria may be unlimited but the undergraduate 
student will need to use a relatively small number, which 
will be discussed in this section. 
Meat Broth (Bouillon).1-This itself is used as a medium 
and as the basis for the preparation of other solid and liquid 
media. 
Finely ground lean beef is selected because it contains the 
necessary food materials. Fat is not desired since it is a 
poor food for most bacteria and in the further processes of 
preparation would be melted and form an undesirable film 
on the surface of the medium. The meat is placed in a 
suitable container and mixed with about twice its weight of 
cold water (not distilled) and allowed to soak overnight or 
longer. The cold water extracts from the meat water- 
soluble proteins, blood, carbohydrates in the form of dex- 
trose (occasionally some glycogen), nitrogenous extractives 
and some of the mineral salts. The fluid is strained or 
pressed free from the meat. This "meat juice" should now 
be thoroughly boiled, which results in a coagulation of a 
large part of the proteins and a precipitation of some of the 
mineral salts, particularly phosphates of calcium and mag- 
nesium, both of which must be filtered off and the water 
loss restored by adding the proper amount of distilled water. 
The boiling is done at this point because the medium must 
later be heated to sterilize it and it is best to get rid of the 
iThe exact laboratory details for preparing various media are not given 
in this chapter. It is the object to explain the choice of different materials 
and the reasons for the various processes to which they are subjected. 174 
CULTURE MEDIA 
coagulable proteins at once. The proteins thus thrown out 
deprive the medium of valuable nitrogenous food material 
which is replaced by adding about 1 per cent, by weight of 
commercial peptone. It is usual also (though not always 
necessary) to add about 0.5 per cent, by weight of common 
salt which helps to restore the proper concentration of min- 
eral ingredients lost by the boiling. The chlorine is also 
an essential element. The reaction is now determined and 
adjusted to the desired end point, "standardized," as it is 
called. The medium is again thoroughly boiled and filtered 
boiling hot. The adjusting of the reaction and the boiling 
ordinarily cause a precipitate to form which is largely phos- 
phates of the alkaline earths with some protein. The 
filtered medium is collected in suitable containers, flasks or 
tubes, which are plugged with well-fitting non-absorbent 
cotton plugs and sterilized, best in the autoclave for twenty 
minutes at 15 pounds pressure, or discontinuously in stream- 
ing steam at 100°. If careful attention is paid to titration 
and to sufficient boiling where indicated, the meat broth 
prepared as above should be clear, only faintly yellowish in 
color and show no precipitate on cooling. 
The conventional method for standardizing an acid med- 
ium is as follows: Take 5 cc of the medium, add 45 cc of 
distilled water and 1 cc of phenolphthalein as indicator. 
Boil the solution and while still hot run in from a burette 
N/20 NaOH solution until a faint pink color appears. 
From the number of cc of N/20 NaOH used to " neutral- 
ize" the 5 cc of medium it is calculated how many cc of 
N/1 NaOH are necessary to give the desired end reaction 
to the volume of medium which is to be standardized. The 
resulting reaction is expressed as % acid'or alkaline to phenol- 
phthalein. If it is necessary to add to each 100 cc of the 
medium lee of N/l NaOH to make it neutral to phenol- 
phthalein, the reaction is called 1% acid: if to each 100 cc 
of medium there is added 1 cc of N/l alkali in addition to 
the quantity necessary to neutralize, the reaction is called 
1% alkaline. 
In order to obtain a pink color when titrating with this 
indicator not only must the "free acid" be neutralized by MEAT BROTH 
175 
the alkali but also loosely combined acid and any other 
substances present which will combine with the alkali rather 
than with the indicator so that in many media more alkali 
is added than is necessary to neutralize the "free acid," 
i. e., the free H ions present. 
It is well established that the controlling factor in the- 
growth of bacteria in so far as "reaction" is concerned is- 
not the titratable substances present but only the "free acid," 
i. e., the number of free H ions, consequently it is better to 
determine the concentration of H ions and to standardize 
to a definite II ion concentration. Phenolphthalein as shown 
above is not a good indicator for this purpose. 
The H ions present can be determined accurately in all 
cases only by electrolytic methods. The apparatus neces- 
sary is usually relatively expensive and scarcely adapted to 
the use of large classes of students. There are a number of 
indicators each of which will show color changes within 
rather narrow ranges of H ion concentration. Standard- 
ization by the use of these indicators, the "colorimetric- 
method," is recommended by the Society of American 
Bacteriologists and is coming into general use. 
The H ion concentration is ordinarily indicated by the- 
conventional symbol Ph, e. g., the concentration in pure- 
yater -which is regarded as neutral is expressed as Ph 7; of 
normal HC1, Ph 0; of normal NaOH, PH14. The 
figure after PH does not in reality represent the concentra- 
tion of H ions in the solution. This, like the concentration 
of acids, is expressed on the basis of normality, i. e., as 
compared with the concentration of a normal solution 
(1 g. equivalent) of H ions. Concentration of H ions in pure 
water is N/10,000,000, i. e., is \/10,000,000 of the con- 
centration in a normal solution of H ions. Expressed in 
other words, it is the concentration in a normal solution of 
H ions diluted ten million times. 10,000,000 = 10 to the 
7th power = 107. Hence the figure after the Ph indicates 
the logarithm of the number of times the solution is diluted. 
Therefore this number increases with the dilution and the 
larger the figure after the Ph, the less acid the solution is. 176 
CULTURE MEDIA 
Most saprophytic organisms and many parasitic ones 
grow within a wide range of H ion concentration so that 
titration with phenolphthalein gives sufficient accuracy for 
media for such organisms. On the other hand, many 
organisms grow within a very narrow range of H ion con- 
centration, hence accurate standardization to a definite 
H ion concentration is necessary. It is also evident that for 
comparative work, such standardization is essential because 
this reaction can be reproduced in other media and by 
other workers.1 
Broth may be prepared from Liebig's or Armour's meat 
extract by adding 5 grams of either, 10 grams peptone and 
5 grams NaCI to 1000 cc of water, boiling to dissolve, then 
titrating and filtering as above. 
The author after much experience finds meat juice prefer- 
able to meat extract for broth and other media for patho- 
genic bacteria, and has abandoned the use of meat extracts 
for these organisms. 
Glycerin Broth.-Glycerin broth is made by adding 4 to 
6 per cent, of glycerin to the broth just previous to the 
sterilization. The glycerin serves as a source of carbon to 
certain bacteria which will not grow on the ordinary broth- 
as Mycobacterium tuberculosis. 
Sugar Broths.-Sugar broths are used for determining 
the action of bacteria on these carbohydrates, since this is a 
valuable means of differentiating certain forms, especially 
those from the intestinal tract. Broth free from sugar must 
first be made. This is done by adding to broth prepared as 
already described, just previous to final filtering and steriliza- 
tion, a culture of some sugar-destroying organism (Bacterium 
coli is ordinarily used), and then allowing the organism to 
grow in the raw broth at body temperature for twenty-four 
hours. Any carbohydrate in the broth is destroyed by the 
1 For a discussion of this method of standardization consult the following: 
Clark & Lubs-J. Bact., 1917, II, 1-34, 109-136, 191-236. 
Committee Report-Ibid., 1919, IV, 107-132. 
Jones-J. Inf. Dis., 1919, 25, 262-268. 
Fennel & Fisher-Ibid., 444-451. 
Additional references will be found in these articles. GELATIN CULTURE MEDIA 
177 
Bacterium coli. This mixture is then boiled to kill the Bac- 
terium coli, restandardized and then 1 per cent, by weight of 
required sugar is added. Dextrose, saccharose and lactose 
are the most used, though many others are used for special 
purposes. After the sugar is added the medium must be 
sterilized by discontinuous heating at 100° for three or four 
successive days, because long boiling or heating in the auto- 
clave splits up the di- and polysaccharids into simpler sugars 
and may even convert the simple sugars (dextrose) into acid. 
Various other modified broths are frequently used for spe- 
cial purposes but need not be discussed here. 
Dunham's peptone solution, frequently used to determine 
indol production, is a solution of 1 per cent, of peptone and 
0.5 per cent, of salt in tap water. It does not need to be 
titrated, but should be boiled and filtered into tubes or 
flasks and sterilized. 
Nitrate Broth.-Nitrate broth for determining nitrate 
reduction is 1 per cent, of peptone, 0.2 per cent, of C. P. 
potassium nitrate dissolved in distilled water and sterilized. 
Milk.-Milk is a natural culture medium much used. It 
should be fresh and thoroughly skimmed, best by a separator 
or centrifuge to get rid of the fat. If the milk is not fresh, 
it should be titrated as for broth and the reaction adjusted. 
The milk should be sterilized discontinuously to avoid split- 
ting up the lactose as well as action on the casein and cal- 
cium phosphate. 
Litmus Milk.-Litmus milk is milk as above to which 
litmus has been added as an acid production indicator. The 
milk should show blue when the litmus is added or be made 
to by the addition of normal NaOH solution. It should be 
sterilized discontinuously. Frequently on heating litmus 
milk the blue color disappears due to a reduction of the 
litmus. This blue color will reappear on shaking with air 
or on standing several days, due to absorption of O and 
oxidation of the reduced litmus, provided the heating has 
produced no other change in the milk, as proper heating will 
not. 
Gelatin Culture Medium.-Gelatin to the extent of 10 to 
15 per cent, is frequently added to broth and gives a culture 178 
CULTURE MEDIA 
medium of many advantages. It is solid at temperatures 
up to about 25° and fluid above this temperature, a property 
which is of great advantage in the isolation of bacteria. 
(See Chapter XVIII.) Further gelatin is liquefied (that is 
digested, converted into gelatin proteose and gelatin pep- 
tone, which are soluble in water and do not gelatinize) by 
many bacteria and not by others, a valuable diagnostic fea- 
ture. The gelatin colonies of many bacteria are very char- 
acteristic in appearance, as is the growth of many on gela- 
tin in culture tubes. 
Gelatin medium may be prepared by adding the proper 
amount of gelatin (10 to 15 per cent, by weight) broken into 
small pieces (powdered gelatin in the same proportion may 
be used) to broth, gently warming until the gelatin is dissolved, 
standardizing as for broth, filtering and sterilizing. It is 
usually cleared before filtering by stirring into the gelatin 
solution, cooled to below 60°, the white of an egg for each 
1000 cc., and then thoroughly boiling before filtering. The 
coagulation of the egg albumen entangles the suspended 
matter so that the gelatin filters perfectly clear, though 
with a slight yellowish color. The filtering may be done 
through filter paper if the gelatin is well boiled and filtered 
boiling hot, but is more conveniently done through absorb- 
ent cotton, wet with boiling water. 
Or, the gelatin may be added to meat juice before it is 
boiled, then this is heated to about body temperature (not 
too hot, or the proteins will be coagulated too soon) until 
the gelatin is dissolved. Then the material is standardized 
and thoroughly boiled and filtered. The proteins of the 
meat juice coagulate and thus clear the medium without 
the addition of egg white. Commercial gelatin is markedly 
acid from the method of manufacture, hence the medium 
requires careful titration, even when made from a standard- 
ized broth. 
Gelatin should be sterilized by discontinuous heating at 
100° on three successive days, because long boiling or heat- 
ing above 100° tends to hydrolyze the gelatin into gelatin 
proteose and peptone and it will not gelatinize on cooling. 
It may be heated in the autoclave for ten to fifteen minutes AGAR MEDIUM 
179 
at 10 pounds' pressure and sometimes not be hydrolyzed, 
but the procedure is uncertain and very resistant spores may 
not be killed. The medium should be put into the culture 
tubes in which it is to be used as soon as filtered, and steril- 
ized in these, since, if put into flasks these must be sterili- 
ized, and then when transferred to tubes for use, it must be 
again sterilized unless great care is taken to have the tubes 
plugged and sterilized first, and in transferring aseptically 
to these tubes. These repeated heatings are very apt to 
decompose the gelatin, so it will not "set" on cooling. The 
prepared and sterilized tubes of gelatin should be kept in 
an ice-box or cool room, as they will melt in overheated 
laboratories in summer or winter. 
Agar Medium.-Agar agar, usually called agar, is a com- 
plex carbohydrate substance of unknown composition ob- 
tained from certain seaweeds along the coast of Japan 
and Southeastern Asia. It occurs in commerce as thin 
translucent strips or as a powder. It resembles gelatin 
only in the property its solutions have of gelatinizing when 
cooled. Gelatin is an albuminoid closely related to the 
proteins, agar a carbohydrate. Agar is much less soluble in 
water, 1 or 1.5 per cent, of agar giving a jelly as dense as 10 
to 15 per cent, of gelatin. It dissolves only in water heated 
to near the boiling-point (98° to 99°) and only after much 
longer heating. This hot solution "jells," "sets" or gela- 
tinizes at about 38° and remains solid until again heated to 
near boiling. Hence bacteria may be grown on agar at the 
body temperature (37°) and above, and the agar will remain 
solid, while gelatin media are fluid above about 25°. No 
pathogenic bacteria and none of the saprophytes liable to 
be met with in the laboratory are able to "liquefy" agar. 
An agar medium is conveniently prepared from broth by 
adding 1 or 1.5 per cent, of the finely divided agar to the 
broth and boiling until dissolved, standardizing, clearing, 
filtering, and sterilizing. The agar must be thoroughly 
boiled, usually for ten to fifteen minutes, and the water loss- 
made up by the addition of distilled water before titration. 
Agar is practically neutral so that there is little difference 
between the titration of the dissolved agar and the original 180 
CULTURE MEDIA 
broth. The agar solution should be kept hot from the begin- 
ning to the end except the cooling down to below 60°, when 
the egg white for clearing is added. Though filtration 
through paper is possible as with gelatin, if the agar solution 
is thoroughly boiled and filtered boiling hot, it is more satis- 
factory for beginners to use absorbent cotton wet with boil- 
ing water and to pour the hot agar through the same filter 
if not clear the first time. The solidified agar medium is 
never perfectly clear, but always more or less opalescent. 
The agar medium may be sterilized in the autoclave for 
fifteen minutes at 15 pounds pressure as the high tempera- 
ture does not injure the agar. 
Potato Media.-Potatoes furnish a natural culture medi- 
um which is very useful for the study of many bacteria. 
The simplest, and for most purposes the best, way to use 
potatoes is in culture tubes as " potato tube cultures " (No. 
8, Fig. 119). These are prepared as follows: Large tubes 
are used. Large healthy potatoes are selected. Each end 
of the potato is sliced off so as to have parallel surfaces. 
With a cork-borer of a size io fit the tubes used, cylinders 
about one and one-half inches long are made. Each cylin- 
der is cut diagonally from base to base. This furnishes two 
pieces each with a circular base and an oval, sloping surface. 
The pieces are then washed clean and dropped for a minute 
into boiling water to destroy the oxidizing enzyme on the 
surface which would otherwise cause a darkening of the 
potato. (The darkening may also be prevented by keeping 
the freshly cut potatoes covered with clean water until 
ready to sterilize.) A bit of cotton one-fourth to one-half 
inch in depth is put into each of the test-tubes to retain 
moisture and a piece of potato dropped in, circular base 
down. The tubes are then plugged with cotton and steril- 
ized in the autoclave at 15 pounds pressure for not less 
than twenty-five minutes, since potatoes usually harbor 
very resistant spores, and it is not unusual for a few tubes 
to spoil even after this thorough heating. 
Potatoes are sometimes used in "potato plate cultures." 
The term "plate culture" is a relic of the time when flat 
glass plates were used for this and other "plate cultures." POTATO MEDIA 
181 
Now glass dishes of the general form shown in Fig. 115, 
called "Petri dishes," or plates are used for practically all 
plate culture work. For "potato plates" slices from pota- 
toes are cut as large and as thick as the relative sizes of 
Fig. 115.-Petri dish with the lid partly raised. X y 
Fig. 116.-A potato plate. X X 
potato and dish permit (Fig. 116). The slices should be 
thin enough not to touch the lid and thick enough to be 
firm. 
It is a good plan to wrap each dish separately in paper to 
retain the lid securely, then sterilize as for potato tubes, 
and leave plates wrapped until wanted. 182 
CULTURE MEDIA 
It sometimes happens that the natural acidity of potatoes 
is too great for the growth of many organisms. The acidity 
is sufficiently corrected by soaking the pieces of potato in a 
1 per cent, solution of sodium carbonate for an hour before 
they are put into the tubes or plates. 
Glycerinized potato tubes are conveniently prepared by 
covering the potato in the tube with glycerin broth, steril- 
izing and pouring off the excess broth immediately after 
sterilizing, taking care that the tubes do not become con- 
taminated which is not very probable if the work is quickly 
done while the tubes are still hot. 
Blood Serum Media.-Blood serum, usually from the 
larger, domestic animals on account of convenience in se- 
curing it in quantity, is used in the study of the bacteria 
causing disease in man and animals. Most commonly the 
serum is collected from the clotted blood after it has well 
separated (usually about forty-eight hours is required for 
this). It is then run into tubes which are plugged with 
cotton and placed in an apparatus for coagulating the serum 
by heat. A copper water bath with a tightly closed air 
compartment or the horizontal autoclave (Fig. 81) is suffi- 
cient for this purpose, though special forms of apparatus are 
to be had. It is important that the temperature be raised 
slowly so that the blood gases escape gradually. Three to 
five hours or longer should be allowed for the temperature 
to reach the boiling-point. If the tubes are heated too 
rapidly, the serum is filled with bubbles and badly torn 
since the gases are driven off suddenly. Loffler's serum is 
made by adding one part of dextrose broth to three parts 
of serum and then coagulating as above. The solidified 
serum in either case is best sterilized discontinuously, 
though with care the autoclave at 15 pounds pressure may 
be used for a single sterilization. This is very apt to cause 
a greater darkening of the serum and frequently also a 
laceration of the solid mass by escaping gases. 
Blood serum is also used in the liquid state. For this 
purpose it is best to collect it aseptically; or it may be 
sterilized discontinuously at a temperature of 55° or 56° on 
seven to ten consecutive days. Novy has recently sug- BLOOD SERUM MEDIA 
183 
gested dialyzing the serum to free it from salts and thus 
prevent its coagulation when heated. Whether the removal 
of the various "extractives" which diffuse out with the salts 
deprives the serum of any of its advantageous properties 
remains to be ascertained. 
From the discussion of the physiological activities of bac- 
teria in Chapters IX-XII it is apparent that a very great 
variety of culture media other than those described is neces- 
sary for the study of special types of bacteria, but such 
media are beyond the scope of the present work. 
The ideal culture media are without a doubt the synthetic 
media, that is media of definite known chemical composition, 
so that the various changes due to the growth of bacteria 
can be accurately determined and thus a means of sharply 
differentiating closely related organisms be secured. Such 
media have been prepared and every bacteriologist believes 
strongly in their future usefulness when media of wider 
application shall have been devised. An example of this 
type of culture media is Uschinsky's synthetic medium, of 
which the following is one of the modifications: 
Distilled water 1000 parts 
Asparagin 4 " 
Ammonium lactate 6 " 
Disodium phosphate . 2 " 
Sodium chloride 5 " 
A criticism of this medium is that the elements K, Ca, 
Mg, Fe, Mn, and S which have been shown to be essential 
are not present if chemically pure salts are used in the 
preparation. METHODS OF USING CULTURE MEDIA. 
CHAPTER XVII. 
The way in which culture media shall be used depends 
on the purpose in view. By far the larger part of bacterio- 
logical work is done with cultures in "bacteriological cul- 
ture tubes." Various laboratories have their own special 
types but all are more or less after the "Board of Health" 
form. They differ from ordinary chemical test-tubes in that 
they are usually longer, have no " lip" and have much thicker 
walls to prevent breakage and consequent loss of the cul- 
ture as well as danger from pathogenic organisms. The 
author finds two sets of tubes most serviceable for student 
use-one size 15 cm. long by 19 mm. outside diameter 
(No. 9, Fig. 119), the other 15 cm. long by 13 mm. (Nos. 
1 to 7, Fig. 119). Culture tubes are conveniently used in 
"wire baskets" circular or square in section and of a size 
to correspond with the length and number of tubes used. 
These baskets are light, do not break, and if made of good 
galvanized wire netting do not readilv rust (Figs. 117 and 
118). 
Liquid media such as broth, milk, litmus milk, indol and 
nitrate broths are used in the above-mentioned tubes when 
small quantities only are to be worked with. The tubes 
are filled approximately one-third full, then plugged with 
non-absorbent cotton and sterilized. Cotton plugs are used 
so much in bacteriological work because they permit a free 
circulation of air and gases and at the same time act as 
filters to keep out the bacteria of the air. 
Sugar broths or other media in which gas may be produced 
are used in fermentation tubes (Smith tubes) of the type 
shown in Fig. 120 so that the gas may be collected in the 
closed arm of the tube, measured (Fig. 121) and tested if 
desired. METHOD OF USING CULTURE MEDIA 
185 
One method of using gelatin and also agar is as "punc- 
ture" or "stab" cultures. The tubes (the narrower tubes 
Fig. 117.-Round wire basket. 
Fig. 118.-Square wire basket 
Fig. 119.-Culture tubes with media in them. X %■ 1 to 7 are the 
smaller tubes mentioned in the text; 9 the larger tube; 8 is extra large for 
potato tubes; 1, plain broth; 2, plain milk; 3, litmus milk; 4, gelatin for 
"stab" or "puncture" culture; 5, agar for "stab" or "puncture" culture; 
6, agar for "slope" or "slant" culture; 7, blood serum; 8, potato tube; 
9, agar for plating. Note the transparency of the broth and gelatin and 
the slight opalescence of the agar. 186 
METHODS OF USING CULTURE MEDIA 
are to be preferred for most "stab" cultures) are filled one- 
third full of the medium while it is still fluid, plugged, steril- 
ized and allowed to cool in the vertical position. The med- 
ium is then "inoculated" with a straight platinum needle by 
plunging this into the center of the surface down to the 
bottom of the tube (Fig. 119, Nos. 4 and 5). 
Tig. 120.-Fermentation tubes. 1, filled ready for use; 2, shows a cloudy 
growth and the development of gas in the closed arm. 
Agar and blood serum are frequently used in the form of 
■"slope" or "slant" cultures. That is, the medium solidifies 
with the tubes lying on their sides which gives a long, slop- 
ing surface on which the bacteria are inoculated (Fig. 119, 
Nos. 6 and 7). 
Potato tubes are likewise used for "slant" or "slope" 
cultures (Fig. 119, No. 8). Potatoes as "plate cultures" 
have been referred to. Agar and gelatin are very largely METHOD OF USING CULTURE MEDIA 1S7 
Fig. 121.-Method of estimating percentage of gas in a fermentation tube 
by means of the "gasometer", the reading is 45 per cent. 
Fig. 122.-A toxin flask showing a large surface growth. 188 
METHODS OF USING CULTURE MEDIA 
used in the form of "plate cultures" also. For this purpose 
Petri dishes are first sterilized, then the melted agar or 
gelatin poured into them and allowed to "set" while the 
plates are kept horizontal. The melted media may be 
"inoculated" before they are poured, or a portion of the 
material to be "plated" may be placed in the dish, then the 
melted medium poured in and distributed over the dish by 
tilting in various directions, or the medium after solidify- 
ing may be inoculated by "strokes" or "streaks" over its 
surface, according to the purpose in view in using the plate. 
The larger sized tubes should be used for making plates in 
order to have sufficient medium in the plate (No. 9, Fig. 119). 
For using large quantities of medium, Florence flasks, 
Ehrlenmeyer flasks, special toxin flasks (Fig. 122) or various 
other devices (Vaughan and Novy's "mass cultures," Figs. 
123 and 124) have been employed. 
For growing anaerobic organisms it is evident that some 
method for removing and excluding the oxygen of the air 
must be used. A very great variety of appliances have 
been devised for these purposes. Some are based on the 
principle of the vacuum, exhausting the air with an air 
pump; some on replacing the air with a stream of hydrogen; 
others on absorbing the oxygen by chemical means, as with 
an alkaline solution of pyrogallic acid, or even by growing 
a vigorous aerobe in the same culture or in the same con- 
tainer with the anaerobe, the aerobe exhausting the oxygen 
so that the anaerobe then develops, or finally by excluding 
the air through the use of deep culture tubes well filled with 
the medium, or in the closed arm of fermentation tubes. 
For many purposes a combination of two or more of the 
above methods gives good results. 
In any event the culture medium should have been freshly 
sterilized just before use, or should, be boiled in order to drive 
out the dissolved oxygen. For most anaerobes the presence 
in the medium of about 1 per cent, of a carbohydrate, as 
dextrose, is advisable. 
A description of all the various devices is unnecessary in 
this work, but the following have answered most of the 
purposes of general work in the author's laboratories. METHOD OF USING CULTURE MEDIA 189 
A. " Vigval tubes" of the style shown (Fig. 125) are made 
from glass tubes of about 6 to 8 mm. outside diameter, 
Fig. 123.-Tank with raised lids. (Vaughan.) 
Fig. 124.-Tank with lids lowered. (Vaughan.) 
Figs. 123 and 124.-Vaughan and Novy's mass culture apparatus 190 
METHODS OF USING CULTURE MEDIA 
sealed at the small end, plugged with cotton above the 
constriction and sterilized. The medium, agar or gelatin,, 
which has been previously inoculated with the anaerobic 
culture, is then drawn up into the tube, after breaking off 
the tip, as far as the constriction. The tube is then sealed 
in the flame at the small end and also at the constriction. 
Since it is full of the medium and sealed, access of air is 
prevented. This forms an excellent means for "isolation" 
(Chapter XVIII); the tube needs merely to be cut with a 
file at the point where colonies appear, then these may be 
readily transferred. 
Fig. 125.-Vignal tubes. X 1, the sterile tube ready for inoculation; 
2, fourth dilution tube showing a few isolated colonies, one near the figure; 
3, third dilution showing colonies isolated but numerous; 4, second dilution 
tube showing colonies still more numerous; 5, first dilution tube showing, 
colonies so numerous and small as to give a cloudy appearance to the tube. 
In use tube 2 would be filed in two at the colony and inoculations made from 
it. 
B. "Fermentation tubes" form a simple means for growing 
liquid cultures of anaerobes, the growth occurring in the 
closed arm only, while with facultative anaerobes, growth 
occurs both in the closed arm and in the open bulb. A little 
paraffin oil (a clear, heavy petroleum derivative) may be 
poured on the fluid in the open bulb as a very efficient 
seal, though it is not usually necessary. 
C. "Deep culture tubes."-The medium, agar, gelatin or 
a liquid is poured into tubes until they are approximately 
one-half full, a little paraffin oil is poured on the surface 
(not essential always), then the tubes are plugged and steril- METHOD OF USING CULTURE MEDIA 
191 
ized. Inoculation is made to the bottom and anaerobes 
grow well (Fig. 126). 
D. For slope or plate, or any type of surface cultures the 
Novy jar (Fig. 127) is the most practical device. It is good 
Fig. 126.-Deep tubes showing anaerobic growth. 1, shows a fewsmall 
gas bubbles; 2, shows the medium broken up by the excessive development 
of gas. 
practice to combine the vacuum method, the hydrogen 
replacement method and the oxygen absorption method in 
using these jars. In operation a solution of 20 per cent. 
NaOH is poured on the bottom of the jar to a depth of 1 
or 2 cm., the cultures are placed on glass supports above 
the alkali and a short wide tube of strong pyrogallol is set 
in on the bottom in such a way that it may be easily upset 192 
MFA1 HODS OF USING CULTURE MEDIA 
and mixed with the alkali when it is desired to do so. The 
cover is now clamped in position with all joints well vase- 
lined. Then the outlet tube is connected with a suction 
pump and the air drawn out. Meanwhile the inlet tube 
has been connected with a hydrogen generator, and after 
the jar is exhausted hydrogen is allowed to flow in, and this 
process is repeated until one is satisfied that the air is re- 
placed. The suction exhausts the air from the tubes or 
plates so that much less time is required to replace the 
air with hydrogen. Finally the stop-cock is closed, and the 
pyrogallol solution is gently shaken down and mixed with 
the alkali so that any remaining oxygen will be absorbed. 
Fig. 127.-Novy jars. 
It must be remembered that facultative anaerobes as well 
as anaerobes will grow under any of the above conditions, 
so that cultures of organisms so obtained must be further 
tested aerobically in order to determine to which group 
the organisms belong. 
Reference has been made above to the "inoculation" of 
culture media, which means introducing into the medium 
used the desired material in the proper way. For small 
quantities this is most conveniently done with platinum 
"needles," that is, pieces of platinum wire inserted into the 
ends of glass rods. The "straight" needle is a piece of 
heavy platinum wire of about 0.022 inch in diameter (Fig. 
128). It is used most frequently to inoculate all forms of METHOD OF USING CULTURE MEDIA 
193 
solid media. The platinum loop is of lighter wire, 0.018 inch. 
The loop in the end is conveniently made by twisting the 
wire around the lead of an ordinary lead-pencil. The " loop 
needle" (Fig. 129) is most used in transferring liquid media. 
On account of the high price of platinum, the author has 
substituted "nichrome" wire for student use. This is 
stiffer, not so easily made into loops and breaks out of the 
rods more easily. The latter defect is remedied to some 
extent by imbedding the wire only slightly for about one- 
Fig, 128.-Straight needle. 
Fig. 129.-Straight and loop needles. 
Fig. 130.-Pasteur flask-" ballon, pipette. ' 
fourth of an inch on the side of the end portion of the rod. 
The low cost, less than one-twentieth of platimum, justifies 
its use. 
Sterile graduated pipettes varying in capacity from 1 cc 
graduated in hundredths, upward, permit the transfer of 
definite amounts of liquids. Large quantities are conven- 
iently transferred by means of Pasteur flasks (Fig. 130). 
The details of inoculation are best derived from laboratory 
practice. CHAPTER XVIII. 
ISOLATION OF BACTERIA IN PURE CULTURE. 
As has been stated, the thorough study of a bacterium 
depends on first getting it in pure culture. In the early 
days of bacteriology supposedly pure cultures were obtained 
by (1) dilution in liquid media. A series of tubes or flasks 
containing sterile liquid media was prepared. Number one 
was inoculated with the material to be examined and thor- 
oughly mixed. A small portion of the mixture was trans- 
ferred to number two, and mixed; from this to number 
three, and so on until a sufficient number were inoculated, 
the last three or four in the series receiving the same amounts 
of a very high dilution of the original material. If one or 
two of these latter showed a growth and the others not, it 
was assumed that the dilution had been carried so far that 
only a single organism was transferred and therefore the 
culture obtained was "pure." The method in this crude 
form is too uncertain to be of value today and recourse is 
had to more exact means. The procedure most widely used 
is that of (2) "plating out" by means of gelatin or agar 
plates. The material to be plated out is diluted by trans- 
ferring to three or more tubes of melted gelatin or agar as 
in the first method and then all the tubes are poured into 
Petri dishes and grown under suitable conditions .By 
proper mixing in the tubes the bacteria are well scattered 
through the medium which holds the individual organisms 
separate when it solidifies. On some of the plates a suffi- 
cient dilution will be reached so that the colonies develop- 
ing from the bacteria will be so few that they are separate 
and pure cultures may be obtained by inoculating from one 
of these a tube of the appropriate medium (Figs. 131 to 134). 
The chief uncertainty with this method is that occasionally 
two kinds of bacteria stick together so closely that even the 
separate colonies contain both organisms. This is not ISOLATION OF BACTERIA IN PURE CULTURE 
195 
common, however, The plate colonies frequently develop 
from groups of bacteria which were not separated, but as 
these are of the same kind the culture is essentially pure. 
Fig. 131.-Dilution plates. X %o- 1, shows the first dilution, the colo- 
nies are so numerous and small that they are invisible (compare Fig. 132); 
2, shows fewer and hence larger colonies, but too crowded to isolate (compare 
Fig. 133); 3, shows the colonies larger and well separated, so that it is easy 
to isolate from them (compare Fig. 134). 
Another method which is frequently applicable with 
material from human or animal sources is to (3) rub the 
material over the surface of a slope tube or of medium solid- 
Fig. 132.-A portion of plate 1 in Fig. 131 as seen under the low-power ob- 
jective. X 100. Very small, closely crowded colonies. 
ified in a Petri dish with a sterile heavy platinum needle, 
glass rod, or cotton swab. If the bacteria are not too numer- 
ous, pure cultures may frequently be obtained. A modifi- 
cation of this method is to make a series of (4) parallel 196 
ISOLATION OF BACTERIA IN PURE CULTURE 
streaks on a slope tube or plate of medium with a needle in- 
serted but once into the material to be plated. On the first 
streak most of the bacteria are rubbed off and a continuous 
growth results, but usually on the last of a series only iso- 
lated colonies appear, which are presumably pure. The 
ideal method for securing pure cultures is to be absolutely 
certain that the culture starts from a single organism. 
Fig. 133.-From the thinnest 
part of plate 2, Fig. 131 as seen 
under the low-power objective. 
X 100. Colonies much larger 
than on plate 1, but still crowded. 
Fig. 134.-The smallest colony 
on plate 3, Fig. 131, as seen under 
the low-power objective. X 100. 
Large, single, isolated colony. 
This may be accomplished by means of the (5) apparatus 
and pipettes devised by Professor Barber of the University of 
Kansas (Figs. 135 and 136). With this instrument a single 
organism is picked out under the microscope and isolated in 
a drop of culture medium and observed until it is seen to 
divide, thus proving its viability. Transfers are then made 
to the proper media. The method requires much practice 
to develop the necessary skill in the making of pipettes, 
determining the proper condition of the large cover-glasses 
used over the isolating box, and in manipulation, but the 
results fully compensate. 
Professor W. A. Starin of the author's department, a 
former student of Professor Barber, has done some excellent 
work with this apparatus. ISOLATION OF BACTERIA IN PURE CULTURE 
197 
A number of procedures may be used to greatly facilitate 
the above methods of isolation by taking advantage of the 
different physiological properties of different organisms in a 
Fig. 135.-Diagram of Barber's isolation apparatus, b, moist chamber;' 
ms, large cover-glass over moist chamber; p, small pipette drawn out to a 
fine point; k, r, g, pipette holder; f, screw for raising and lowering k, r. g,; s, 
screw for lateral motion of k, r, g; n, screw for clamp on pipette which allows 
it to be moved in or out; m, mechanical stage of microscope; t, rubber tube 
held in the mouth and used to move the liquid culture medium in the pipette. 
(Journal of Infectious Diseases, October 20, 1908, vol. 5, No. 4, p. 381.) 198 
ISOLATION OF BACTERIA IN PURE CULTURE 
mixture such as ability to form spores, different resistance 
to antiseptics, special food requirements, and pathogenic 
properties, (a) If material contains resistant spores, it may 
be heated to temperatures high enough to kill all of the organ- 
isms except the spores (80° for half an hour, for example) 
Fig. 136.-Photograph of microscope with Barber's isolation apparatus 
set up to use. 
and then plated out. Or (6) an antiseptic which restrains 
the growth of some organisms and not others may be placed 
in the culture media (carbolic acid, various anilin dyes, (p. 
162), excess acid, or alkali, ox bile, etc.), when the more re- 
sistant organisms grow on the final plates, the others not. ISOLATION OF BACTERIA IN PURE CULTURE 
199 
(c) Special food substances (various carbohydrates) from 
which the organism desired forms special products (acids, 
aldehydes) that may be shown on the plates by various in- 
dicators, is one of the commonest means. Or media in 
which certain organisms thrive and others not, so that the 
former soon "crowd out" the latter (unsterilized milk for. 
lactic acid bacteria, inorganic media in soil bacteriology) 
may be used. A combination of the general methods (&) 
and (c) is much used in the separation of the organisms of 
the "intestinal group" in human practice, (d) The inocul- 
ation of a susceptible animal with a mixture suspected to 
contain a given pathogenic bacterium frequently] results 
in the development of the latter in pure culture in the body 
of an animal, from which it may be readily recovered. In 
all of the above methods (except Barber's) the first "pure 
culture" obtained should be "purified" by replating in a 
series of dilution plates to make sure that it is pure. CHAPTER XIX. 
STUDY OF INDIVIDUAL BACTERIA-STAINING. 
When an organism has been obtained in pure culture by 
any of the methods described in the preceding chapter the 
next step is the study of its morphology as discussed in 
Chapters II-IV. This involves the use of the microscope, 
and since bacteria are so small, objectives of higher power 
than the student has presumably used will be needed. 
Doubtless only the two-thirds inch or 16 mm. and the one- 
sixth inch or 4 mm. objectives are all that have been used 
in previous microscopic work, while for examining bacteria 
a one-twelfth inch or 2 mm. is necessary. It will have 
been observed that the higher the power of the objective 
the smaller is the front lens or object glass and consequently 
the less is the amount of light which enters. With the use 
of the one-twelfth inch or 2 mm. objective it is necessary to 
employ two devices for increasing the amount of light enter- 
ing it, with which the student is probably not familiar. One 
of these is to place a drop of cedar oil between the front 
lens and the object and to immerse the lens in this oil- 
hence the term "oil-immersion objective;" the other is the 
substage or Abbe condenser. The latter is a system of 
lenses placed below the stage and so constructed as to bring 
parallel rays of light-daylight-from an area much larger 
than the face of the front lens of the objective to a focus 
on the object to be examined, thus adding very greatly to 
the amount of light entering the objective. Since the con- 
denser brings parallel rays to a focus on the object the flat- 
mirror is always used with the condenser when working 
with daylight. With artificial light close to the microscope, 
the concave mirror may be used to make the divergent rays 
more nearly parallel and thus give better illumination. HANGING DROP SLIDE 
201 
The function of immersion oil is to prevent the dispersion 
of considerable light that would otherwise occur owing to 
refraction as the light passes up through the slide and into 
the air. The accompanying diagram will help to make this 
clearer (Fig. 137). A ray of light (A B) coming through the 
slide will be refracted in the direction B C if the medium 
has a lower refractive index than the slide, as air has, and 
hence will not enter the objective 0. If, however, there is 
interposed between the objective and the slide a medium 
which has the same refractive index as the slide, as immer- 
sion oil has, then the ray will continue in the same direction 
(B D) at the point B and hence enter the objective. Evi- 
-Front lens of objective 
Immersion oil 
Slide 
dently the immersion oil causes much more light to enter 
the front lens and makes the field brighter and at the same 
time prevents considerable refraction and dispersion of light 
from the object seen and hence this appears more distinct 
and sharply defined. The Abbe condenser and the oil- 
immersion objective are practically always used in the 
microscopic study of bacteria (Fig. 138). 
Fig. 137.-Diagram of use of immersion oil. 
HANGING DROP SLIDE. 
It is sometimes necessary to examine living bacteria and 
for this purpose the device known as the "hanging drop Fig. 138.-Diagram of paths of rays of microscope. HANGING DROP SLIDE 
203 
slide" is used (Fig. 139). The slide has a slight concave 
depression ground in the middle of one face. A ring of 
vaseline is placed around this depression with the loop 
needle. On a clean cover-glass, large enough to fit over the 
ring of vaseline, several drops of a broth culture, or of ma- 
terial from a solid culture suspended in broth or physiologi- 
cal normal salt solution are placed. The slide is inverted on 
the cover-glass in such a way that the ring of vaseline seals 
the latter to the slide. When the whole preparation is 
quickly turned cover side up, the drops are seen "hanging" 
to the under side of the cover over the depression in the 
slide. In examining such a preparation with the micro- 
scope great care is necessary in order to focus on the bac- 
teria without breaking the cover. To see the organisms 
distinctly the lower iris diaphragm of the condenser must be 
nearly closed, so that the light coming through consists 
mainly of parallel vertical rays, otherwise the transparent 
Fig. 139.-Hanging drop slide. 
bacteria themselves refract and diffract the light and appear 
blurred and indistinct. By studying living bacteria with 
this device it can be determined whether they are motile or 
not. The motility should not be confounded with the 
familiar "Brownian movement" of all minute insoluble 
inert particles which non-motile living bacteria and also 
dead bacteria show. The hanging drop slide is of value 
in the measurement of bacteria, since this is properly done 
on the living organism. Measurement is done with a cali- 
brated ocular micrometer as in other kinds of measurement 
with the microscope with which the student is presumably 
familiar. The direct effect of various agents on living bac- 
teria as light, electricity, heat, etc., in the study of "trop- 
isms" and "taxes" has been investigated on various modi- 
fications of the above-described hanging drop slide. 
Cell forms and cell groupings may be studied in the same 
way but these features are best determined on stained 
preparations in many instances. 204 
STUDY OF INDIVIDUAL BAC TERI A-STAIN ING 
"Dark field" illumination and the ultramicroscope are 
of great value in the study of living bacteria and other 
minute objects, but apparatus of this type would scarcely 
be used by the student in an introductory course, so that 
they will not be discussed in the present volume. 
STAINING. 
The main use of the microscope in bacteriology is in the 
study of stained preparations of the organisms. Staining 
makes bacteria opaque and hence more easily seen than the 
transparent unstained forms. Some methods ,of staining 
also show morphological structures which are either imper- 
fectly recognized in the unstained cell, spores, or are not 
visible at all-capsules, metachromatic granules, flagella. 
Finally certain bacteria are colored by special methods of 
staining which do not affect others, so that under proper 
conditions these bacteria may be recognized by staining 
methods alone-tubercle bacilli in the organs of animals. 
The phenomena of staining are essentially chemical, 
though sometimes the chemical union is a very weak one, 
even resembling an absorption of the dye rather than true 
chemical union--most watery stains. In other cases the 
chemical compounds formed are decidedly stable and are 
not decomposed even by strong mineral acids-staining of 
tubercle bacilli and other "acid-fast" organisms. In still 
other cases the principal action is a precipitation on the 
surface of the object stained-methods for staining flagella. 
In many methods of staining in addition to the dyes used 
other substances are added to the solution which assist in 
fixing the dye in or on the organism stained. Such sub- 
stances are called mordants. The principal mordants used 
are alkalies, anilin, carbolic acid, iodine, metallic salts, 
tannic acid. 
While it is true that some bacteria may be stained by that 
standard histological nuclear dye, hematoxylin, it is of little 
value for this purpose. Practically all bacteriological stains 
are solutions of the anilin dyes. These dyes, as is well 
known, are of nearly every conceivable color and shade but STAINING 
205 
relatively very few are used in bacteriological work. The 
beginning student will rarely use solutions of other than 
the three dyes fuchsin (red), methylene blue and gentian 
violet for staining bacteria, with occasionally Bismarck 
brown, or eosin, or safranin as tissue contrast stains. 
The bacteriological dyes are kept "in stock" as saturated 
solutions in 95 per cent, alcohol which are never used as 
stains, but merely for convenience in making the various 
staining solutions. 
The approximate percentages of the three common dyes 
in such solutions are indicated in the following table adapted 
from Woods Chemical and Microscopical Diagnosis, Third 
Edition, 1917, Appendix: 
Fuchsin . . . 3.0 % 
Gentian Violet . 4.8 % 
Methylene Blue . 2.0 % 
The stains made from these dyes which are in most com- 
mon use are the following: 
1. Aqueous (watery) gentian violet solution. 
Saturated alcoholic solution of gentian violet . 1 part 
Distilled water ...... .20 parts 
Mix well and filter. 
2. Anilin gentian violet. 
Saturated alcoholic solution of gentian violet . 1 part 
Anilin water (see below) ..... 10 parts 
Mix well and Alter. 
3. Anilin Fuchsin. 
Saturated alcoholic solution of fuchsin . . .1 part 
Anilin water (see below) . . . . .10 parts 
Mix and Alter. 
These stains rarely keep longer than ten days in the 
laboratory (unless kept in the ice-box) and must be made 
fresh on the first sign of a deposit on the glass of the con- 
tainer. 
Anilin Water.-Anilin water is made by putting 3 or 4 cc 
of anilin "oil" in a 120cc. flask, adding 100 cc of distilled 
water, shaking vigorously for a minute or so and filtering 206 
STUDY OF INDIVIDUAL BACTERIA-STAINING 
through a wet filter, in other words, a saturated solution 
of anilin in water. 
4. Loffler's (methylene) blue. 
Saturated alcoholic solution of methylene blue . 3 parts 
Aqueous solution of NaOH (or KOH), 1 to 10,000 . 10 
Mix and filter. 
5. Carbol-fuchsin (Ziehl's solution). 
Saturated alcoholic solution of fuchsin . . .1 part 
5 per cent, aqueous solution of carbolic acid . 10 parts 
Mix and filter. 
6. Gabbet's (methylene) blue (solution). 
Dry methylene blue ...... 4 parts 
Concentrated H2SO4 ...... 25 " 
Distilled water . . . .... 75 " 
Dissolve the dry dye in the acid and add the solution to the 
distilled water and filter. 
Fig. 140.-Author's staining set. Square bottles are set in square holes in 
the block. The capacity of each bottle is 30 cc. 
Staining solutions are conveniently kept in square drop- 
ping bottles inserted in a block as shown in Fig. 140. This 
form of holder necessitates the use of one hand only in secur- 
ing the stain and dropping it on the preparation. STAINING 
207 
The actual staining of bacteriological preparations can 
be learned only by repeated laboratory practice, yet the 
following methods have given such uniform results in class 
work that it is felt they are not out of place in a text-book. 
Preparation of the "Film.''-The author learned to stain 
bacteria on the "cover-glass" but does not recall having 
used this method in fifteen years and does not teach it to 
his students. All staining is done on the slide. To prepare 
a film from a solid culture medium the procedure is as 
follows: 
First, be sure the slide is clean and free from grease. This- 
is accomplished most readily by scouring a few minutes with 
finely ground pumice stone and a little water, then washing 
and drying with a grease-free cloth, handerchief, or piece 
of cheese-cloth. With the " loop " needle place in the middle 
of the slide a small loop of water. This is best done by 
filling the loop by dipping in water, then tapping it gently 
so that all that remains is the water that just fills the loop- 
level full, and this amount is placed on the slide by touching 
the flat side of the loop to the glass. Then the straight 
needle is sterilized, dipped into the culture and just touched 
once into the small drop of water on the slide. The remain- 
der of the culture on the straight needle is then burned off 
and the needle is used to spread the drop of water contain- 
ing the bacteria into a thin even film, which will result, pro- 
vided the slide is free from grease. This is dried and then 
"fixed" by passing three times through the Bunsen flame at 
intervals of about one second, passing through slowly for 
thick slides and a little more rapidly for thin ones. If the 
culture is in a liquid medium, the use of the loop of water is 
unnecessary; a loop of the fluid from the surface, middle or 
bottom as the culture indicates is spread out to a thin film,, 
dried and fixed. 
After the film is fixed the stain desired is dropped on,, 
allowed to act for the proper time, which will depend on the 
stain and the preparation, washed in water, dried thoroughly 
and examined with the oil-immersion lens, without a cover. 
If it is desired to preserve the preparation it may then be 
mounted in balsam. This is not necessary, as they keep- 208 
STUDY OF INDIVIDUAL BACTERIA-STAINING 
just as well, provided the immersion oil is removed. To 
do this, fold a piece of filter paper so that at least three 
thicknesses result. Lay this on the slide and press firmly 
several times, when the surplus oil will be taken up by the 
paper. Slides not mounted in balsam are more apt to 
become dusty than those that are. This is the only disad- 
vantage. 
Gram's Method of Staining.-It has been ascertained 
that some bacteria contain a substance, possibly a protein, 
which forms a compound with gentian violet and iodine, 
which compound is insoluble in alcohol, and other bacteria 
do not contain this substance. Consequently when bac- 
teria are stained by Gram's method (given below), those that 
contain this chemical remain colored, while if it is not present 
the dye is washed out by the alcohol and the bacteria are 
colorless and may be stained by a contrast stain. The bac- 
teria which stain by this method are said to "take Gram's" 
or to be "Gram-positive," while those that decolorize are 
called "Gram-negative." The method is: 
1. Prepare the film as above given. 
2. Stain with fresh anilin gentian violet 1 minute. 
3. Wash in tap water. 
4. Cover with Gram's solution 1 minute. 
5. Wash in tap water. 
6. Wash with 95 per cent, alcohol three times or until no 
more color comes out. 
7. Dry and examine. 
Gram's solution is: 
I .......... 1 part 
KI . . >• . . . . . . . .2 parts 
H2O 300 " 
This method is excellent for differentiating Gram-posi- 
tive and Gram-negative organisms on the same slide. First 
stain by this method and after washing with alcohol stain 
with a counter-stain, carbol-fuchsin diluted ten to fifteen 
times with water is excellent. The Gram-positive bacteria 
are violet and the Gram-negative are red. 
It is also of great value in staining Gram-positive bac- 
teria in tissues, but the sections should be stained about five STAINING 
209 
minutes in the anilin gentian violet and be left about two 
minutes in the Gram's solution. Sections are to be counter- 
stained in Bismarck brown, dilute eosin or safranin solutions 
and cleared in oil of bergamot, lavender or origanum and 
not in clove oil or carbol-xylol, as these latter dissolve out 
the dye from the bacteria. 
Staining of Spores in the Rod.-Prepare the films as usual. 
Cover with carbol-fuchsin, using plenty of stain so that it 
will not dry on the slide; heat until vapor arises, not to boil- 
ing; cool until the stain becomes cloudy and heat again 
until the stain clears, and repeat once more; wash in tap 
water and then wash in 1 per cent. H2SO4 three times, 
dropping on plenty of acid, tilting and running this over the 
slide three times and then pour off and use fresh acid and 
repeat this once. Wash thoroughly in distilled water, then 
stain with Loffler's blue one to three minutes. Wash, dry 
and examine. The spores should be bright red in a blue 
rod. 
This method will give good results if care is taken to 
secure cultures of the right age. If the culture is too old 
the spores will all be free outside the rods, while if too'young 
they will decolorize with the acid. For Bacillus subtilis and 
Bacillus anthracis, cultures on agar slants forty-eight hours 
in the 37° incubator are just right. For the spores of Clos- 
tridium tetani, the culture should be three days old, but may 
be as old as a week. 
Staining of "Acid-fast" Bacilli.-Mycobacterium tubercu- 
losis, Mycobacterium of Johne's disease, "grass" and "butter 
bacilli," Mycobacterium leprae, Mycobacterium smegmatis. 
Gobbet's method: 
1. Prepare the film as usual. 
2. Stain with carbol-fuchsin as given above for spores. 
3. Wash with tap water. 
4. Decolorize and stain at the same time with Gabbet's 
blue, two or three minutes. 
5. Wash, dry and examine. 
The sulphuric acid in Gabbet's blue removes the carbol- 
fuchsin from everything except the "acid-fast" bacteria, 210 
STUDY OF INDIVIDUAL BAG TERI A-STAINING 
which remain red, and the blue stains the decolorized bac- 
teria and nuclei of any tissue cells present. 
Ziehl-Neelson method: 
1, 2, 3, as in Gabbet's method. 
4. Decolorize with 10 per cent. HC1 until washing with 
water shows only a faint pink color left on slide. 
5. Wash thoroughly. 
6. Stain with Loffller's blue one or two minutes. 
7. Wash, dry and examine. 
The results are the same as with Gabbet's method. 
Staining of Capsules.-Rabiger's Method.-Films of the 
organism to show capsules should be freshly prepared, dried 
but not fixed. Material is usually obtained from milk or 
blood. A drop of the fluid is placed on the middle of a slide 
about one-fourth of the distance from one end. The nar- 
row edge of another clean slide is placed in this drop and then 
drawn lengthwise across the slide with firm pressure. This 
gives a thin layer which is necessary if good results are to be 
expected. The preparation is covered with a freshly pre- 
pared, saturated solution of gentian violet in formalin and 
this allowed to stain for 30 seconds. Then wash lightly, 
dry and examine. The organisms appear deeply violet 
and much larger than with ordinary stains and capsules 
are well stained and show well. 
Welch's Method.-Prepare films as in the above method. 
Cover with glacial acetic acid for 10 to 20 seconds. Wash 
off the acid with carbol-fuchsin. Wash the stain off with 
physiological normal salt solution (0.85 per cent.) until all- 
surplus stain is removed. Dry and examine. Capsules and 
bacteria are red. 
Staining of Flagella.-The rendering of flagella visible is 
considered one of the most difficult processes in staining. 
Experience of a number of years during which whole classes 
numbering from one hundred to three hundred students 
accomplish this result shows that it is no more difficult than 
many other staining processes. The essentials are: (1) 
clean slides, (2) young cultures on agar slopes, (3) freshly 
prepared mordant and stain which are kept free from pre- STAINING 
211 
cipitate, (4) gentle heating. The author's students are fur- 
nished only stock materials and make their own cultures, 
mordants and stains. 
The slides are cleaned with pumice in the usual way. An 
agar slope culture of the organism to be stained from six to 
twenty-four hours old is selected. A bit of the culture is 
removed and placed in a watch-glass of water. The bac- 
teria are allowed to diffuse of themselves without stirring. 
After several minutes a loop of this water is removed and 
three streaks are made across the slide, one in the middle 
and one on each side of this about one-quarter of an inch 
from it. This gives well scattered bacteria in one of the 
three streaks at least and very little other material on the 
slide to cause preciptates. The slide is carefully dried and 
fixed and then covered with an abundance of the mordant 
by filtering through a small filter onto the slide so that the 
mordant shows transparent on the slide. The preparation 
is then gently warmed and cooled three times, adding mor- 
dant if necessary. Do not heat to steaming. After mordant- 
i ng for about five minutes the excess is washed off under the 
tap. It is a good plan to hold the slide level and allow the 
water to run into the center of the mordant and flow it off. 
Inclining the slide is apt to cause the film on the surface of 
the mordant to settle down on the slide and spoil the prepa- 
ration. After the mordant is washed off and all traces of it 
removed with a clean cloth if necessary the stain is applied 
and gently heated and cooled the same way for from three 
to five minutes. The preparation is then washed, dried and 
examined. 
The mordant used is a modification of Loffler's which is 
somewhat simpler in preparation since the stock solution of 
FeCh is more permanent than FeSCh solution. 
Mordant sufficient for one student: 
5 per cent solution of FeCh .... . 20.0 cc 
25 per cent, solution of tannic acid . . , 20.0 cc 
Anilin fuchsin ...... . 4.0 cc 
Normal NaOH ...... . 1.5 cc 
The solution of FeCk is made up in the cold and must be 
perfectly clear. The tannic acid solution must be thor- 212 
STUDY OF INDIVIDUAL BACTERIA-STAINING 
oughly boiled and filtered until clear. The iron and the acid 
are carefully mixed, boiled and filtered clear. The anilin 
fuchsin must be added slowly with constant stirring and the 
mixture boiled and filtered. The NaOH is added in the 
same way and this mixture boiled and filtered. The final 
mordant should not leave a film on a clean slide when 
poured on and allowed to run off. Unless the mordant is in 
this condition and perfectly clear, it should not be used, but 
a new one must be made up. Time and care in the prepar- 
ation of the mordant are essential. 
The stain to follow this mordant is anilin fuchsin. 
Staining of Metachromatic Granules.-Neisser's Method. 
Prepare the film in the usual way. Stain with Neisser's 
stain a few seconds only. Wash and stain with Bismarck 
brown a few seconds only. 
Neisser's Stain: 
Sat. alcoholic solution of methylene blue 1.0 part 
Glacial acetic acid 2.5 parts 
Distilled water 50.0 parts 
Bismarck Brown: 
Bismarck brown (dry dye) 2 parts 
Distilled Water 1000 parts 
By the use of the hanging drop slide and the methods of 
staining just described all the various morphological fea- 
tures of the bacterial cell may be ascertained. 
It is necessary when cell groupings as characteristic of 
definite modes of division are to be determined to make 
slides from a liquid culture, as broth. Place a drop of the 
material, preferably from the bottom of the tube in most 
instances, from the top in case a pellicle or scum is formed 
on the surface, on the slide and allow this to dry without 
spreading it out, fix, wash gently with water, then slain 
lightly with Lbffler's blue. Such slides also show charac- 
teristic cell forms as well. Slides should be made from solid 
media to show variations in form and size and involution 
forms. These latter are especially apt to occur on potato 
media. CHAPTER XX. 
STUDY OF THE PHYSIOLOGY OF BACTERIA. 
Of the environmental conditions influencing the growth 
of bacteria the following are the chief ones ordinarily deter- 
mined : 
A. Temperature.-The optimum temperature for growth 
is usually about the temperature of the natural environment 
and ordinarily one determines merely whether the organism 
grows at body temperature (37° and at room temperature 
(20°) or not. For exact work the maximum, minimum and 
optimum temperature must be ascertained by growing in 
"incubators" with varying temperatures. 
A bacteriological incubator is an apparatus for growing 
bacteria at a constant temperature. This may be any tem- 
perature within the limits for bacterial growth. If tempera- 
tures above that of an ordinary room are desired, some 
source of artificial heat is needed. Electricity, gas or oil 
may be used. A necessary adjunct is some device for main- 
taining the temperature constant, a "thermoregulator" or 
"thermostat." For lower temperatures a cooling arrange- 
ment must be installed. For the great part of bacteriologi- 
cal work only two temperatures are used, 20° so-called " room 
temperature" (this applies to European "rooms" not to 
American) and 37° or body temperature. Incubators for 
37° of almost any size and style desired may be secured from 
supply houses and need not be further described. Figs. 141 
and 142 illustrate some of the types. 
For use with large classes "incubator rooms" are to be 
preferred. The author has one such room for 37° work with 
200 compartments for student use which did not cost over 
S60 to install. 
The styles of incubators for lower temperatures, 20° and 
below, are not so numerous nor so satisfactory. The author Fig. 141.-Small laboratory incubator, gas heated. 
Fig. 142.-Electric incubator. STUDY OF THE PHYSIOLOGY OF BACTERIA 
215 
has constructed a device which answers every purpose for a 
small class. The diagram, Fig. 143, explains it. 
The thermal death-point is determined by exposing the 
organisms in thin tubes of broth at varying temperatures 
for ten-minute periods and then plating out to determine 
Fig. 143.-Diagram of fittings for a cold incubator. 1, small tank for 
constant head, about 1 foot in each dimension, a, inflow; b, overflow; 
c, lead pipe. 2, refrigerator, a', ice; b', flat coil under ice; c', outflow 
to incubator. 3, incubator, a", cold water inflow; b", overflow; ther- 
mometer and burner omitted. The diagram explains the construction. The 
water cooled to about 14° with artificial ice by flowing through the lead coil 
under the ice, flows into the incubator which may be heated and regulated 
in the usual way. 
growth. The effect of heat may also be determined by 
exposing at a given temperature, e. g., 60°, for varying 
lengths of time and plating out. 
B. Oxygen relations-whether the organism is aerobic, 
anaerobic, or facultative is determined by inoculation in 
gelatin or agar puncture or stab cultures and noting whether 216 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
the most abundant growth is at the top, the bottom or all' 
along the line of inoculation. 
C. Reaction of the medium-acid, alkaline or neutral as 
influencing the rate and amount of growth. 
D. The kind of medium on which the organism grows best. 
E. The effect of injurious chemicals, as various disinfec- 
tants, on the growth. 
F. Osmotic pressure conditions, though modifying decid- 
edly the growth of bacteria, are not usually studied as aids 
in their recognition, nor are the effects of various forms of 
energy, such as light, electricity, x-rays, etc. 
Among the "Physiological Activities" discussed in Chap- 
ters IX-XII those which, in addition to the staining reac- 
tions described, are of most use in the identification of 
non-pathogenic bacteria are the first ten listed below. For 
pathogenic bacteria the entire thirteen are needed. 
1. Liquefaction of gelatin. 
2. Digestion of blood serum. 
3. Coagulation and digestion of milk. 
4. Acid or gaseous fermentation in milk, or both. 
5. Acid or gaseous fermentation of various carbohydrates 
in carbohydrate broth, or both. 
6. Production of indol in "indol solution." 
7. Production of pigments on various media. 
8. Reduction of nitrates to nitrites, ammonia, or free 
nitrogen. 
9. Production of enzymes as illustrated in the above 
activities. 
10. Appearance of growth on different culture media. 
11. Production of free toxins as determined by injection 
of animals with broth cultures filtered free from bacteria. 
12. Causation of disease as ascertained by the injection 
of animals with the bacteria themselves, and recovery of the 
organism from the animals. 
13. Formation of specific antibodies as determined by the 
proper injection of animals with the organism or its products 
and the subsequent testing of the blood serum of the inocu- 
lated animals. 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. 
Endorsed by the Society for general use at the Annual Meeting, December, 1907. 
GLOSSARY OF TERMS. 
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. 
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 semiconfluent colonies 
along the lines 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 pseudozooglese. 
COAGULATION,1 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 o$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. 
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, colojiies 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 forming either 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. 
PSEUDOZOOGLEZE, 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 exposure. 
TRANSIENT, a few days. 
TURBID, cloudy with flocculent particles; cloudy plus flocculenoe. 
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. 
ZOOGLEZE, firm gelatinous masses of bacteria, one of the most tyj ical 
examples of which is the Streptococcus mesenterioides of 4 gar 
vats (Leuconostoc mesenterioides), the bacterial chains h fing 
surrounded by an enormously thickened, firm covering it fide 
of which there may be one or many groups of the bacteria. 
NOTES. 
(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 morphological 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 time 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. 
TABLE I. 
A NUMERICAL SYSTEM OF RECORDING THE SALIENT 
CHARACTERS OF AN ORGANISM. (GROUP NUMBER.) 
100 Endospores produced 
200 Endospores not produced 
10 Aerobic (strict) 
20 Facultative anaerobic 
30 An'aerobic (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 
0.01...- 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 
0.09003 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 propel 
symbol which precedes the number thus: (7) 
Bacillus coli (Esch.) Mig. becomes B. 222.111102 
Bacillus alcaligenes Petr. becomes B. 212.333102 
Pseudomonas campestbis (Pam.) Sm. becomes Ps. 211.333151 
Bacterium suicida Mig. becomes Bact. 222.232103 
1 Term also applied to the solidification of serum in media: e. g.. the Hiss inulin medium for the differentiation: of pneumococci (see diploeoccus of pneumonia). Source Date of Isolation Name Group No. 0 
DETAILED FEATURES. 
NOTE-Underscore required terms. Observe notes and 
glossary of terms on opposite side of card. 
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. 
(Orientation (grouping) 
Chains (No. of elements) 
Short chains,-long chains 
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 
. o_ I Orientation (grouping) 
i Chains (No. of elements) 
nangmg-mocK 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 
II. 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, iri- 
descent. 
Chromogenesis (8) 
Odor, absent, decided, resembling 
Consistency, slimy, butyrous, viscid, membranous, cori- 
aceous, brittle. 
Medium grayed, browned, reddened, blued, greened. 
2. Potato. 
Growth scanty, moderate, abundant, transient, persistent. 
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. 
Chromogenesis (8) Pigment in water insol- 
uble, soluble: othe* solvents 
Odor, absent, decided, resembling 
Consistency, slimy, butyrous, viscid, membranous, 
.coriaceous, brittle. 
Medium, prayed, 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. 
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. ..., lOd...., 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, mycelioid, 
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, mycelioid, 
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-fluorescent. 
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 
III. PHYSICAL AND BIOCHEMICAL FEATURES. 
a; 
CQ 
O O q 
1. Fermentation-tubes con- o s ® "'E •" 
taining peptone-water or h © 5 g § 
sugar-free bouillon and S o o "3 >> g 
Gas production, in per cent. 
^CO? 
Growth in closed arm 
Amount of acid produced Id. 
Amount of acid produced 2d. 
Amount of acid produced 3d. 
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 0 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: 
>> 5^ 
'•S 'Tj 
£ a g 2 
Substance Method used x tf c- rc 
£ - m2? 
s * 
5 i S £ 
S Eh M < 
i ~ 
j n_ 
j । 
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. 
BRIEF CHARACTERIZATION. 
Mark + or 0, and when two terms 
occur on a line erase the one which 
does not apply unless both apply. 
MORPHOLOGY C) 
Diameter over 1/4 
Chains, filaments 
Endospores 
Capsules 
Zooglea, Pseudozooglea 
Motile 
Involution forms 
Gram's stain 
CULTURAL FEATURES (3) 
Broth 
Cloudy, turbid 
Ring 
Pellicle 
Sediment 
Agar 
Shining 
Dull 
Wrinkled 
- 
Chromogenic 
Gel. plate 
Round 
- 
Proteus-like 
Rhizoid 
Filamentous 
Curled 
Gel. 
Stab. 
Surface growth 
Needle growth 
- 
Potato 
Moderate, absent 
Abundant 
Discolored 
Starch destroyed 
Grows at 37° C. 
Grow's in Cohn's sol. 
- 
Grows in Uschinsky's sol. 
BIOCHEMICAL FEATURES 
|| 
Gelatin (4) 
Blood-serum 
Casein 
Milk 
Acid curd 
Rennet curd 
Casein peptonized 
Indol (6) 
- 
Hydrogen sulphide 
Ammonia (5) 
• 
Nitrates reduced (5) 
- 
Fluorescent 
Luminous 
DISTRIBUTION 
Animal pathogen, epizoon 
Plant pathogen, epiphyte 
Soil 
Milk 
Fresh water 
Salt water 
Sewage 
Iron bacterium 
Sulphur bacterium GROWTH ON DIFFERENT CULTURE MEDIA 
217 
For special kinds of bacteria other activities must be 
determined (oxidation, nitrate and nitrite formation, action 
of sulphur and iron bacteria, etc.). 
The first nine activities are determined by inoculating 
the different culture media already described and observ- 
ing the phenomena indicated, making chemical tests where 
necessary. 
APPEARANCE OF GROWTH ON DIFFERENT CULTURE 
MEDIA. 
In addition to those changes that are associated with the 
manifestation of different physiological activities, many 
bacteria, show characteristic appearances on the various cul- 
ture media which are of value in their identification. 
Too much stress should not be laid on these appearances 
alone, however, since slight variations, particularly in solid 
media due especially to the age of the medium, may change 
decidedly the appearance of a colony. This is true of 
variations in the amount of moisture on agar plates. Colo- 
nies which are ordinarily round and regular may assume 
very diverse shapes, if there chance to be an excess of mois- 
ture on the surface. 
Also in slope and puncture cultures on the various solid 
media much variation results from the amount of material 
on the inoculation needle and just how the puncture is 
made, or the needle drawn over the slope. These variations 
are largely prevented by the use of standard media and by 
inoculating by standard methods. The Laboratory Com- 
mittee of the American Public Health Association has pro- 
posed standard methods for all culture media and tests and 
for methods of inoculation, and these have been generally 
adopted in this country for comparative work. 
Likewise the Society of American Bacteriologists has 
at different times (1904, 1914, 1917) adopted "descriptive 
charts" for detailing all the characteristics of a given or- 
ganism. A committee is at present working on a revission 
of the 1917 chart to be presented at the 1920 meeting. One 
of the earlier charts which includes a glossary of descriptive 
terms is inserted in this chapter. 218 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
Among the cultural appearances the following are of most 
importance: 
Fig. 144.-Broth cultures x %. 1, uninoculated transparent broth; 2, 
broth cloudy from growth of organisms; 3, broth slightly cloudy with a 
deposit in bottom; 4, broth slightly cloudy with a heavy membrane at the 
surface. 
In broth cultures the presence or absence of growth on 
the surface and the amount of the same. Whether the GROWTH ON DIFFERENT CULTURE MEDIA 
219 
broth is rendered cloudy or remains clear, and whether there 
is a deposit at the bottom or not (Fig. 144). An abundant 
surface growth with little or nothing below indicates a strict 
aerobe, while a growth or deposit at bottom and a clear or 
nearly clear medium above, an anaerobe. These appear- 
Fig. 145.-A fili- 
form stab or punc- 
ture culture, x %• 
Fig. 146.-A beaded 
stab or puncture cul- 
ture. x 
Fig. 147.-A vill- 
ous stab or puncture 
culture, x K 
ances are for the first few days only of growth. If the broth 
is disturbed, or after the culture stands for several days 
many surface growths tend to sink to the bottom. So an 
actively motile organism causes in general a cloudiness, 
■especially if the organism is a facultative anaerobe, which 
tends to clear up by precipitation after several days when 
the organisms lose their motility. Non-motile facultative 220 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
anaerobes usually cloud the broth also, but settle out more 
rapidly than the motile ones. 
In gelatin and agar punctures the oxygen relationship is 
shown by surface growth for aerobes, growth near the 
Fig. 148 
Fig. 148.-Crateriform liquefaction of gelatin, x y2. 
Fig. 149.-Funnelform liquefaction of gelatin, x 
Fig. 150.-Saccate liquefaction of gelatin, x Vz 
Fig. 151.-Stratiform liquefaction of gelatin, x 
Fig. 149 
Fig. 150 
Fig. 151 
bottom of the puncture for anaerobes, and a fairly uniform 
growth all along the line of inoculation for facultative 
anerobes. In the case of these last organisms, a preference 
for more or less oxygen is indicated by the approach to the 
aerobic or anaerobic type of growth. GROWTH ON DIFFERENT CULTURE MEDIA 
221 
Along the line of puncture the commonest types are 
filiform (Fig. 145), which indicates a uniform growth; beaded 
(Fig. 146), or small separate colonies; villous (Fig. 147), deli- 
Fig. 152.-Filiform 
slope culture, x K 
Fig. 153.-Filiform, 
slightly spreading, slope 
culture, x K- 
Fig. 154.-Beaded 
slope culture, x /• 
cate lateral outgrowths which do not branch; arborescent, 
tree-like growths branching laterally from the line. In agar 
these branchings are usually short and stubby, or technically, 
papillate. 
Further, in the gelatin puncture the liquefaction which 
occurs is frequently characteristic. It may be crateriform 
(Fig. 148), a shallow saucer at the surface; or funnel-shaped 222 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
(Fig. 149); or it may be of uniform width all along the 
puncture, i. e., saccate (Fig. 150); or it may be stratiform, 
(Fig. 151), i. e., the liquefaction extends to the sides of the 
tube and proceeds uniformly downward. 
Fig. 156 
Fig. 155 
Fig. 155.--Effuse slope culture. x y2. 
Fig. 156.-Rhizoid slope culture, x ¥2- 
Fig. 157.-Rugose slope culture, x ¥2- 
Fig. 158.-Verrucose slope culture, x ¥2- 
Fig. 157 
Fig. 158 
On agar, potato and blood serum slope tubes the amount 
of growth, its form and elevation, the character of the sur- 
face, and the consistency should be carefully noted, and in 
some few cases the character of the edge. Figures 152 to* 
158 show some of the commoner types. GROWTH ON DIFFERENT CULTURE MEDIA 
223 
Fig. 159.--Punctiform colonies on a plate, x 
Fig. 160.-A rhizoid colony on a plate. Natural size. 224 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
On agar and gelatin plates made so that the colonies are 
well isolated, the form of the latter, the rate of their growth, 
the character of the edge and of the surface, the elevation 
Fig. 161.-Ameboid colonies on a plate, x 
Fig. 162.-Large effuse colony on a plate. The edge is lacerated. Inci- 
dentally the colony shows the rate of growth for six successive days. X %• GROWTH ON DIFFERENT CULTURE MEDIA 
225 
Fig. 163.-Colony with edge en- 
tire !as seen under the low-power 
objective. X 100. 
Fig. 164.-Colony with edge 
coarsely granular as seen under the 
low-power objective, x 100. 
Fig. 165.-Colony with edge 
curled as seen under the low-power 
objective, x 100. 
Fig. 166.-Colony with edge 
rhizoid as seen under the low-power 
objective, x 100. 
Fig. 167.-A small deep rhizoid colony as seen under the low-power objective. 
X 100. 226 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
and the internal structure as determined by a low-power 
lens are often of almost diagnostic value. Also in the case 
of the gelatin plates, the character of the liquefaction is 
important. Figs. 159 to 167 show some of the commoner 
characteristics to be noted. 
Fig. 168.-A small mold colony 
natural size as viewed by trans- 
mitted light. 
Fig. 169.-The same colony as 
viewed by reflected light. 
Fig. 170.-A portion of the thin 
edge of the same colony as seen 
with the lower-power objective. 
X 100. 
Fig. 171.-A single fruiting body 
(sporangium) from the same colony 
as seen under the lower-power ob- 
jective. X 100. 
Colonies of mold frequently appear on plates. These are 
readily differentiated from bacterial colonies after a little 
experience. With the naked eye usually the fine radiations 
of the edge of the colony are apparent. The surface appears 
duller and by reflected light more or less "fuzzy." With 
the low-power objective the relatively large, branching 
threads of the mold (mycelia) show distinctly. Also the 
large fruiting bodies (sporangia) are easily distinguished. 
Figs. 168 to 171 illustrate a common black mold {Rhizopus 
nigricans'). CHAPTER XXI. 
ANIMAL INOCULATION. 
Animal inoculation has been referred to (1) as a method 
of assisting in the preparation of pure cultures of patho- 
genic organisms; (2) as a means of testing the poisonous 
properties of substances produced in bacterial cultures; (3) 
in order to test the ability of an organism to cause a disease; 
(4) for the production of various antibodies; it may be added 
(5) that some bacteria produce in the smaller experimental 
animals lesions which do not occur in animals naturally 
infected, but which nevertheless are characteristic for the 
given organism. The best illustration is the testicular reac- 
tion of young male guinea-pigs to intraperitoneal injections 
of glanders bacilli. Experimental animals are also inocu- 
lated (6) to test the potency of various bacterial and other 
biological products, as toxins, antitoxins, etc. 
Guinea-pigs are the most widely used experimental ani- 
mals because they are easily kept and are susceptible to 
so many diseases on artificial inoculation. Rabbits are used 
very largely also, as are white mice. For special purposes 
white rats, pigeons, goats and swine are necessary. For 
commercial products horses (antitoxins) and cattle (small- 
pox vaccine) are employed. In the study of many human 
diseases the higher monkeys and even the anthropoid apes 
are necessary, since none of the lower animals are susceptible. 
The commonest method of animal inoculation is undoubt- 
edly the subcutaneous. This is accomplished most readily 
with the hypodermic needle. The skin at the point selected 
(usually in guinea-pigs the lateral posterior half of the 
abdominal surface, in mice the back near the root of the 
tail) is pinched up to avoid entering the muscles and the 
needle quickly inserted. Clipping the hairs and washing 
with an antiseptic solution should precede the inoculation 
as routine practice. Frequently a small "skin pocket" is 
all that is needed. The hair is clipped off, the skin pinched 
up with small forceps and a slight snip with sharp scissors 228 
ANIMAL INOCULATION 
is made. The material may be inserted into this pocket 
with a heavy platinum needle. Cutaneous inoculation is 
made by shaving the skin and rubbing the material onto 
the shaved surface or scratching with a scalpel or special 
scarifier, but without drawing blood, and then rubbing in 
the material to be inoculated. 
Intravenous injections are made with larger animals. In 
rabbits the posterior external auricular is a convenient vein. 
In larger animals the external jugular is used. 
Intraperitoneal, -thoracic, -cardiac, -ocular, -muscular injec- 
tions, and injections into the parenchyma of internal organs 
are accomplished with the hypodermic needle. In the case 
of the first two, injury to contained organs should be care- 
fully avoided. Intracardiac injection, or aspiration of the 
heart to secure blood, requires considerable practice to be 
successful without causing the death of the animal at once 
through internal hemorrhage. In siibdural injections into 
the cranial cavity it is necessary to trephine the skull first, 
while such injections into the spinal canal may be accom- 
plished between the vertebra with needles longer and 
stronger than the usual hypodermic needle. Occasionally 
animals are caused to inhale the organisms, or are fed cultures 
mixed with the feed. 
SECURING AND TRANSPORTING MATERIAL FROM 
ANIMALS FOR BACTERIOLOGICAL EXAMINATION. 
If the site of the lesion is readily accessible from the 
exterior, material from the living animal should be collected 
with sterile instruments and kept in sterile utensils until the 
necessary tests can be made. Testing should be done on 
material as soon after collection as possible, in all cases, to 
avoid the effects of "decomposition" bacteria. 
If the blood is to be investigated it may be aspirated from 
a peripheral vein with a sterile hypodermic syringe of appro- 
priate size or allowed to flow through a sterile canula into 
sterile receptacles. The site of the puncture should be 
shaved and disinfected before the instrument is introduced. 
Discharges of whatever kind should likewise be collected 
in sterile receptacles and examined as soon as may be. ANIMAL INOCULATION 
229 
If internal organs are to be examined it is best to kill a 
moribund animal than to wait for death, since after death, 
and in severe infections even sometimes before, the tissues 
are rapidly invaded by saprophytic bacteria from the ali- 
mentary and respiratory tracts which complicate greatly 
the isolation of the specific organism. Hence the search for 
specific bacteria in carcasses or organs several hours after 
death is frequently negative. Animal inoculation with such 
material is very often followed by sepsis or septicemia in a 
few hours, so that the specific organism has no opportunity 
to manifest itself. 
In securing material for cultures from internal organs it 
is a good plan to burn the surface of the organ with a gas 
or alcohol flame, or to sear it with a hot instrument to kill 
surface organisms, then make the incision or puncture 
through the burned area and secure material from the inte- 
rior of the organ. Such punctures made with a stiff plati- 
num needle frequently give pure cultures of the organism 
sought. Slides may be made from such material and cul- 
ture media inoculated at once. 
Since a bacteriological diagnosis depends most commonly 
on growing the organisms, it is evident that material sent 
for examination must never be treated with an antiseptic or pre- 
servative. If decomposition is to be feared the only safe pro- 
cedure is to pack the material in ice and forward in this way. 
Tuberculous material from the parenchyma of internal 
organs may be forwarded in a preservative (not formalin, 
since this makes it very difficult to stain the bacteria) as in 
this special case a very positive diagnosis may be made by 
staining alone. Even here it is better to pack in ice in 
order that the diagnosis by staining may be confirmed by 
inoculating the living organisms into guinea-pigs. 
In the case of material from a rabid animal and many 
protozoal diseases the rule against preservatives is not abso- 
lute, since staining is a reliable diagnostic means. Even 
in these cases it is often desirable to inoculate animals,, 
hence, as before stated, it is best to make it a uniform prac- 
tice to pack material for examination in ice and use no pre- 
servatives. PART IV. 
GENERAL PATHOGENIC BACTERI- 
OLOGY 
CHAPTER XXII. 
INTRODUCTION. 
Pathogenic Bacteriology treats of the unicellular micro- 
organisms which are responsible for disease conditions, i. e., 
pathological changes in other organisms. Hence not only 
are bacteria considered, but also other low vegetable forms, 
as yeasts and molds, likewise protozoa in so far as they 
may be pathogenic. For this reason the term pathogenic 
"Microbiology" has been introduced to include all these 
organisms. It is largely for the reason that the methods 
devised for the study of bacteria have been applied to the 
investigation of other microorganisms that the term "bac- 
teriology" was extended to cover the entire field. The 
general discussion in this chapter is intended to include, 
therefore, microorganisms of whatever kind pathogenic to 
animals. 
The term pathogenic as applied to an organism must be 
understood in a purely relative sense, since there is no single 
organism that can cause disease in all of a certain class, but 
each is limited to a more or less narrow range. Some form 
of tuberculosis attacks nearly all vertebrates, but no other 
classes of animals and no plants. Lockjaw or tetanus 
attacks most mammals, but not any other vertebrates 
naturally. Typhoid fever affects human beings; hog chol- 
era, swine, etc. This point is more fully discussed in Chap- 
ter XXIII but can not be too greatly insisted upon. 
"The greatest enemy to mankind is man." 
Exceptions to this statement do occur and are important 
and must be considered in efforts to protect completely 232 
INTRODUCTION 
human beings from disease (tuberculosis from cattle, glan- 
ders from horses, poisoning from spoiled canned goods, an- 
thrax from hair, hides, wool, of animals dead of the disease), 
but the most common human diseases are derived from other 
human beings directly or indirectly. 
Diseases which are due to unicellular pathogenic micro- 
organisms are called infectious diseases, while if such diseases 
are transmitted under natural conditions from organism to 
organism they are spoken of as contagious diseases. Most 
infectious diseases are contagious but not all. Tetanus is 
a good illustration of a non-contagious infectious disease. 
There are very few such diseases. 
When a unicellular microorganism gains entrance into the 
body and brings about any pathological changes there, the 
result is an infection. Undoubtedly many pathogenic organ- 
isms get into the body but never manifest their presence by 
causing disease conditions, hence do not cause an infection. 
It is the pathological conditions which result that constitute 
the infection, and not the mere invasion. 
The time that elapses between the entrance of the organ- 
ism and the appearance of symptoms is called the period of 
incubation and varies greatly in different diseases. 
The term infestation is used to denote pathological condi- 
tions due to multicellular parasites. Thus an animal is 
infested (not infected) with tapeworms, roundworms, lice, 
mites, etc. Many of these conditions, probably all, are 
contagious, i. e., transmissable naturally from animal to 
animal. The word contagious has been used in a variety 
of ways to mean communicated by direct contact, communi- 
cated by a living something (contagium) that might be car- 
ried to a distance and finally communicable in any manner, 
transmissable. The agency of transmission may be very 
roundabout-as through a special tick in Texas fever, a 
mosquito in malaria, etc.,-or by direct personal contact, as 
generally in venereal diseases. After all, though exactness 
is necessary, it is better to learn all possible about the means 
of transmission of diseases, than quibble as to the terms to 
be used. INTRODUCTION 
233 
An infectious disease may be acute or chronic. An acute 
infection is one which runs for a relatively short time and is 
"self-limited," so-called, i. e., the organisms cease to mani- 
fest their presence after a time. In some acute infections 
the time is very short-German measles usually runs five 
or six days. Typhoid fever may continue eight to ten 
weeks, sometimes longer, yet it is an acute infectious dis- 
ease. It is not so much the time as the fact of self-limita- 
ion that characterizes acute infections. 
In chronic infections there is little or no evidence of limi- 
tation of the progress of the disease which may continue for 
years. Tuberculosis is usually chronic. Leprosy in man is 
practically always so. Glanders in horses is most commonly 
chronic; in mules and in man it is more apt to be acute. 
Many infections begin acutely and later change to the 
chronic type. Syphilis in man is a good illustration. 
The differences between acute and chronic infections are 
partly due to the nature of the organism, partly to the num- 
ber of organisms introduced and the point of their intro- 
duction and partly to the resistance of the animal infected. 
An infectious disease is said to be specific when one kind 
of organism is responsible for its manifestations-as diph- 
theria due to the Corynebacterium diphtherias, lockjaw due to 
Clostridium tetani, Texas fever due to the Piroplasma. 
bigeminum, etc. It is non-specific when it may be due to a 
variety of organisms, as enteritis (generally), bronchopneu- 
monia, wound infections. 
Henle, as early as 1840, stated certain principles that 
must be established before a given organism can be accepted 
as the cause of a specific disease. These were afterward 
restated by Koch, and have come to be known as "Koch's 
postulates." They may be stated as follows: 
1. The given organism must be found in all cases of the 
disease in question. 
2. No other organism must be found in all cases. 
3. The organism must, when obtained in pure culture, 
reproduce the disease in susceptible animals. 
4. It must be recovered from such animals in pure cul- 
ture and this culture likewise reproduce the disease. 234 
INTRODUCTION 
These postulates have not been fully met with reference 
to any disease, but the principles embodied have been applied 
as far as possible in all those infections which we recognize 
as specific, and whose causative agent is accepted. In many 
diseases recognized as infectious and contagious no organism 
has been found which is regarded as the specific cause. In 
some of these the organism appears to be too small to be 
seen with the highest powers of the microscope, hence they 
are called "ultramicroscopic" organisms. Because these 
agents pass through the finest bacterial filters, they are also 
frequently called "filterable." The term "virus" or "filter- 
able virus" is likewise applied to these "ultramicroscopic" 
and "filterable" agents. 
The term primary infection is sometimes applied to the 
first manifestation of a disease, either specific or non-specific, 
while secondary refers to later developments. For example, 
a secondary general infection may follow a primary wound 
infection, or primary lung tuberculosis be followed by second- 
ary generalized tuberculosis, or primary typhoid fever by a 
secondary typhoid pneumonia. The terms primary and 
secondary are also used where the body is invaded by one 
kind of an organism and later on by another kind; thus a 
primary measles may be followed by secondary infection of 
the middle ear, or a primary influenza may be followed by a 
secondary pneumonia, or a primary scarlet fever by a second- 
ary nephritis (inflammation of the kidney). Where several 
organisms seem to be associated simultaneously in causing 
the condition then the term mixed. inf ection is used- in 
severe diphtheria, streptococci are commonly associated with 
the Corynebacterium diphtherias. In many cases of hog-cholera, 
mixed infections in the lungs and in the intestines are com- 
mon. Wound infections are usually mixed. Auto-infection 
refers to those conditions in which an organism commonly 
present in or on the body in a latent or harmless condition 
gives rise to an infectious process. If the Bacterium coli nor- 
mal to the intestine escapes into the peritoneal cavity, or 
passes into the bladder, a severe peritonitis or cystitis, re- 
spectively, is apt to result. "Boils" and "pimples" are 
frequently autoinfections. Such infections are also spoken INTRODUCTION 
235 
of as endogenous to distinguish them from those due to the 
entrance of organisms from without-exogenous infections. 
Relapses are usually instances of autoinfection. 
Those types of secondary infection where the infecting 
agent is transferred from one disease focus to another or 
several other points and sets up the infection there are 
sometimes called metastases. Such are the transfer of tub- 
ercle bacilli from lung to intestine, spleen, etc., the forma- 
tion of abscesses in internal organs following a primary sur- 
face abscess, the appearance of glanders nodules through- 
out various organs following pulmonary glanders, etc. 
The characteristic of a pathogenic microorganism which 
indicates its ability to cause disease is called its virulence. 
If slightly virulent, the effect is slight; if highly virulent, the 
effect is severe, and may be fatal. 
On the other hand, the characteristic of the host which 
indicates its capacity for infection is called susceptibility. 
If slightly susceptible, infection is slight, if highly suscep- 
tible, the infection is severe. 
Evidently the degree of infection is dependent in large 
measure on the relation between the virulence of the invad- 
ing organism and the susceptibility of the host. High viru- 
lence and great susceptibility mean a severe infection; low 
virulence and little susceptibility a slight infection; while 
high virulence and little susceptibility or low virulence 
and great susceptibility might mean a moderate infection 
varying in either direction. Other factors influencing the 
degree of infection are the number of organisms introduced, 
the point where they are introduced and various condi- 
tions. These will be discussed in another connection (Chap- 
ter XXV). 
The study of pathogenic bacteriology includes the thor- 
ough study of the individual organisms according to the 
methods already given (Chapters XVIII-XXI) as an aid to 
diagnosis and subsequent treatment, bacteriological or other, 
in a given disease. Of far greater importance than the 
treatment, which in most infectious diseases is not specific, 
is the prevention and ultimate eradication of all infectious 
diseases. To accomplish these objects involves further a 236 
INTRODUCTION 
study of the conditions under which pathogenic organisms 
exist outside the body, the paths of entrance into and elimina- 
tion from the body and those agencies within the body itself 
which make it less susceptible to infection or overcome the 
infective agent after its introduction. That condition of the 
body itself which prevents any manifestation of a virulent 
pathogenic organism after it has been once introduced is 
spoken of as immunity in the modern sense. Immunity is 
thus the opposite of susceptibility and may exist in varying 
degrees. 
That scientists are and have been for some years in posses- 
sion of sufficient knowledge to permit of the prevention and 
eradication of most, if not all, of our infectious diseases can 
scarcely be questioned. The practical application of this 
knowledge presents many difficulties, the chief of which is 
the absence of a public sufficiently enlightened to permit the 
expenditure of the necessary funds. Time and educative 
effort alone can surmount this difficulty. It will probably 
be years yet, but it will certainly be accomplished. CHAPTER XXIII. 
PATHOGENIC BACTERIA OUTSIDE THE BODY. 
Pathogenic bacteria may exist outside the body of the 
host under a variety of conditions as follows: 
I. In or on inanimate objects or material. 
(a) As true saprophytes. 
(6) As facultative saprophytes. 
(c) Though obligate parasites, they exist in a latent 
state. 
II. In or on other animals, or products from them: 
A. Susceptible to the disease. 
(a) Sick themselves. 
(As far as human beings are concerned these are 
mainly: 
1. Other human beings for most diseases. 
2. Rats for plague. 
3. Dogs for rabies. 
4. Horses for glanders. 
5. Cattle, swine, parrots for tuberculosis). 
(6) Recovered from illness. 
(c) Never sick but "carriers." 
B. Not susceptible. 
(d) Accidental carriers. 
(e) Serving as necessary intermediate hosts for certain 
stages of the parasite-this applies to protozoal 
diseases only, as yet. ' 
I. 
(a) The bacilli of tetanus, malignant edema and the 
organisms of "gas gangrene" are widely distributed. There 
is no evidence that their entrance into the body is at all 
necessary for the continuation of their life processes, or that  PATHOGENIC BACTERIA OUTSIDE THE BODY 
238 
one case of either of these diseases ever has any connection 
with any other case; they are true saprophytes. Manifestly 
it would be futile to attempt to prevent or eradicate such 
diseases by attacking the organism in its natural habitat. 
Clostridium botulinum, which causes a type of food poisoning in 
man, does not even multiply in the body, but the disease 
symptoms are due to a soluble toxin which is produced dur- 
ing its growth outside the body. 
(b) Organisms like the bacterium of anthrax and the 
bacillus of black-leg from their local occurrence seem to be 
distributed from animals infected, though capable of a. 
saprophytic existence outside the body for years. These 
can no more be attacked during their saprophytic existence 
than those just mentioned. Doubtless in warm seasons of 
the year and in the tropics other organisms pathogenic to 
animals may live and multiply in water or in damp soil 
where conditions are favorable, just as the cholera organism 
in India, and occasionally the typhoid bacillus in temperate 
climates do. 
(c) Most pathogenic organisms, how'ever, when they are 
thrown off from the bodies of animals, remain quiescent, do 
not multiply, in fact always tend to die out from lack of all 
that is implied in a "favorable environment," food, moisture, 
temperature, light, etc. Disinfection is sometimes effective- 
in this class of diseases in preventing new cases. 
II. A. 
(a) The most common infectious diseases of animals 
are transmitted more or less directly from other animals of 
the same species. Human beings get nearly all their dis- 
eases from other human beings who are sick; horses, from 
other horses; cattle, from other cattle; swine, from swine, 
etc. Occasionally transmission from one species to another 
occurs. Tuberculosis of swine most frequently results from 
feeding them milk of tuberculous cattle or from their eating 
the droppings of such cattle. Human beings occasionally 
contract anthrax from wool, hair and hides of animals dead 
of the disease or from postmortems on such animals; glanders- 
from horses; tuberculosis (in children) from tuberculous- PATHOGENIC BACTERIA OUTSIDE THE BODY 23ff 
milk; bubonic plague from rats; rabies practically always 
from the bites of dogs and other rabid animals, etc. The 
mode of limiting this class of diseases is evidently to isolate- 
the sick, disinfect their discharges and their immediate sur- 
roundings, sterilize such products as must be handled or 
used, kill lower animals that are dangerous, and disinfect, 
bury properly, or destroy their carcasses. 
Classes of the sick that are especially dangerous for the 
spread of disease are the mild cases and the undetected cases. 
These individuals do not come under observation and hence- 
not under control. 
(6) This class of carriers offers a difficult problem in the 
prevention of infectious diseases since they may continue to 
give off the organisms indefinitely and thus infect others. 
Typhoid carriers have been known to do so for fifty-five 
years. Cholera, diphtheria, meningitis and other carriers 
are well known in human practice. Carriers among animals, 
have not been so frequently demonstrated, but there is 
every reason for thinking that hog-cholera, distemper, roup, 
influenza and other carriers are common. Carriers furnish 
the explanation for many of the so-called "spontaneous" 
outbreaks of disease among men and animals. 
It is the general rule that those who are sick cease to carry 
the organisms on recovery and it is the occasional ones who- 
do not that are the exceptions. In those diseases in which 
the organism is known it can be determined by examination 
of the patient or his discharges how long he continues to 
give off the causative agent. In those in which the cause- 
is unknown (in human beings, the commonest and most 
easily transmitted diseases, scarlet fever, measles, German 
measles, mumps, chicken-pox, small-pox, influenza), no such 
check is possible. It is not known how long such individ- 
uals remain carriers. Hence isolation and quarantine of 
such convalescents is based partly on experience and partly 
on theory. It is highly probable that in the diseases just 
mentioned transmission occurs in the early stages only, ex- 
cept in small-pox and chicken-pox where the organism seems 
to be in the pustules and transmission by means of material 
from these is possible, though only by direct contact with it. 240 
PATHOGENIC BACTERIA OUTSIDE THE BODY 
The fact that such individuals are known to have had the 
disease is a guide for control. The methods to be used are 
essentially the same as for the sick,, (a), though obviously 
such human carriers are much more difficult to deal with 
since they are well. 
(c) Another class of carriers is those who have never had 
the disease. Such individuals are common and are very 
dangerous sources of infection. Many of them have assoc- 
iated with the sick or with convalescents and these should 
always be suspected of harboring the organisms. Their 
control differs in no way from that of class (6). Unfortu- 
nately a history of such association is too often not available. 
Modern transportation and modern social habits are largely 
responsible for the nearly universal distribution of this type 
of carrier. Their detection is probably the largest single 
problem in the prevention of infectious diseases. A partial 
solution would be universal bacteriological examination. 
In our present stage of progress this is impossible and would 
not detect carriers of diseases of unknown cause. 
The various classes of carriers just discussed are in a 
large part responsible for the continued presence of the 
commoner diseases throughout the country. The difficult- 
ies in control have been mentioned. A complete solution 
of the problem is not yet obtained. The army experience 
of the past few years in the control of infectious diseases 
shows what may be done. 
There is another class of carriers which might be called 
the "universal carrier," i. e., there are certain organisms 
which seem to be constantly or almost constantly present 
in or on the human body. These are micrococci, strepto- 
cocci and pneumococci, all Gram positive organisms. They 
are ordinarily harmless parasites, but on occasion may give 
rise to serious, even fatal, infection. Infected wounds, 
pimples, boils, "common colds," most "sore throats," 
bronchitis, pneumonia are pathological conditions that come 
in this class. Such infections are usually autogenous. 
There is a constant interchange of these organisms among 
individuals closely associated, so that all of a group usually 
harbor the same type though no one individual can be PATHOGENIC BACTERIA OUTSIDE THE BODY 
241 
called the carrier. Whenever, for any reason, the resistance 
of an individual (see Chaps. XXV et seq.) is lowered either 
locally or generally some of these organisms are liable to 
gain a foothold and cause infection. It sometimes happens 
that a strain of dangerous organisms may be developed in 
an individual in this way which is passed around to others 
with its virulence increased and thus cause an epidemic. 
Or, since all of the group are living under the same conditions 
the resistance of all or many of them may be lowered from 
the same general cause and an epidemic result from the 
organism common to all (pneumonia after measles, scarlet 
fever and influenza in camps). Protection of the individual 
is chiefly a personal question, i. e., by keeping up the "nor- 
mal healthy tone" in all possible ways. The use of protect- 
ive vaccines (Chap. XXX) appears to be advisable in such 
instances (colds, pneumonia after measles and influenza, 
inflammation of throat and middle ear following scarlet 
fever and measles). Results obtained in this country dur- 
ing the recent influenza epidemic have been conflicting but 
on the whole appear to show that preventive vaccination 
against pneumonia liable to follow should be practiced. 
It would seem that among groups of individuals where 
infection may be expected the proper procedure would be 
to prepare autogenous vaccines (Chapter XXX) from mem- 
bers of the group and vaccinate all with the object of pro- 
tecting them. 
II. B. 
(d) In this class come the "accidental carriers" like flies, 
fleas, lice, bed-bugs, ticks, and other biting and blood- 
sucking insects, vultures, buzzards, foxes, rats, and carrion- 
eating animals generally; pet animals in the household, etc. 
Here the animals are not susceptible to the given disease 
but become contaminated with the organisms and then 
through defilement of the food or drink or contact with 
individuals or with utensils pass the organisms on to the 
susceptible. Some biting and blood-sucking insects trans- 
mit the organisms through biting infected and non-infected 
animals successively. The spirilloses and trypanosomiases 242 
PATHOGENIC BACTERIA OUTSIDE THE BODY 
seem to be transmitted in this way, though there is evidence- 
accumulating which may place these diseases in the next 
class. Anthrax is considered in some instances to be trans- 
mitted by flies and by vultures in the southern United States. 
Transmission of typhoid, dysentery, cholera and other 
diseases by flies is well established in man. Why not hog- 
cholera from farm to farm by flies, English sparrows, pigeons 
feeding, or by turkey buzzards? Though this would not 
be easy to prove, it seems reasonable. 
Preventing contact of such animals with the discharges 
or with the carcasses of those dead of the disease, destruc- 
tion of insect carriers, screening and prevention of fly breed- 
ing are obvious protective measures. 
(e) In this class come certain diseases for which particu- 
lar insects are necessary for the parasite in question, so that 
certain stages in its life history may be passed therein. The 
surest means for eradicating such diseases is the destruction 
of the insects concerned. Up to the present no bacterial 
disease is known in which this condition exists, unless Rocky 
Mountain spotted fever and typhus fever shall prove to be 
due to bacteria. Such diseases are all due to protozoa. 
Among them are Texas fever, due to Piroplasma bigeminum 
in this country which has been eradicated in entire districts 
by destruction of the cattle tick (Margaropus annulatus.') 
Piroplasmoses in South Africa among cattle and horses, 
and in other countries are transmitted in similar ways. 
Probably many of the diseases due to spirochetes and trypan- 
osomes are likewise transmitted by necessary insect inter- 
mediaries. In human medicine the eradication of yellow 
fever from Panama and Cuba is due to successful warfare 
against a certain mosquito {Stegomyia}. So the freeing of 
large areas in different parts of the world from malaria 
follows the destruction of the mosquitoes. The prevention 
of typhus fever and of trench fever by "delousing" methods 
is familiar from recent army experience though for typhus 
this method has been practiced in Russia for more than ten 
years to the author's personal knowledge. The campaign 
against disease in animals and man from insect sources 
must be considered as still in its infancy. The full utiliza- 
tion of tropical lands depends largely on the solution of this 
problem. CHAPTER XXIV. 
PATHS OF ENTRANCE OF PATHOGENIC 
ORGANISMS, 
OR 
CHANNELS OF INFECTION. 
A. The Skin.-If the skin is healthy there is no oppor- 
tunity for bacteria to penetrate it. It is protected not 
only by the stratified epithelium, but also in various animals, 
by coats of hair, wool, feathers, etc. The secretion pressure 
of the healthy sweat and oil glands acts as an effective bar 
even to motile bacteria. Nevertheless a very slight injury 
only is sufficient to give normal surface parasites and other 
pathogenies, accidentally or purposely brought in contact 
with it, an opportunity for more rapid growth and even 
entrance for general infection. Certain diseases due to 
higher fungi are characteristically "skin diseases" and rarely 
become general-various forms of favus, trichophyton in- 
fections, etc. A few disease organisms, tetanus, malignant 
edema, usually get in through the skin; others, black-leg, 
anthrax, quite commonly; and those diseases transmitted 
by biting and blood-sucking insects, piroplasmoses, trypano- 
somiases, spirilloses, scarcely in any other way. Defective 
secretion in the skin glands from other causes, may permit 
lodgment and growth of bacteria in them or in the hair 
follicles. "Pimples" and boils in man and local abscesses- 
occasionally in animals are illustrations. Sharp-edged and 
freely bleeding wounds are less liable to be infected than 
contusions, ragged wounds, burns, etc. The flowing blood 
washes out the wound and the clotting seals it, while there 
is less material to be repaired by the leukocytes and they are 
free to care for invading organisms (phagocytosis). Patho- 244 
ENTRANCE OF PATHOGENIC ORGANISMS 
genic organisms, especially pus cocci, frequently gain lodg- 
ment in the milk glands and cause local (mastitis) or general 
infection. 
f. Mucosae directly continuous with the skin and lined 
with stratified epithelium are commonly well protected 
thereby and by the secretions. 
(a) The external auditory meatus is rarely the seat even of 
local infection. The tympanic cavity is normally sterile, 
though it may become infected by extension through the 
Eustachian tube from the pharynx (otitis media). 
(b) The conjunctiva is frequently the seat of localized, 
very rarely the point of entrance for a generalized infection, 
except after severe injury. Those diseases whose path of 
entrance is generally assumed to be the respiratory tract 
(see "Lungs" below) might also be admitted through the 
eye. Material containing such organisms might get on the 
conjunctiva and be washed down through the lachrymal 
canal into the nose. Experiment has shown that bacteria 
may pass in this way in a few minutes. In case masks are 
worn to avoid infection from patients suffering with these 
diseases, the eyes should therefore be protected as well as 
the nose and mouth. 
(c) The nasal cavity on account of its anatomical struc- 
ture retains pathogenic organisms which give rise to local 
infections more frequently than other mucosse of its charac- 
ter. These may extend from here to middle ear, neighbor- 
ing sinuses, or along the lymph spaces of the olfactory nerve 
into the cranial cavity (meningitis). Acute coryza ("colds" 
in man) is characteristic. Glanders, occasionally, is pri- 
mary in the nose, as is probably roup in chickens, leprosy 
in man. The meningococcus and the virus of poliomyel- 
itis pass from the nose into the cranial cavity without local 
lesions in the former. 
(d) The mouth cavity is ordinarily protected by its epi- 
thelium and secretions, though the injured mucosa is a 
common source of actinomycosis infection, as well as thrush. 
In foot-and-mouth disease no visible lesions seem necessary 
to permit the localization of the unknown infective agent. LUNGS 
245 
(e) The tonsils afford a ready point of entrance for ever- 
present micrococci and streptococci whenever occasion offers 
(follicular tonsillitis, "quinsy"), and articular rheumatism is 
not an uncommon sequel. The diphtheria bacillus charac- 
teristically seeks these structures for its development. 
Tubercle and anthrax organisms occasionally enter here. 
(/) The pharynx is the seat of localized infection as in 
micrococcal, strepococcal and diphtherial "sore throat" in 
human beings, but both it and the esophagus are rarely 
infected in animals except as the result of injury. 
{g} The external genitalia are the usual points of entrance 
for the venereal organisms in man (gonococcus, Treponema 
pallidum, and Ducrey's bacillus). The bacillus of conta- 
gious abortion and probably the trypanosome of dourine 
are commonly introduced through these channels in animals. 
C. Lungs.-The varied types of pneumonia due to many 
different organisms (tubercle, glanders, influenza, plague 
bacilli, pneumococcus, streptococcus, micrococcus and many 
others) show how frequently these organs are the seat of a 
localized infection, which may or may not be general. 
Whether the lungs are the actual point of entrance in these 
cases is a question which is much discussed at the present 
time, particularly with reference to tuberculosis. The 
mucous secretion of the respiratory tract tends to catch 
incoming bacteria and other small particles and the ciliary 
movement along bronchial tubes and trachea tends to carry 
such material out. "Foreign body pneumonia" shows clini- 
cally, and many observers have shown experimentally that 
microorganisms may reach the alveoli even though the 
exchange of air between them and the bronchioles and larger 
bronchi takes place ordinarily only by diffusion. The pres- 
ence of carbon particles in the walls of the alveoli in older 
animals and human beings and in those that breathe dusty 
air for long periods indicates strongly, though it does not 
prove absolutely, that these came in with inspired air. On 
the other hand, experiment has shown that tubercle bacilli 
introduced into the intestine may appear in the lungs and 
cause disease there and not in the intestine. It is probably 
safe to assume that in those diseases which are transmitted 246 
ENTRANCE OF PATHOGENIC ORGANISMS 
most readily through close association though not neces- 
sarily actual contact, the commonest path is through the 
respiratory tract, which may or may not show lesions (small- 
pox, scarlet fever, measles, chicken-pox, whooping-cough, 
pneumonic plague in man, lobar and bronchopneumonias 
and influenza in man and animals, some cases of glanders 
and tuberculosis). On the other hand, the fact that the 
Bacterium typhosum and Bacterium coli may cause pneumonia 
when they evidently have reached the lung from the intes- 
tinal tract, and the experimental evidence of lung tubercu- 
losis above mentioned show that this route cannot be ex- 
cluded in inflammations of the lung. 
D. Alimentary Tract.-The alimentary tract affords the 
ordinary path of entrance for the causal microbes of many 
of the diseases of animals and man, since they are carried 
into the body most commonly and most abundantly in the 
food and drink. 
h (a) The stomach is rarely the seat of local infection, even 
in ruminants, except as the result of trauma. The character 
of the epithelium in the rumen, reticulum and omasum in 
ruminants, the hydrochloric acid in the abomasum and in 
the stomachs of animals generally are usually sufficient pro- 
tection. Occasionally anthrax "pustules" develop in the 
gastric mucosa. (The author saw nine such pustules in a 
case of anthrax in a man.) 
(6) The intestines are frequently the seat of localized 
infections, as various "choleras" and "dysenteries" in men 
and many animals, anthrax, tuberculosis, Johne's disease. 
Here doubtless enter the organisms causing "hemorrhagic 
septicemias" in many classes of animals, and numerous 
others. These various organisms must have passed through 
the stomach and the question at once arises, why did the 
HC1 not destroy them? It must be remembered that the 
acid is present only during stomach digestion, and that 
liquids taken on an "empty stomach" pass through rapidly 
and any organisms present are not subjected to the action of 
the acid. Also spores generally resist the acid. Other 
organisms may pass through the stomach within masses of 
undigested food. The fact that digestion is going on in the MECHANISM OF ENTRANCE OF ORGANISMS 
247 
stomach of ruminants practically all the time may explain 
the relative freedom of adult animals of this class from 
■"choleras" and "dysenteries." 
MECHANISM OF ENTRANCE OF ORGANISMS. 
In the preceding chapters statements have been made 
that "bacteria enter" at various places or they "pass 
through" different mucous membranes, skin, etc. Strictly 
speaking such statements are incorrect-bacteria do not 
"enter" or "pass through" of themselves. It is true that 
some of the intestinal organisms are motile, but most of the 
bacteria which are pathogenic are non-motile. Even the 
motile ones can not make their way against fluids secreted 
nr excreted on free surfaces. Bacteria cannot pass by dif- 
fusion through membranes since they are finite particles and 
not in solution. 
In the case of penetrating wounds bacteria may be carried 
mechanically into the tissues, but this is exceptional in most 
infections. Also after gaining lodgment they may gradually 
grow through by destroying tissue as they grow, but this 
is a minor factor. Evidently, there must be some mechan- 
ism by which they are carried through. The known mechan- 
isms for this in the body are ameboid cells, especially the 
phagocytes. It is most probable that these are the chief 
agents in getting bacteria into the tissues through various 
free surfaces. The phagocytes engulf bacteria, carry them 
into the tissues and either destroy them, are destroyed by 
them, or may disgorge or excrete them free in the tissues or in 
the blood. 
DISSEMINATION OF ORGANISMS. 
Dissemination of organisms within the tissues occurs either 
through the lymph channels or the bloodvessels or both. 
If through the lymph vessels only it is usually much more 
restricted in extent, or much more slowly disseminated, 
while blood dissemination is characterized by the number 
of organs involved simultaneously. 248 
ENTRANCE OF PATHOGENIC ORGANISMS 
PATHS OF ELIMINATION OF PATHOGENIC MICRO- 
ORGANISMS. 
I. Directly from the point of injury. This is true in 
infected wounds open to the surface, skin glanders (farcy), 
black-leg, surface anthrax, exanthemata in man and animals 
(scarlet fever (?), measles (?), smallpox; hog erysipelas, foot- 
and-mouth disease): also in case of disease of mucous mem- 
branes continuous with the skin-from nasal discharges 
(glanders), saliva (foot-and-mouth disease), material coughed 
or sneezed out (tuberculosis, influenza, pneumonias), ure- 
thral and vaginal discharges (gonorrhea and syphilis in man, 
contagious abortion and dourine in animals), intestinal dis- 
charges (typhoid fever, "choleras," "dysenteries," anthrax, 
tuberculosis, Johne's disease). Material from nose, mouth 
and lungs may be swallowed and the organisms passed out 
through the intestines. 
II. Indirectly through the secretions and the excretions 
where the internal organs are involved. The saliva of rabid 
animals contains the ultramicroscopic virus of rabies (the 
sympathetic ganglia within the salivary glands, and pan- 
creas also, are affected in this disease as well as the cells of 
the central nervous system). The gall-bladder in man is 
known to harbor colon and typhoid bacilli, as that of hog- 
cholera hogs does the virus of this disease. It may harbor 
analogous organisms in other animals, though such knowl- 
edge is scanty. The kidneys have been shown experimen- 
tally to excrete certain organisms introduced into the circu- 
lation within a few minutes (micrococci, colon and typhoid 
bacilli, anthrax). Typhoid bacilli occur in the urine of 
typhoid-fever patients in about 25 per cent, of all cases and 
the urine of hogs with hog cholera is highly virulent. Most 
observers are of the opinion, however, that under natural 
conditions the kidneys do not excrete bacteria unless they 
themselves are infected. 
The milk both of tuberculous cattle and tuberculous 
women has been shown to contain tubercle bacilli even when 
the mammary glands are not involved. Doubtless such bacteria 
are carried through the walls of the secreting tubules or of 
the smaller ducts by phagocytes and are then set free in 
the milk. LOCATION OF INFECTIVE ORGANISMS 
249 
SPECIFICITY OF LOCATION OF INFECTIVE ORGANISMS. 
It is readily apparent that certain disease organisms tend 
to locate themselves in definite regions and the question 
arises, Is this due to any specific relationship between organ- 
ism and tissue or not? Diphtheria in man usually attacks 
the tonsils first, gonorrhea and syphilis the external geni- 
tals, tuberculosis the lung, "choleras" the small intestine, 
"dysenteries" the large intestine, influenza the lungs. In 
these cases the explanation is probably that the points 
attacked are the places where the organism is most commonly 
carried, with no specific relationship, since all of these organ- 
isms (Asiatic cholera excepted) also produce lesions in other 
parts of the body when they reach them. On the other hand, 
the virus of hydrophobia attacks nerve cells, leprosy fre- 
quently singles out nerves, glanders bacilli introduced into 
the abdominal cavity of a young male guinea-pig cause an 
inflammation of the testicle, malarial parasites and piro- 
plasms attack the red blood corpuscles, etc. In fact, most 
pathogenic protozoa are specific in their localization either 
in certain tissue cells or in the blood or lymph. In these 
cases there is apparently a real chemical relationship, as 
there is also between the toxins of bacteria and certain tissue 
cells (tetanus toxin and nerve cells). Whether "chemo- 
therapy" will ever profit from a knowledge of such chemical 
relationships remains to be developed. It appears that a 
search for these specific chemical substances with the object 
of combining poisons with them so that the organisms might 
in this way be destroyed, would be a profitable line of re- 
search. CHAPTER XXV. 
IMMUNITY. 
Immunity, as has already been stated, implies such a con- 
dition of the body that pathogenic organisms after they 
have been introduced are in'capableof manifesting themselves, 
and are unable to cause disease. The word has come to 
have a more specific meaning than resistance in many in- 
stances, in other cases the terms are used synonymously. 
It is the opposite of susceptibility. The term must be under- 
stood always in a relative sense, since no animal is immune 
to all pathogenic organisms, and conceivably not entirely 
so to anyone, because there is no question that a sufficient 
number of bacteria of any kind might be injected into the 
circulation to kill an animal, even though it did it purely 
mechanically. 
Immunity may be considered with reference to a single 
individual or to entire divisions of the organic world, with 
all grades between. Thus plants are immune to the diseases 
affecting animals; invertebrates to vertebrate diseases; cold- 
blooded animals to those of warm blood; man is immune 
to most of the diseases affecting other mammals; the rat to 
anthrax, which affects other rodents and most mammals; 
the well-known race of Algerian sheep is likewise immune 
to anthrax while other sheep are susceptible; the negro 
appears more resistant to yellow fever than the white; some 
few individuals in a herd of hogs always escape an epizootic 
of hog cholera, etc. 
Immunity within a given species is modified by a number 
of factors-age, state of nutrition, extremes of heat or cold, 
fatigue, excesses of any kind, in fact, anything which tends 
to lower the "normal healthy tone" of an animal also tehds 
to lower its resistance. Children appear more susceptible 
to scarlet fever, measles, whooping-cough, etc., than adults; IMMUNITY 
251 
young cattle more frequently have black-leg than older ones 
(these apparently greater susceptibilities may be due in part 
to the fact that most of the older individuals have had the 
diseases when young and are immune for this reason). Ani- 
mals weakened by hunger or thirst succumb to infection 
more readily. Frogs and chickens are immune to tetanus, 
but if the former be put in water and warmed up to and 
kept at about 37°, and the latter be chilled for several hours 
in ice-water, then each may be infected. Pneumonia fre- 
quently follows exposure to cold. The immune rat may be 
given anthrax if first he is made to run in a "squirrel cage" 
until exhausted. Alcoholics are far less resistant to infec- 
tion than temperate individuals. "Worry," mental an- 
guish, tend to predispose to infection. 
The following outlines summarize the different classifi- 
cations of immunity so far as mammals are concerned for 
the purposes of discussion. 
Immunity. 
1. Inherited'through the germ cell or cells. 
A. Congenital 
(a) By having the 
disease in utero. 
(&) By absorption 
of immune sub- 
stances from the 
mother. 
I. Natural 
2. Acquired in utero. 
B. Acquired by having the disease. 
II. Artificial-acquired through human agency by: 
1. Introduction of the organism or its products. 
2. Introduction of the blood serum of an immune animal. 
Immunity. 
I. Active-due to the introduction of the organism or due to the intro- 
duction of the products of the organism. 
A. Naturally by having the disease. 
B. Artificially. 
1. By introducing the organism: 
f 1. Passage through another animal. 
| 2. Drying. 
| 3. Growing at a higher temperature. 
! 4. Heating the cultures. 
| 5. Treating with chemicals. 
[ 6. Sensitizing. 
.7. Cultivation on artificial media. 
(a) Alive and virulent. 
(&) Alive and virulence 
reduced by 
(c) Dead. 
2. By introducing the products of the organism. 
II. Passive-due to the introduction of the blood serum of an actively 
1 immunized animal. 252 
IMMUNITY 
Immunity present in an animal and not due to human 
interference is to be regarded as natural immunity, while if 
brought about by man's effort it is considered artificial. 
Those cases of natural immunity mentioned above which 
are common to divisions, classes, orders, families, species or 
races of organisms and to those few individuals where no 
special cause is discoverable, must be regarded as instances 
of true inheritance through the germ cell as other char- 
acteristics are. All other kinds of immunity are acquired. 
Occasionally young are born with every evidence that they 
have had a disease in utero and are thereafter as immune as 
though the attack had occurred after birth ("small pox 
babies," "hog-cholera pigs"). Experiment has shown that 
immune substances may pass from the blood of the mother 
to the fetus in utero and the young be immune for a time 
after birth (tetanus) This is of no practical value as yet. 
It is a familiar fact that with most infectious diseases re- 
covery from one attack confers a more or less lasting im- 
munity, though there are marked exceptions. 
Active Immunity.-By active immunity is meant that 
which is due to the actual introduction of the organism, or 
in some cases of its products. The term active is used be- 
cause the body cells of the animal immunized perform the 
real work of bringing about the immunity as will be dis- 
cussed later. In passive immunity the blood serum of an 
actively immunized animal is introduced into a second ani- 
mal, which thereupon becomes immune, though its cells are 
not concerned in the process. The animal is passive, just 
as a test-tube, in which a reaction takes place, plays no other 
part than that of a passive container for the reagents. 
In active immunity the organism may be introduced in 
what is to be considered a natural manner, as when an ani- 
mal becomes infected, has a disease, without human inter- 
ference. Or the organism may be purposely introduced to 
bring about the immunity. For certain purposes the intro- 
duction of the products of the organism (toxins) is used to 
bring about active immunity (preparation of diphtheria and 
tetanus antitoxin from the horse). The method of produc- 
ing active immunity by the artificial introduction of the ACTIVE IMMUNITY 
253 
organism is called vaccination, and a vaccine must therefore 
contain the organism. Vaccines for bacterial diseases are 
frequently called bacterins. The use of the blood serum of 
an immunized animal to confer passive immunity on a 
second animal is properly called serum therapy, and the 
serum so used is spoken of as an antiserum, though the latter 
word is also used to denote any serum containing any kind 
of an antibody (Chapters XXVII-XXXI). In a few in- 
stances both the organism and an antiserum are used to 
cause both active and passive immunity (serum-simultan- 
eous method in immunizing against hog cholera). 
In producing active immunity the organism may be intro- 
duced (a) alive and virulent, but in very small doses, or in 
combination with an immune serum, as just mentioned for 
hog cholera. The introduction of the live virulent organism 
alone is done only experimentally as yet, as it is obviously 
too dangerous to do in practice, except under the strictest 
control (introduction of a single tubercle bacillus, followed 
by gradually increasing numbers-Barber and Webb). 
More commonly the organisms are introduced (b) alive but 
with their virulence reduced ("attenuated") in one of several 
ways: (1) By passing the organism through another animal 
as is the case with smallpox vaccine derived from a calf or 
heifer. This method was first introduced by Jenner in 
1795 and was the first practical means of preventing disease 
by vaccination. This word was used because material was 
derived from a cow-Latin vacca. (2) By drying the organ- 
ism, as is done in the preparation of the vaccine for the Pas- 
teur treatment of rabies, where the spinal cords of rabbits are 
dried for varying lengths of time-one to four days, Russian 
method, one to three days, German method, longer in this 
country. (It is probable that the passage of the "fixed 
virus" through the rabbit is as important in this procedure 
as the drying, since it is doubtful if the "fixed virus" is 
pathogenic for man.) It would be more correct to speak of 
this as a preventive vaccination against rabies, since the latter 
is one of the few diseases which is not amenable to treatment. 
The patient always dies if the disease develops. (3) The 
organism may be attenuated by growing at a temperature 254 
IMMUNITY 
above the normal. This is the method used in preparing- 
anthrax vaccine as done by Pasteur originally. (4) Instead 
of growing at a higher temperature the culture may be 
heated in such a way that it is not killed but merely weak- 
ened. Black-leg vaccines are made by this method. (5) 
Chemicals are sometimes added to attentuate the organisms, 
as was formerly done in the preparation of black-leg vaccine 
by Kruse's method in Germany. The use of toxin-anti- 
toxin mixtures in immunizing against diphtheria and in the 
preparation of diphtheria antitoxin from horses is an appli- 
cation of the same principle, though here it is the product 
of the organism and not the organism whose action is weak- 
ened. (6) Within the past few years the workers in the Pas- 
teur Institute in Paris have been experimenting with vac- 
cines prepared by treating living virulent bacteria with 
antisera ("sensitizing them") so that they are no longer 
capable of causing the disease when introduced, but do 
cause the production of an active immunity. The method 
has been used with typhoid fever bacilli in man and seems 
to be successful. It remains to be tried out further before 
its worth is demonstrated (the procedure is more compli- 
cated and the chance for infection apparently much greater 
than by the use of killed cultures). The term sero-bacterins 
is used by manufacturers in this country to designate such 
bacterial vaccines. (7) Growing on artificial culture media 
reduces the virulence of most organisms after a longer or 
shorter time. This method has been tried with many or- 
ganisms in the laboratory, but is not now used in practice. 
The difficulties are that the attenuation is very uncertain 
and that the organisms tend to regain their virulence when 
introduced into the body. 
In producing active immunity against many bacterial 
diseases the organisms are introduced (c) dead. They are 
killed by heat or by chemicals, or by using both methods 
(Chapter XXX). 
When the products of an organism are introduced the 
resulting immunity is against the products only and not 
against the organism. If the organism itself is introduced 
there results an immunity against it and in some cases also ACTIVE IMMUNITY 
255 
against the products, though the latter does not necessarily 
follow. Hence the immunity may be antibacterial or anti- 
toxic or both. 
Investigation as to the causes of immunity and the various 
methods by which it is produced has not resulted in the dis- 
covery of specific methods of treatment for as many dis- 
eases as was hoped for at one time. Just at present progress 
in serum therapy appears to be at a standstill, though vac- 
cines are giving good results in many instances not believed 
possible a few years ago. As a consequence workers in all 
parts of the world are giving more and more attention to 
the search for specific chemical substances, which will destroy 
invading parasites and not injure the host (chemotherapy). 
Nevertheless, in the study of immunity very much of value 
in the treatment and prevention of disease has been learned. 
Also much knowledge which is of the greatest use in other 
lines has been accumulated. Methods of diagnosis of great 
exactness have resulted, applicable in numerous diseases. 
Ways of detecting adulteration in foods, particularly foods 
from animal sources, and of differentiating proteins of varied 
origin, as well as means of establishing biological relation- 
ships and differences among groups of animals through 
"immunity reactions" of blood serums have followed from 
knowledge gained by application of the facts or the methods 
of immunity research. Hence the study of "immunity 
problems" has come to include much more than merely the 
study of those factors which prevent the development of 
disease in an animal or result in its spontaneous recovery. 
A proper understanding of the principles of immunity neces- 
sitates a study of these various features and they will be 
considered in the discussion to follow. CHAPTER XXVI. 
THEORIES OF IMMUNITY. 
Pasteur and the bacteriologists of his time discovered 
that bacteria cease to grow in artificial culture media after 
a time, because of the exhaustion of the food material in 
some cases and because of the injurious action of their own 
products in other instances. These facts were brought for- 
ward to explain immunity shortly after bacteria were shown 
to be the cause of certain diseases. Theories based on these 
observations were called (1) "Exhaustion Theory"of Pasteur, 
and (2) "Noxious Retention Theory" of Chauveau respec- 
tively. The fact, soon discovered, that virulent pathogenic 
bacteria are not uncommonly present in perfectly healthy 
animals, and the later discovery that immunity may be 
conferred by the injection of dead bacteria have led to the 
abandonment of both these older ideas. The (3) " Unfavor- 
able Environment" theory of Baumgartner, i. e., bacteria 
do not grow in the body and produce disease because their 
surroundings are not suitable, in a sense covers the whole 
ground, though it is not true as to the first part, as was 
pointed out above, and is of no value as a working basis, 
since it offers no explanation as to what the factors are that 
constitute the "unfavorable environment." Metchnikoff 
brought forward a rational explanation of immunity with 
his (4) "Cellular or Phagocytosis Theory." As first pro- 
pounded it based immunity on the observed fact that cer- 
tain white blood corpuscles, phagocytes, engulf and destroy 
bacteria. Metchinkoff has since elaborated the original 
theory to explain facts of later discovery. Ehrlich soon after 
published his (5) "Chemical or Side-chain Theory" which 
seeks to explain immunity on the basis of chemical substances 
in the body which may in part destroy pathogenic organ- 
isms or in part neutralize their products; or in some instances PLATE VI 
PAUL EHRLICH THEORIES OF IMMUNITY 
257 
there may be an absence of certain chemical substances 
in the body cells so that bacteria or their products cannot 
unite with the cells and hence can do no damage. 
At the present time it is generally accepted, in this coun- 
try at least, that Ehrlich's theory explains immunity in 
many diseases as well as many of the phenomena related to 
immunity, and in other diseases the phagocytes, frequently 
assisted by chemical substances, are the chief factors. Spe- 
cific instances are discussed in Pathogenic Bacteriologies 
which should be consulted. It is essential that the student 
should be familiar with the basic ideas of the chemical theory, 
not only from the standpoint of immunity, but also in order 
to understand the principles of a number of valuable methods 
of diagnosis. 
The chemical theory rests on three fundamental physi- 
ological principles: (1) the response of cells to stimuli, in 
this connection specific chemical stimuli, (2) the presence 
within cells of specific chemical groups which combine with 
chemical stimuli and thus enable them to act on the cell, 
which groups Ehrlich has named receptors, and (3) the 
"over-production" activity of cells as announced by Weigert. 
1. That cells respond to stimuli is fundamental in physi- 
ology. These stimuli may be of many kinds as mechanical, 
electrical, light, thermal, chemical, etc. The body possesses 
groups of cells specially developed to receive some of these 
stimuli-touch cells for mechanical stimuli, retinal cells for 
light, temperature nerve endings for thermal, olfactory and 
gustatory cells for certain chemical stimuli. Response to 
chemical stimuli is well illustrated along the digestive tract. 
That the chemical stimuli in digestion may be more or less 
specific is shown by the observed differences in the enzymes 
of the pancreatic juice dependent on the relative amounts 
of carbohydrates, fats, or proteins in the food, the specific 
enzyme in each case being increased in the juice with the 
increase of its corresponding foodstuff. The cells of the 
body, or certain of them at least, seem to respond in a spe- 
cific way when substances are brought into direct contact 
with them, that is, without having been subjected to diges- 
tion in the alimentary tract, but injected directly into the 258 
THEORIES OF IMMUNITY 
blood or lymph stream. Cells may be affected by stimuli 
in one of three ways: if the stimulus is too weak, there is 
no effect (in reality there is no "stimulus" acting); if the 
stimulus is too strong, the cell is injured, or may be destroyed; 
if the stimulus is of proper amount then it excites the cell 
to increased activity, and in the case of specific chemical 
stimuli the increased activity, as mentioned for the pan- 
creas, shows itself in an increased production of whatever 
is called forth by the chemical stimulus. In the case of many 
organic chemicals, the substances produced by the cells 
under their direct stimulation are markedly specific for the 
particular substance introduced. 
2. Since chemical action always implies at least two 
bodies to react, Ehrlich assumes that in every cell which is 
affected by a chemical stimulus there must therefore be a 
chemical group to unite with this stimulus. He further 
states that there must be as many different kinds of these 
groups as there are different kinds of chemicals which stimu- 
late the cell. Since these groups are present in the body 
cells to take up different kinds of chemical substances, 
Ehrlich calls them receptors. Since these groups must be 
small as compared with the cell as a whole, and must be 
more or less on the surface and unite readily with chemical 
substances he further speaks of them as "side-chains" after 
the analogy of compounds of the aromatic series especially. 
The term receptors is now generally used. As was stated 
above, the effect of specific chemical stimuli is to cause the 
production of more of the particular substance for which it is 
specific and in the class of bodies under discussion, the 
particular product is these cell receptors with which the chemi- 
cal may unite. 
3. Weigert first called attention to the practically con- 
stant phenomenon that cells ordinarily respond by doing 
more of a particular response than is actually called for by 
the stimulus, that there is always an "overproduction" of 
activity. In the case of chemical stimuli this means an 
increased production of the specific substance over and above 
the amount actuallv needed. THEORIES OF IMMUNITY 
259 
The student will better understand this theory if he re- 
calls his fundamental physiology. Living substance is char- 
acterized, among other things, by irritability which is in- 
stability. It is in a constant state of unstable equilibrium. 
Whenever the equilibrium becomes permanently stable the 
substance is dead. It is also continually attempting to 
restore disturbances in its equilibrium. Whenever a chem- 
ical substance unites with a chemical substance in the cell, 
a receptor, the latter is, so far as the cell is concerned, 
thrown out of function for that cell. The chemical equi- 
librium of the latter is upset. It attempts to restore this 
and does so by making a new receptor to take the place 
of the one thrown out of function. If this process is con- 
tinued, i. e., if the new receptor is similarly "used up" 
and others similarly formed are also, then the cell will pre- 
pare a supply of these and even an excess, according to 
Weigert's theory. Whenever a cell accumulates an excess 
of products the normal result is that it excretes them from 
its own substance into the surrounding lymph, whence they 
reach the blood stream to be either carried to the true 
excretory organs, utilized by other cells or remain for a 
longer or shorter time in the blood. Hence the excess of 
receptors is excreted from the cell that forms them and they 
become free in the blood. These free receptors are termed 
antibodies. They are receptors but* instead of being retained 
in the cell are free in solution in the blood. One function 
of the free receptor, the antibody, is always to unite with 
the chemical substance which caused it to be formed. It may 
have additional functions. The chemical substance which 
caused the excess formation of receptors, antibodies, is 
termed an antigen for that particular kind of antibody. 
To recapitulate, Ehrlich's theory postulates specific chemi- 
cal stimuli, which react with specific chemical substances in 
the body cells, named receptors, and that these receptors, 
according to Weigert, are produced in excess and hence are 
excreted from the cell and become free receptors in the blood 
and lymph. These free receptors are the various kinds of 
antibodies, the kind depending on the nature of the stimulus, 
antigen, the substance introduced. Any substance which 260 
THEORIES OF IMMUNITY 
when introduced into the body causes the formation of an 
antibody of any kind whatsoever is called an antigen,1 i. e., 
anti (body) former. 
The foregoing discussion explains Ehrlich's theory of im- 
munity. According to this theory the manner of formation 
of all antibodies is the same. The kind of antibody and 
the manner of its action will differ with the different kinds 
of antigens used. 
The succeeding chapters discuss some of the kinds of 
antibodies, the theory of their action and some practical 
applications. It must be borne in mind throughout the 
study of these, as has been stated, that every antibody has 
the property of uniting with its antigen whether it has any 
property in addition or not. 
Just what antibodies are chemically has not been deter- 
mined because no one has as yet succeeded in isolating them 
chemically pure. To the author they appear to be en- 
zymes. 
Antigens were considered by Ehrlich to be proteins or to 
be related to proteins. Most workers since Ehrlich have 
held similar views. Dr. Carl Warden of the University of 
Michigan has been doing much work in recent years in which 
he is attempting to show that the antigens are not proteins 
but are fats or fatty acids. Mr. E. E. H. Boyer, in his 
work (not yet published) in the author's laboratory for the 
degree of Ph.D., received in June, 1920, succeeded in pro- 
ducing various antibodies from Bacterium coli antigens. In 
these antigens he could detect only fatty acids or salts of 
fatty acids. If the work of these men is confirmed, it will 
open up a most interesting and extremely important field 
in immunity and in preventive medicine. It is not apparent 
that the nature of the antigen would affect Ehrlich's theory 
of the formation of antibodies. 
The author has no doubt that eventually the formation 
of antibodies and the reactions between them and their 
antigens will be explained on the basis of physical-chemical 
laws, but this probably awaits the discovery of their nature. 
'The term "antigen" is also used to designate substances which may take 
the place of what are supposed to be the true antigens in certain diagnostic 
reactions (Chapter XXIX, Complement Fixation Test for Syphilis). CHAPTER XXVII. 
RECEPTORS OF THE FIRST ORDER. 
ANTITOXINS-ANTIENZYMES. 
' The general characteristics of toxins have been described 
(Chapter XII). It has been stated that they are more or 
less specific in their action on cells. In order to affect a cell 
it is evident that a toxin must enter into chemical combina- 
tion with it. This implies that the toxin molecule possesses 
a chemical group which can combine with a receptor of the 
cell. This group is called the haptophore or combining 
group. The toxic or injurious portion of the toxin molecule 
is likewise spoken of as the toxophore group. When a toxin 
is introduced into the body its ha/ptophore group combines 
with suitable receptors in different cells of the body. If not 
too much of the toxin is given, instead of injuring, it acts as a 
chemical stimulus to the cell in the manner already described. 
The cell in response produces more of the specific thing, 
which in this instance is more receptors which can combine 
with the toxin, i. e., with its haptophore group. If the stim- 
ulus is kept up, more and more of these receptors are pro- 
duced until an excess for the cell accumulates, which excess 
is excreted from the individual cell and becomes free in the 
blood. These free receptors have, of course, the capacity 
to combine with toxin through its haptophore group. When 
the toxin is combined with these free receptors, it cannot, 
combine with any other receptors, e. g., those in another 
cell and hence cannot injure another cell. These free recep- 
tors constitute, in this case, antitoxin, so-called because they 
can combine with toxin and hence neutralize it. Anti- 
toxins are specific-that is, an antitoxin which will combine 
with the toxin of Clostridium tetani will not combine with 
that of Coryn ebacterium diphtherias, or of Clostridium botu- 
linum, or of any other toxin, vegetable or animal. 262 
RECEPTORS OF THE FIRST ORDER 
When a toxin is kept in solution for some time or when it 
is heated above a certain temperature (different for each 
toxin) it loses its poisonous character. It may be shown, 
however, that it is still capable of uniting with antitoxin, 
and preventing the latter from uniting with a fresh toxin. 
This confirms the hypothesis that a toxin molecule has at 
least two groups: a combining or haptophore, and a poison- 
ing or toxophore group. A toxin which has lost its poisonous 
property, its toxophore group, is spoken of as a toxoid. The 
theory of antitoxin formation is further supported by the 
fact that the proper introduction of toxoid, the haptophore 
group, and hence the real stimulus, can cause the production 
of antitoxin to a certain extent at least. 
The close relationship between toxins and enzymes has 
already been pointed out. This is still further illustrated 
by the fact that when enzymes are properly introduced into 
the tissues of an animal there is formed in the animal an 
antienzyme specific for the enzyme in question which can 
prevent its action. The structure of enzymes, as composed 
of a haptophore, or uniting, and a zymophore or digesting 
(or other activity) group, is similar to that of toxins, and 
enzymoids or enzymes which can combine with the substance 
acted on but not affect it further, have been demonstrated. 
These free cell receptors, antitoxins or antienzymes, which 
are produced in the body by the proper introduction of 
toxins or enzymes, respectively, have the function of com- 
bining with these bodies but no other action. As was pointed 
out above, this is sufficient to neutralize the toxin or enzyme 
and prevent any injurious effect since they can unite with 
nothing else. Since these receptors are the simplest type 
which has been studied as yet, they are spoken of by Ehrlich 
as receptors of the first order. Other antibodies which are 
likewise free receptors of the first order and have the func- 
tion of combining only have been prepared and will be 
referred to in their proper connection. They are mainly of 
theoretical interest. 
Ehrlich did a large part of his work on toxins and anti- 
toxins with ricin, the toxin of the castor-oil bean, abrin, 
from the jequirity bean, robin from the locust tree, and with A N TI TOXINS-AN TIENZ YMES 
263 
the toxins and antitoxins for diphtheria and tetanus. Anti- 
toxins have been prepared experimentally for a large num- 
ber of both animal and vegetable poisons, including a num- 
ber for bacterial toxins. The only ones which, as yet, are of 
much practical importance are antivenin for snake poison, 
(not a true toxin, however, see p. 275), antipollenin (sup- 
posed to be for the toxin of hay fever) and the antitoxins 
for the true bacterial toxins of Coryn ebacterium diphtheria 
and Clostridium tetani. 
. The method of preparing antitoxins is essentially the same 
in all cases, though differing in minor details. For com- 
mercial purposes large animals are selected, usually horses, so 
that the yield of serum may be large. The animals must, of 
course, be vigorous, free from all infectious disease. The 
first injection given is either a relatively small amount of a 
solution of toxin or of a mixture of toxin and antitoxin. 
The animal shows more or less reaction, increased temper- 
ature, pulse and respiration and frequently an edema at the 
point of injection, unless this is made intravenously. After 
several days to a week or more, when the animal has recov- 
ered from the first injection, a second stronger dose is given, 
usually with less reaction. Increasingly large doses are 
given at proper intervals until the animal may take several 
hundred times the amount which would have been fatal if 
given at first. The process of immunizing a horse for diph- 
theria or tetanus toxin usually takes several months. Varia- 
tions in time and in yield of antitoxin are individual and 
not predictable in any given case. 
After several injections a few hundred cubic centimeters 
of blood are withdrawn from the jugular vein and serum 
from this is tested for the amount of antitoxin it contains. 
When the amount is found sufficiently large (250 "units" 
at least for diphtheria per cc.)1 then the maximum amount 
of blood is collected from the jugular with sterile trocar and 
cannula. The serum from this blood with the addition of an 
antiseptic (0.5 per cent, phenol, tricresol, etc.) constitutes 
"antidiphtheritic serum" or "antitetanic serum," etc. All 
1 If the antitoxin is later concentrated (see last paragraph in this chapter) a 
serum containing as little as 175 units per cc. may be commercially profitable. 264 
RECEPTORS OF THE FIRST ORDER 
sera which are put on the market must conform to definite 
standards of strength expressed in "units" as determined 
by the U. S. Hygienic Laboratory. In reality a "unit" of 
diphtheria antitoxin in the United States is an amount 
equivalent to 1 cc. of a given solution of a standard diph- 
theria antitoxin which is kept at the above-mentioned 
laboratory. This statement, of course, gives no definite 
idea as to the amount of antitoxin actually in a "unit." 
Specifically stated, a "unit" of antitoxin contains approxi- 
mately the amount which would protect a 250 gram guinea- 
pig from 100 minimum lethal doses of diphtheria toxin, or 
protect 100 guinea-pigs weighing 250 grams each from one 
minimum lethal dose each. The minimum lethal dose (M. 
L. D.) of diphtheria toxin is the least amount that will kill 
a guinea-pig of the size mentioned within four days. Since 
toxins on standing change into toxoids to a great extent, the 
amount of antitoxin in a "unit," though protecting against 
100 M. L. D., in reality would protect against about 200 
M. L. D. of toxin containing no toxoid. 
The official unit for tetanus antitoxin is somewhat dif- 
ferent, since it is standardized against a standard toxin which 
is likewise kept at the Hygienic Laboratory. The unit is 
defined as "ten times the amount of antitoxin necessary to 
protect a 350 g. guinea-pig for 96 hours against the stan- 
dard test dose" of the standard toxin. The standard test 
dose is 100 M. L. D. of toxin for a 350 g. guinea-pig. To 
express it another way, one could say that a "unit" of te- 
tanus antitoxin would protect one thousand 350 g. guinea- 
pigs from 1 M. L. D. each of standard tetanus toxin. 
Various methods have been devised for increasing the 
amount of antitoxin in 1 cc. of solution by precipitating 
out portions of the blood-serum proteins and at the same 
time concentrating the antitoxin in smaller volume. It is 
not considered necessary in a work of this character to enter 
into these details nor to discuss the process of standardizing 
antitoxin so that the exact amount of "units" per cc. may 
be known. CHAPTER XXVIII. 
RECEPTORS OF THE SECOND ORDER. 
AGGLUTININS. 
Charrin and Rogers appear to have been the first (1889) 
to observe the clumping together of bacteria (Pseudomonas 
pyocyanea) when mixed with the blood serum of an animal 
immunized against them. Gruber and Durham (1896) first 
used the term "agglutination" in this connection and called 
the substance in the blood-serum "agglutinin." Widal 
(1896) showed the importance of the reaction for diagnosis 
by testing the blood serum of an infected person against a 
known culture (typhoid fever). 
It is now a well-known phenomenon that the proper injec- 
tion of cells of any kind foreign to a given animal will lead 
to the accumulation in the animal's blood of substances 
which will cause a clumping together of the cells used when 
suspended in a suitable liquid. The cells settle out of such 
suspension much more rapidly than they would otherwise 
do. This clumping is spoken of as "agglutination" and the 
substances produced in the animal are called "agglutinins." 
If blood cells are injected then "hemagglutinins" result: if 
bacterial cells "bacterial agglutinins" for the particular 
organism used as "glanders agglutinin" for Pfeifferella mallei, 
"abortion agglutinin" for Bacterium abortus, "typhoid 
agglutinin" for Bacterium typhosum, etc. 
The phenomenon may be observed either under the micro- 
scope or in small test-tubes, that is, either 'microscopically or 
m acroscopically. 
In this case the cells introduced, or more properly, some 
substances within the cells, act as stimuli to the body cells 
of the animal injected to cause them to produce more of the 266 
RECEPTORS OF THE SECOND ORDER 
specific cell receptors which respond to the stimulus. The 
substance within the introduced cell which acts as a stimulus 
{antigen) to the body cells is called an "agglutinogen. " 
That " agglutinogen" is present in the cell has been shown by 
injecting animals experimentally with extracts of cells (bac- 
terial and other cells) and the blood serum of the animal in- 
jected showed the presence of agglutinin for the given cell. 
It will be noticed that the receptors which become the free 
agglutinins have at least two functions, hence at least two 
chemical groups. They must combine with the foreign cells 
and also bring about their clumping together, their aggluti- 
nation. Hence it can be stated technically that an agglu- 
tinin possesses a haptophore group and an agglutinating 
group. 
It is probable that the agglutination, the clumping, is a 
secondary phenomenon depending on the presence of certain 
salts and that the agglutinin acts on its antigen as an enzyme, 
possibly a "splitting" enzyme. This is analagous to what 
occurs in the curdling of milk by rennet and in the coagula- 
tion of blood. This probability is substantiated by the 
fact that suspensions of bacteria may be "agglutinated" 
by appropriate strengths of various acids. 
The formation of agglutinin in the body for different bac- 
teria does not as yet appear to be of any special significance 
in protecting the animal from the organism, since the bac- 
teria are not killed, even though they are rendered non- 
motile, if of the class provided with flagella, and are clumped 
together. The fact that such bodies are formed, however, 
is of decided value in the diagnosis of disease, and also in 
the identification of unknown bacteria. 
In many bacterial diseases, agglutinins for the particular 
organism are present in the blood serum of the affected 
animal. Consequently if the blood serum of the animal be 
mixed with a suspension of the organism supposed to be the 
cause of the disease and the latter be agglutinated, one is 
justified in considering it the causative agent, provided cer- 
tain necessary conditions are fulfilled. In the first place it 
must be remembered that the blood of normal animals fre- 
quently contains agglutinins ("normal agglutinins") for AGGLUTININS 
267 
many different bacteria when mixed with them in full 
strength. Hence the serum must always be diluted with 
physiological salt solution (0.85 per cent.). Further, closely 
related bacteria may be agglutinated to some extent by the 
same serum. It is evident that if they are closely related, 
their protoplasm must contain some substances of the same 
kind to account for this relationship. Since some of these 
substances may be agglutinogens, their introduction into 
the animal body will give rise to agglutinins for the related 
cells, as well as for the cell introduced. The agglutinins 
for the cell introduced "chief agglutinins," will be formed 
in larger quantity, since a given bacterial cell must contain 
more of its own agglutinogen than that of any other cell. By 
diluting the blood serum from the animal to be tested the 
agglutinins for the related organisms (so-called "coagglu- 
tinins" or "partial agglutinins") will become so much 
diminished as to show no action, while the agglutinin for 
the specific organism is still present in an amount sufficient 
to cause its clumping. Agglutinins are specific for their 
particular agglutinogens, but since a given blood serum may 
contain many agglutinins, the serum's specificity for a given 
bacterium can be determined only by diluting it until this 
bacterium alone is agglutinated. Hence the necessity of 
diluting the unknown serum in varying amounts when test- 
ing against several known bacteria to determine for which 
it is specific, i. e., which is the cause of the disease in the 
animal. 
The agglutinins in the serum may be removed from it 
by treating it with a suspension of the cells for which agglu- 
tinins are present. If the "chief" cell is used all the agglu- 
tinins will be absorbed. If related cells are used, only the 
agglutinins for this particular kind are removed. These 
"absorption tests" furnish another means of determining- 
specificity of serum, or rather of determining the "chief 
agglutinin" present. 
Just as an unidentified disease in an animal may be deter- 
mined by testing its serum as above described against known 
kinds of bacteria, so unknown bacteria isolated from an 
animal, from water, etc., may be identified by testing them 268 
RECEPTORS OF THE SECOND ORDER 
against the blood sera of different animals, each of which has 
been properly inoculated with a different kind of known 
bacteria. If the unknown organism is agglutinated by the 
blood of one of the animals in high dilution, and not by the 
others, evidently the bacterium is the same as that with 
which the animal has been inoculated, or immunized, as is 
usually stated. This method of identifying cultures of bac- 
teria is of wide application, but is used practically only in 
those cases where other methods of identification are not 
readily applied, and especially where other methods are 
not sufficient as in the "intestinal group" of organisms in 
human practice. 
The diagnosis of disease in an animal by testing its serum 
is also a valuable and much used procedure. This is the 
method of the "Widal" or "Gruber-Widal" test for typhoid 
fever in man and is used in veterinary practice in testing 
for glanders, contagious abortion, etc. In some cases a dilu- 
tion of the serum of from 20 to 50 times is sufficient for 
diagnosis (Malta fever), in most cases, however, 50 times 
is the lowest limit. Evidently the greater the dilution, that 
is, the higher the "titer," the more specific is the reaction. 
PRECIPITINS. 
Since agglutinins act on bacteria, probably through the 
presence of substances within the bacterial cell, it is reason- 
able to expect that if these substances be dissolved out of the 
cell, there would be some reaction between their (colloidal) 
solution and the same serum. As a matter of fact Kraus 
(1897) showed that broth cultures freed from bacteria by 
porcelain filters do show a precipitate when mixed with the 
serum of an animal immunized against the particular bac- 
terium and that the reaction is specific under proper condi- 
tions of dilution. ■ It was not long after Kraus's work until 
the experiments were tried of "immunizing" an animal not 
against a bacterium or its filtered culture, but against 
(colloidal) solutions of proteins, such as white of egg, casein 
of milk, proteins of meat and of blood serum, vegetable 
proteins, etc. It was ascertained that in all these cases the 
animal's serum contains a substance which causes a preci- PRECI PI TINS 
269 
pitate with solutions of the protein used for immunization. 
The number of such precipitating serums that have been 
made experimentally is very large and it appears that pro- 
tein from any source when properly introduced into the 
blood or tissues of an animal will cause the formation of a 
precipitating substance for its solutions. This substance is 
known, technically as a "precipitin." The protein used as 
antigen to stimulate its formation, or some part of the pro- 
tein molecule (haptophore group), which acts as stimulus 
to the cell is spoken of as a "precipitinogen," both terms 
after the analogy of "agglutinin" and "agglutinogen." 
In fact the specific precipitation and agglutination are strict- 
ly analogous phenomena. Precipitins act on proteins in 
(colloidal) solution and cause them to settle out, agglutinins 
act on substances within cells which cells are in suspension 
in a fluid and cause the cells to settle out. Ehrlich's theory 
of the formation of precipitins is similar to that of aggluti- 
nins, and need not be repeated. Substitute the correspond- 
ing words in the theory of formation of agglutinins as above 
given and the theory applies. 
The precipitin reaction has not found much practical use 
in bacteriology largely because the "agglutination test" 
takes its place as simpler of performance and just as ac- 
curate. The reaction is, however, generally applicable to 
filtrates of bacterial cultures and could be used if needed. 
The so-called "mallease" reaction in glanders is an instance. 
Precipitins find their greatest usefulness in legal medi- 
cine and in food adulteration work. As was noted above, 
if animals, rabbits for example, are immunized with the 
blood of another animal (human beings) precipitins are 
developed which are specific for the injected blood with 
proper dilution. This forms an extremely valuable means 
of determining the kind of blood present in a given spot 
shown by chemical and spectroscopic tests to be blood and 
has been adopted as a legal test in countries where such rules 
of procedure are applied. Similarly the test has been used 
to identify the different kinds of meat in sausage, and dif- 
ferent kinds of milk in a mixture. An extract of the sau- 
sage is made and tested against the serum of an animal 270 
RECEPTORS OF THE SECOND ORDER 
previously treated with extract of horse meat, or hog meat, 
or beef, etc., the specific precipitate occurring with the 
specific serum. Such reactions have been obtained where 
the protein to be tested was diluted 100,000 times and more. 
Biological relationships and differences have been detected 
by the reaction. Human immune serum shows no reaction 
with the blood of any animals except to a slight extent with 
that of various monkeys, most with the higher, very slight 
with the lower Old World and scarcely any with New World 
monkeys. 
It is a fact of theoretical interest mainly that if agglutinins 
and precipitins themselves be injected into an animal they 
will act as antigens and cause the formation of antiagglu- 
tinins or antiprecipitins, which are therefore receptors of 
the first order since they simply combine with these immune 
bodies to neutralize their action, have only a combining or 
haptophore group. Also if agglutinins or precipitins be 
heated to the proper temperature they may retain their 
combining power but cause no agglutination or precipita- 
tion, i. e., they are converted into agglutinoid or precipi- 
tinoid respectively after the analogy of toxin and toxoid. 
Precipitins like agglutinins possess at least two groups- 
a combining or haptophore group and a precipitating (some- 
times called zymophore) group. Hence they are somewhat 
more complex than antitoxins or antienzymes which have 
a combining group only. For this reason Ehrlich classes 
agglutinins and precipitins as receptors of the second order. CHAPTER XXIX 
RECEPTORS OF THE THIRD ORDER. 
CYTOLYSINS. 
Before Koch definitely proved bacteria capable of caus- 
ing disease several physiologists had noted that the red cor- 
puscles of certain animals were destroyed by the blood of 
other animals (Creite, 1869, Landois, 1875), and Traube and 
Gescheidel had shown that freshly drawn blood destroys 
bacteria (1874). It was not until about ten years afterward 
that this action of the blood began to be investigated in 
connection with the subject of immunity. Von Fodor 
(1885) showed that saprophytic bacteria injected into the 
blood are rapidly destroyed. Fliigge and his pupils, especi- 
ally Nuttall in combating Metchnikoff's theory of phago- 
cytosis, announced in 1883, studied the action of the blood 
on bacteria and showed its destructive effect (1885-87). 
Nuttall also showed that the blood lost this power if heated 
to 56°. Buchner (1889) gave the name "alexin" (from the 
Greek "to wTard off") to the destroying substance and showed 
that the substance was present in the blood serum as well as 
in the whole blood, and that when the serum lost its power 
to dissolve, this could be restored by adding fresh blood. 
Pfeiffer (1894) showed that the destructive power of the 
blood of animals immunized against bacteria (cholera and 
typhoid) was markedly specific for the bacteria used. He 
introduced a mixture of the blood and the bacteria into the 
abdominal cavity of the immunized animal or of a normal 
one of the same species and noted the rapid solution of the 
bacteria by withdrawing portions of the peritoneal fluid and 
examining them ("Pfeiffer's phenomenon"). Belfanti and 
Carbone and especially Bordet (1898) showed the specific 
dissolving action of the serum of one animal on the blood 
corpuscles of another animal with which it had been injected. 272 
RECEPTORS OF THE THIRD ORDER 
Since this time the phenomenon has been observed with a 
great variety of cells other than red blood corpuscles and 
bacteria-leukocytes, spermatozoa, cells from liver, kidney, 
brain, epithelia, etc., protozoa, and many vegetable cells. 
It is therefore a well-established fact that the proper 
injection of an animal with almost any cell foreign to it 
will lead to the blood of the animal injected acquiring the 
power to injure or destroy cells of the same kind as those 
introduced. The destroying power of the blood has been 
variously called its "cytotoxic" or "cytolytic" power, 
though the terms are not strictly synonymous since "cyto- 
toxic" means "cell poisoning" or "injuring," while "cyto- 
lytic" means "cell dissolving." The latter term is the one 
generally used and there is said to be present in the blood a 
specific "cytolysin. " The term is a general one and a given 
cytolysin is named from the cellf which is dissolved, as a 
bacteriolysin, a hemolysin (red-corpuscle-lysin), epitheliolysin, 
nephrolysin (for kidney cells), etc. If the cell is killed but 
not dissolved the suffix "cidin" or "toxin" is frequently used 
as "bacteriocidin," "spermotoxin," "neurotoxin," etc. 
The use of the term "cytolysin" is also not strictly cor- 
rect, though convenient, for the process is more complex 
than if one substance only were employed. As was stated 
above, the immune serum loses its power to dissolve the cell 
if it is heated to 55° to 56° for half an hour, it is inactivated. 
But if there be added to the heated or inactivated serum a 
small amount of normal serum (which contains only a very 
little cytolytic substance, so that it has no dissolving power 
when so diluted) the mixture again becomes cytolytic. 
It is evident then that in cytolysis there are two distinct 
substances involved, one which is present in all serum, normal 
or immune, and the other present only in the immune cytolytic 
serum. This may be more apparent if the facts are arranged 
in the following form: 
I. Immune serum dissolves cells in high dilution. 
II. Heated immune serum does not dissolve cells. 
III. Normal serum in high dilution does not dissolve cells. 
II. + III., i. e., Heated immune serum plus diluted normal 
serum dissolves cells. AMBOCEPTORS 
273 
Therefore, there is something in heated immune serum neces- 
sary for cell dissolving and something different in diluted 
normal serum which is necessary. This latter something is 
present in unheated immune serum also, and is destroyed 
by heat. Experiment has shown that it is the substance 
present in all serum both normal and immune that is the 
true dissolving body, while the immune substance serves to 
unite this body to the cell to be destroyed, i. e., to the anti- 
gen. Since the immune body has therefore two uniting 
groups, one for the dissolving substance and one for the cell 
to be dissolved, Ehrlich calls it the "amboceptor." He also 
uses the word "complement" to denote the dissolving sub- 
stance, giving the idea that it completes the action of dis- 
solving after it has been united to the cell by the amboceptor, 
thus replacing Buchner's older term "alexin" for the same 
dissolving body. 
The theory of formation of amboceptors is similar to that 
for the formation of the other types of antibodies. The 
cell introduced contains some substance, which acts as a 
chemical stimulus to some of the body cells provided with 
proper receptors so that more of these special receptors are 
produced, and eventually in excess so that they become free 
in the blood and constitute the free amboceptors. It will 
be noticed that these free receptors differ from either of the 
two kinds already described in that they have two uniting 
groups, one for the antigen (cell introduced) named cytophil- 
haptophore, the other for the complement, complementophil 
haptophore. Hence amboceptors are spoken of as receptors 
of the third order. They have no other function than that 
of this double combining power. The action which results 
is due to the third body-the complement. It will be readily 
seen that complement must possess at least two groups, a 
combining or haptophore group which unites with the ambo- 
ceptor, and an active group which is usually called the zy- 
mophore or toxophore group. Complements thus resemble 
either toxins, where the specific cell (antigen) is injured or 
AMBOCEPTORS. 274 
RECEPTORS OF THE THIRD ORDER 
killed, or enzymes, in case the cell is likewise dissolved. 
This action again shows the close relation between toxins- 
and enzymes. Complement may lose its active group in the 
same way that toxin does and becomes then complementoid. 
Complement is readily destroyed in blood or serum by 
heating it to 55° to 56° for half an hour, and is also destroyed 
spontaneously when serum stands for a day or two, less 
rapidly at low temperature than at room temperature. 
Amboceptors appear to be specific in the same sense that 
agglutinins are. That is, if a given cell is used to immunize 
an animal, the animal's blood will contain amboceptors for 
the cell used and also for others closely related to it. Im- 
munization with spermatozoa or with epithelial or liver cells 
gives rise to amboceptors for these cells and also for red blood 
corpuscles and other body cells. A typhoid bactericidal 
serum has also some dissolving effect on colon bacilli, etc. 
Hence a given serum may contain a chief amboceptor and a 
variety of "coamboceptors," or one amboceptor made up 
of a number of "partial amboceptors" each specific for its 
own antigen ("amboceptorogen"). Amboceptors may com- 
bine with other substances than antigen and complement, as- 
is shown by their union with lecithin and other "lipoids," 
though these substances seem capable of acting as comple- 
ment in causing solution of blood corpuscles. 
COMPLEMENTS. 
As to whether complements are numerous, as Ehrlich 
claims, or there is only one complement, according to Buch- 
ner and others, need not be discussed here. In the practi- 
cal applications given later as means of diagnosis it is appar- 
ent that all the complement or complements are capable of 
uniting with at least two kinds of amboceptors. 
If complement be injected into an animal it may act as 
an antigen and give rise to the formation of anticomplement 
which may combine with it and prevent its action and is 
consequently analogous to antitoxin. If amboceptors as 
antigen are injected into an animal there will be formed by FAILURE OF CYTOLYTIC SERUMS 
275 
the animal's cells antiamboceptors. As one would expect, 
there are two kinds of antiamboceptors, one for each of its 
combining groups, since it has been stated that it is always 
the combining group of any given antigen that acts as th'e 
cell stimulus. Hence we may have an "anticytophil ambo- 
ceptor" or an " anticomplementophil amboceptor." These 
antiamboceptors and the anticomplements are analogous to 
antitoxin, antiagglutinin, etc., and hence are receptors of 
the first order. 
ANTISNAKE VENOMS. 
A practical use of antiamboceptors is in antisnake venoms. 
Snake poisons appear to contain only amboceptors for differ- 
ent cells of the body. In the most deadly the amboceptor 
is specific for nerve cells (cobra), in others for red corpuscles, 
or for endothelial cells of the bloodvessels (rattlesnake). 
The complement is furnished by the blood of the individual 
bitten, that is, in a sense the individual poisons himself, 
since he furnishes the destroying element. The antisera 
contain antiamboceptors which unite with the amboceptor 
introduced and prevent it joining to cells and thus binding 
the complement to the cells and destroying them. With 
this exception these antibodies are chiefly of theoretical 
interest. 
The discovery of the possibility of producing a strongly 
bactericidal serum in the manner above described aroused 
the hope that such sera would prove of great value in passive 
immunization and serum treatment of bacterial diseases. 
Unfortunately such expectations have not been realized and 
no serum of this character of much practical importance has 
been developed as yet (with the possible exception of Flex- 
ner's antimeningococcus serum in human practice. What 
hog cholera serum is remains to be discovered). 
The reasons for the failure of such sera are not entirely 
clear. The following are some that have been offered: (1) 
FAILURE OF CYTOLYTIC SERUMS. 276 
RECEPTORS OF THE THIRD ORDER 
Amboceptors do not appear to be present in very large 
amount so that relatively large injections of blood are neces- 
sary, which is not without risk in itself. (2) Since the com- 
plement is furnished by the blood of the animal to be treated, 
there may not be enough of this to unite with a sufficient 
quantity of amboceptor to destroy all the bacteria present, 
hence the disease is continued by those that escape. (3) Or 
the complement may not be of the right kind to unite with 
the amboceptor introduced, since this is derived from the 
blood of a heterologous ("other kind") species. In hog- 
cholera serum, if it is bactericidal, this difficulty is removed 
by using blood of a homologous ("same kind") animal. 
Hence Ehrlich suggested the use of apes for preparing bac- 
tericidal sera for human beings. The good results which 
have been reported in the treatment of human beings with 
the serum of persons convalescing from the same disease 
indicate that this lack of proper complement for the given 
amboceptor is probably a chief factor in the failure of sera 
from lower animals. (4) The bacteria may be localized in 
tissues (lymph glands), within cavities (cranial, peritoneal), 
in hollow organs (alimentary tract), etc., so that it is not 
possible to get at them with sufficient serum to destroy all. 
This difficulty is obviated by injecting directly into the 
spinal canal when Flexner's antimeningococcus serum is 
used. (5) Even if the bacteria are dissolved it does not 
necessarily follow that their endotoxins are destroyed. 
These may be merely liberated and add to the danger of the 
patient, though this does not appear to be a valid objection. 
,(6) Complement which is present in such a large excess 
•of amboceptor may just as well unite with amboceptor 
which is not united to the bacteria to be destroyed as with 
that which is, and hence be actually prevented from dissolv- 
ing the bacteria. Therefore it is difficult to standardize the 
serum to get a proper amount of amboceptor for the com- 
plement present. 
COMPLEMENT-FIXATION TEST. 
Although little practical use has been made of bactericidal 
sera, the discovery of receptors of this class and the peculiar COMPLEMEN T-FIXA TION TES TS 
277 
relations between the antigen, amboceptor and complement 
have resulted in developing one of the most delicate and 
accurate methods for the diagnosis of disease and for the 
recognition of small amounts of specific protein that is in use 
today. 
This method is usually spoken of as the "complement- 
fixation" or the "complement-deviation test" ("Wasser- 
mann test" in syphilis) and is applicable in a great variety 
of microbial diseases, but it is of practical importance in 
those diseases only where other methods are uncertain- 
syphilis in man, concealed glanders in horses, contagious 
abortion in cattle, etc. A better name would be the "Un- 
known Amboceptor Test" since it is the amboceptor that 
is searched for in the test by making use of its power to 
"fix" complement. 
The principle is the same in all cases. The method 
depends, as indicated above, on the ability of complement 
to combine with at least two amboceptor-antigen systems, 
and on the further fact that if the combination with one 
amboceptor-antigen system is once formed, it does not dis- 
sociate so as to liberate the complement for union with the 
second amboceptor-antigen system. If an animal is in- 
fected with a microorganism and a part of its defensive 
action consists in destroying the organisms in its blood or 
lymph, then it follows from the above discussion of cytoly- 
sins that there will be developed in the blood of the animal 
amboceptor specific for the organism in question. If the 
presence of this specific amboceptor can be detected, the 
conclusion is warranted that the organism for which it is 
specific is the cause of the disease. Consequently what is 
sought in the "complement-fixation test" is a specific ambo- 
ceptor. In carrying out the test, blood serum from the sus- 
pected animal is collected, heated at 56° for half an hour to 
destroy any complement it contains and mixed in definite 
proportions with the specific antigen and with complement. 
The antigen is an extract of a diseased organ (syphilitic 
fetal liver, in syphilis), a suspension of the known bacteria, 
or an extract of these bacteria. Complement is usually 
derived from a guinea-pig, since the serum of this animal is 278 
RECEPTORS OF THE THIRD ORDER 
higher in complement than that of most animals. The 
blood of the gray rat contains practically as much. If the 
specific amboceptor is present, that is, if the animal is in- 
fected with the suspected disease, the complement will unite 
with the antigen-amboceptor system and be "fixed," that is, 
be no longer capable of uniting with any other amboceptor- 
antigen system. No chemical or physical means of telling 
whether this union has occurred or not, except as given 
below, has been discovered as yet, though doubtless will be 
by physico-chemical tests, nor can the combination be seen. 
Hence an "indicator," as is so frequently used in chemistry, 
is put into the mixture of antigen-amboceptor-complement 
after it has been allowed to stand in the incubator for one- 
half to one hour to permit the union to become complete. 
The "indicator" used is a mixture of sheep's corpuscles and 
the heated ("inactivated") blood serum of a rabbit which 
has been injected with sheep's blood corpuscles and there- 
fore contains a hemolytic amboceptor specific for the corpuscles 
which is capable also of uniting with complement. The 
indicator is put into the first mixture and the whole is again 
incubated and examined. If the mixture is clear and color- 
less with a deposit of red corpuscles at the bottom, that 
would mean that the complement had been bound to the 
first complex, since it was not free to unite with the second 
sheep's corpuscles (antigen)-rabbit serum (hemolytic am- 
boceptor) complex-and destroy the corpuscles. Hence if 
the complement is bound in the first instance, the specific 
amboceptor for the first antigen must have been present 
in the blood, that is, the animal was infected with the organ- 
ism in question. Such a reaction is called a "positive" 
test. 
On the other hand, if the final solution is clear but of a red 
color, that would mean that complement must have united 
with the corpuscles-hemolytic amboceptor system-and 
destroyed the corpuscles in order to cause the clear red 
solution of hemoglobin. If complement united with this 
system it could not have united with the first system, hence 
there was no specific amboceptor there to bind it; no specific 
amboceptor in the animal's blood, means no infection. 
Hence a red solution is a "negative test." COMPLEMEN T-FIXA TION TES TS 
279 
The scheme for the test may be outlined as follows: 
Antigen + Patient's Serum, heated + Complement 
(specific for the (unknown amboceptor) (derived from 
amboceptor sought) guinea pig's serum) 
Incubate one-half hour in a water bath or one hour in an 
incubator. 
Then add the indicator which is 
Antigen + Amboceptor 
(red blood corpuscles) (for corpuscles, serum of 
a rabbit immunized against 
the red corpuscles) 
Incubate as above. 
In practice all the different ingredients must be accurately 
tested, standardized and used in exact quantities, and 
tests must also be run as controls with a known normal 
blood of an animal of the same species as the one examined 
and with a known positive blood. 
It should be stated that in one variety of the Complement Fixation Test, 
namely, the "Wassermann Test for Syphilis" in human beings, an antigen 
is used which is not derived from the specific organism (Treponema pallidum') 
which causes the disease nor even from syphilitic tissue. It has been deter- 
mined that alcohol will extract from certain tissues, human or animal, sub- 
stances which act specifically in combining with the syphilitic amboceptor 
present in the blood. Alcoholic extracts of beef heart are most commonly 
used. Details of this test may be learned in the advanced course in Immunity 
and Serum Therapy. 
The complement-fixation test might be applied to the 
determination of unknown bacteria, using the unknown cul- 
ture as antigen and trying it with the sera of different ani- 
mals immunized against a variety of organisms, some one 
of which might prove to furnish specific amboceptor for the 
unknown organism and hence indicate what it is. The test 
used in this way has not been shown to be a practical neces- 
sity and hence is rarely employed. It has been used, how- 
ever, to detect traces of unknown proteins, particularly 
blood-serum proteins, in medico-legal cases in exactly the 
above outlined manner and is very delicate and accurate. CHAPTER XXX. 
PHAGOCYTOSIS-OPSONINS. 
It has been mentioned that Metchnikoff, in a publication 
in 1883, attempted to explain immunity on a purely cellular 
basis. It has been known since Haeckel's first observation 
in 1858 that certain of the white corpuscles do engulf solid 
particles that may get into the body, and among them bac- 
teria. Metchnikoff at first thought that this engulfing and 
subsequent intracellular digestion of the microorganisms 
were sufficient to protect the body from infection. The 
later discoveries (discussed in considering Ehrlich's theory 
of immunity) of substances present in the blood serum and 
even in the blood plasma which either destroy the bacteria 
or neutralize their action have caused Metchnikoff to modify 
his theory to a great extent. He admitted the presence of 
these substances, though giving them other names, but as- 
scribed their formation to the phagocytes or to the same 
organs which form the leukocytes-lymphoid tissue gener- 
ally, bone marrow. It is not within the province of this 
work to attempt to reconcile these theories, but it may be 
well to point out that Ehrlich's theory is one of chemical 
substances and that the origin of these substances is not an 
essential part of the theory, so that the two theories, except 
in some minor details, are not necessarily mutually exclu- 
sive. 
Sir A. E. Wright and Douglas, in 1903, showed that even 
in those instances where immunity depends on phagocytosis, 
as it certainly does in many cases, the phagocytes are more 
or less inactive unless they are aided by chemical substances 
present in the blood. These substances act on the bacteria, 
not on the leukocytes, and change them in such a way that 
they are more readily taken up by the phagocytes. Wright PLATE V 
ELIE METCHNIKOFF PHA GOC F TOSIS-OP SO NINS 
281 
proposed for these bodies the name opsonin, derived from a 
Greek word signifying "to prepare a meal for." Neufeld 
and Rimpau at about the same time (1904), in studying 
immune sera, observed substances of similar action in these 
sera and proposed the name bacteriotropins, or bacteriotropic 
substances. There is scarcely a doubt that the two names 
are applied to identical substances and that Wright's name 
opsonin should have preference. 
The chemical nature of opsonins is not certainly deter- 
mined, but they appear to be a distinct'class of antibodies 
and to possess two groups, a combining or haptophore and 
a preparing or opsonic group and hence are similar to anti- 
bodies of Ehrlich's second order-agglutinins and precipi- 
tins. Wright also showed that opsonins are just as specific 
as agglutinins are-that is, a micrococcus opsonin prepares 
micrococci only for phagocytosis and not streptococci or any 
other bacteria. 
Wright showed that opsonins for many bacteria are pres- 
ent in normal serum and that in the serum of an animal 
which has been immunized against such bacteria the opso- 
nins are increased in amount. Also that in a person infected 
with certain bacteria the opsonins are either increased or 
diminished, depending on whether the progress of the infec- 
tion is favorable or unfavorable. The opsonic power of a 
serum normal or otherwise is determined by mixing an 
emulsion of fresh leukocytes in normal saline solution with 
a suspension of the bacteria and with the serum to be tested. 
The leukocytes must first be washed in several changes of 
normal salt solution to free them from any adherent plasma 
or serum. The mixture is incubated for about fifteen min- 
utes and then slides are made, stained with a good differ- 
ential blood stain, Wright's or other, and the average num- 
ber of bacteria taken up by at least fifty phagocytes taken 
in order in a field is determined by counting under the 
microscope. The number so obtained Wright calls the 
phagocytic index of the serum tested. The phagocytic in- 
dex of a given serum divided by the phagocytic index of a 
normal serum gives the opsonic index of the serum tested. 
Assuming the normal opsonic index to be 1, Wright asserts 282 
PHA GOC Y TOSIS-OPSONINS 
that in healthy individuals the range should be not more 
than from 0.8 to 1.2, and that an index below 0.8 may show 
a great susceptibility for the organism tested, infection with 
the given organism if derived from the individual, or im- 
proper dosage in case attempts have been made to immun- 
ize by using killed cultures, vaccines, of the organism. 
On the occasion of the author's visit to Wright's clinic 
(1911) he stated that he used the determination of the 
opsonic index chiefly as a guide to the dosage in the use of 
vaccines. 
Most workers outside the Wright school have failed to 
recognize any essential value of determinations of the 
opsonic index in the use of vaccines. Some of the reasons 
for this are as follows: The limit of error in phagocytic 
counts may be as great as 50 per cent, in different series of 
fifty, hence several hundred must be counted, which adds 
greatly to the tediousness and time involved; the variation 
in apparently healthy individuals is frequently great, hence 
the "normal" is too uncertain; finally the opsonic index 
and the clinical course of the disease do not by any means 
run parallel. Undoubtedly the method has decided value 
in the hands of an individual who makes opsonic determina- 
tions his chief work, as Wright's assistants do, but it can 
scarcely be maintained at the present time that such deter- 
minations are necessary in vaccine therapy. Nevertheless 
that opsonins actually exist and that they play an essential 
part in phagocytosis, and hence in immunity, is now gener- 
ally recognized. 
BACTERIAL VACCINES. 
Whether determinations of opsonic index are useful or 
not is largely a matter of individual opinion, but there is 
scarcely room to doubt that Wright has conferred a lasting 
benefit by his revival of the use of dead cultures of bacteria, 
bacterial vaccines, both for protective inoculation and for 
treatment. It is perhaps better to use the older terms 
"vaccination" and "vaccine" (though the cow, vacca, is not 
concerned) than to use Wright's term "opsonic method" in BACTERIAL VACCINES 
283 
this connection, bearing in mind that the idea of a vaccine 
is that it contains the causative organism of the infection as 
indicated on p. 253. 
As early as 1880 Touissant proposed the use of dead cul- 
tures of bacteria to produce immunity. But because injec- 
tions of such cultures were so frequently followed by abscess 
formation, doubtless due to the high temperatures used to 
kill the bacteria, the method was abandoned. Further, Pas- 
teur and the French school persistently denied the possi- 
bility of success with such a procedure, and some of them 
even maintain this attitude at the present time. The suc- 
cesses of Wright and the English school which are being 
repeated so generally wherever properly attempted, leave 
no doubt in the unprejudiced of the very great value of the 
method and have unquestionably opened a most promising 
field both for preventive inoculation and for treatment in 
many infectious diseases. That the practice is no more 
universally applicable than are immune serums and that it 
has been and is still being grossly overexploited is un- 
doubted. 
The use of a vaccine is based on two fundamental prin- 
ciples. The first of these is that the cell introduced must 
not be in a condition to cause serious injury to the animal by 
its multiplication and consequent elaboration of injurious 
substances. The second is that, on the other hand, it must 
contain antigens in such condition that they will act as 
stimuli to the body cells to produce the necessary antibodies, 
whether these be opsonins, bactericidal substances, or anti- 
endotoxins. In the introduction of living organisms there 
is always more or less risk of the organism not being suffi- 
ciently attenuated and hence of the possibility of its pro- 
ducing too severe an infection. In using killed cultures, 
great care must be exercised in destroying the organisms, 
so that the antigens are not at the same time rendered inactive. 
Hence in the preparation of bacterial vaccines by Wright's 
method the temperature and the length of time used to kill the 
bacteria are most important factors. This method is in gen- 
eral to grow the organisms on an agar medium, rub off the 
culture and emulsify in sterile normal salt solution (0.85 284 
PHA GOC F TOSIS-OPSONINS 
per cent. NaCl). The number of bacteria per cc. is deter- 
mined by staining a slide made from a small volume of the 
emulsion mixed with an equal volume of human blood drawn 
from the finger and counting the relative number of bac- 
teria and of red blood corpuscles. Since the corpuscles are 
normally 5,000,000 per c.mm., a simple calculation gives 
the number of bacteria. The emulsion of bacteria is then 
diluted so that a certain number of millions shall be contained 
in each cc., "standardized" as it is called, then heated to 
the proper temperature for the necessary time and it is 
ready for use. A preservative, as 0.5 per cent, phenol, tri- 
cresol, etc., is added unless the vaccine is to be used up at 
once. The amounts of culture, salt solution, etc., vary with 
the purpose for which the vaccine is to be used, from one 
or two agar slant cultures and a few cc. of solution, wThen a 
single animal is to be treated, to bulk agar cultures and 
liters of solution as in preparing antityphoid vaccine on a 
large scale. 
Agar surface cultures are used so that there will be as 
little admixture of foreign protein as possible (see Anaphy- 
laxis, p. 289 et seq.\ Normal saline solution is isotonic with 
the body cells and hence is employed as the vehicle. 
Lipovaccines.-The suspension of bacteria in neutral oil 
was first used by Le Moignac and Pinoy who gave the name 
"lipovaccines" (Xtxo; = fat) to them. It was claimed 
that the reaction following injection of these vaccines was 
less severe than with saline vaccines in many instances; 
also, that the bacteria were much more slowly absorbed. 
For these two reasons it was hoped that much larger num- 
bers of bacteria could be injected at one dose and one in- 
jection would suffice instead of three or more as ordinarily 
used. The technique of preparation, standardization and 
killing of the organisms has not as yet been sufficiently 
well established to warrant the general substitution of lipo- 
vaccines for ordinary saline suspensions. 
Vaccines are either "autogenous" or "stock." An "auto- 
genous" vaccine is a vaccine that is made from bacteria 
derived from the individual or animal which it is desired to 
vaccinate and contains not only the particular organism but BACTERIAL VACCINES 
285 
the particular strain of that organism which is responsible 
for the lesion. Stock vaccines are made up from organisms 
like the infective agent in a given case but derived from 
some other person or animal or from laboratory cultures. 
Commercial vaccines are "stock" vaccines and are usually 
"polyvalent" or even "mixed." A "polyvalent" vaccine 
contains several strains of the infective agent and a "mixed" 
contains several different organisms. 
Stock vaccines have shown their value when used as pre- 
ventive inoculations, notably so in typhoid fever in man, 
anthrax and black-leg in cattle. The author is strongly 
of the opinion, not only from the extended literature on the 
subject, but also from his own experience in animal, and 
especially in human cases, that stock vaccines are much 
inferior and much more uncertain in their action when used 
in the treatment of an infection, than are autogenous vaccines. 
This applies particularly to those instances in which pneu- 
mococci, streptococci, micrococci, and colon bacilli are the cau- 
sative agents but to others as well. The following are some 
of the reasons for this opinion: The above organisms are 
notoriously extremely variable in their virulence. While 
there is no necessarily close connection between virulence 
and antigenic property, yet since virulence is so variable, it 
is rational to assume that antigenic property is also extremely 
variable. Individuals vary just as much in susceptibility 
and hence in reactive power, and generally speaking, an 
individual will react better in the production of antibodies 
to a stimulus to which he has been more or less subjected, 
i. e., to organisms derived from his own body. 
In the preparation of a vaccine great care must be used 
in heating so that the organisms are killed, but the antigens 
are not destroyed. Many of the enzymes present in bac- 
teria, especially the proteolytic ones, are not any more sen- 
sitive to heat than are the antigens, hence are not destroyed 
entirely. Therefore a vaccine kept in stock for a long time 
gradually has some of its antigens destroyed by the unin- 
jured enzymes present with them, and so loses in potency. 
Therefore in treating a given infection it is well to make up a 
vaccine from the lesion, use three or four doses and if more 
are necessary make up a new vaccine. 286 
PHA GOC Y TOSIS-OPSONINS 
If the above statements are borne in mind and vaccines 
are made and administered accordingly, the author is well 
satisfied that much better results will be secured. 
In accordance with the theory on which the use of vac- 
cines is based, i. e., that they stimulate the body cells to 
produce immunizing antibodies, it is clear that they are espe- 
cially suitable in those infections in which the process is 
localized and should not be of much value in general infec- 
tions. In the latter case the cells of the body are stimulated 
to produce antibodies by the circulating organisms, prob- 
ably nearly to their limit, hence the introduction of more of 
the same organisms, capable of stimulating though dead, is 
apt to overtax the cells and do more harm than good. It is 
not possible to tell accurately when this limit is reached, but 
the clinical symptoms are a guide. If vaccines are used at 
all in general infections they should be given in the early 
stages and in small doses at first with close watch as to the 
effect. In localized infections only the cells in the immedi- 
ate neighborhood are much stimulated, hence the introduc- 
tion of a vaccine calls to their aid cells in the body gener- 
ally, and much more of the resulting antibodies are carried 
to the lesion in question. Manifestly surgical procedures 
such as incision, drainage, washing away of dead and ne- 
crotic tissue with normal saline solution, not necessarily 
antiseptics, will aid the antibodies in their action and are to 
be recommended where indicated. 
In the practical application of any remedy the dosage is 
most important. Unfortunately there is no accurate method 
of determining this with a vaccine. Wright recommended 
determining the number of the organisms per cc. as before 
mentioned, and his method or some modification of it is 
still in general use. From what was said with regard to 
variation, both in organisms and in individuals, it can be 
seen that the number of organisms is at least only a very 
rough guide. This is further illustrated by the doses of 
micrococcus (staphylococcus) vaccines recommended by 
different writers, which vary from 50,000,000 to 2,000,000,- 
000 per cc. The author is decidedly of the opinion that 
there is no way of determining the dosage of a vaccine in the BACTERIAL VACCINES 
287 
treatment of any given case except by the result of the first dose. 
Hence it is his practice to make vaccines of a particular 
organism of the same approximate strength, and to give a 
dose of a measured portion of a cubic centimeter, judging 
the amount by what the individual or animal can apparently 
withstand, without too violent a reaction. If there is no 
local or general reaction or if it is very slight and there is no 
effect on the lesion, the dose is too small. If there is a 
violent local reaction with severe constitutional symptoms 
clinically, and the lesion appears worse, the dose is too 
large. There should be some local reaction and some 
general, but not enough to cause more than a slight disturb- 
ance, easy to judge in human subjects, more difficult in 
animals. In cases suitable for vaccine treatment no serious 
results should follow from a properly prepared vaccine, 
though the process of healing may be delayed temporarily. 
Wright claimed, and many have substantiated him, that 
always following a vaccination there is a period when the 
resistance of the animal is diminished. This is called the 
"negative phase," and Wright considered this to last as 
long as the opsonic index remained low, and when this 
latter began to increase the stage of the "positive" or 
favorable phase was reached. As has been stated the op- 
sonic index is pretty generally regarded as of doubtful value, 
though the existence of a period of lowered resistance is 
theoretically probable from the fact that antibodies already 
present in the blood will be partially used up in uniting with 
the vaccine introduced and that the body cells are called 
upon suddenly to do an extra amount of work and it takes 
them some time to adapt themselves. This time, the 
"negative phase," is much better determined by the clini- 
cal symptoms, general and especially local. It is good 
practice to begin with a dose relatively small. The result 
of this is an indication of the proper dosage and also pre- 
pares the patient for a larger one. The second dose should 
follow the first not sooner than three or four days, and 
should be five to seven days if the first reaction is severe. 
These directions are not very definite, but clinical experi- 
ence to date justifies them. It is worth the time and money 288 
PHAGOC Y TOSIS-OPSONINS 
to one who wishes to use vaccines to learn from one who 
has had experience both in making and administering them, 
and then to remember that each patient is an individual 
case for the use of vaccines as well as for any other kind of 
treatment. 
AGGRESSIN. 
Opsonins have been shown to be specific substances which 
act on bacteria in such a way as to render them more read- 
ily taken up by the leukocytes. By analogy one might 
expect to find bacteria secreting specific substances which 
would tend to counteract the destructive action of the 
phagocytes and bactericidal substances. Bail and his co- 
workers claim to have demonstrated such substances in 
exudates in certain diseases and have given the distinctive 
name "aggressins" to them. By injecting an animal with 
"aggressins," antiaggressins are produced which counteract 
their effects and thus enable the bacteria to be destroyed. 
The existence of such specific bodies is not generally accepted 
as proved. The prevailing idea is that bacteria protect 
themselves in any given case by the various toxic substances 
that they produce, and that "aggressins" as a special class 
of substances are not formed. CHAPTER XXXI. 
ANAPHYLAXIS. 
Dallera, in 1874, and a number of physiologists of that 
period, observed peculiar skin eruptions following the trans- 
fusion of blood, that is, the introduction of foreign proteins. 
In the years subsequent to the introduction of diphtheria 
■antitoxin (1890) characteristic "serum rashes" were not 
infrequently reported, sometimes accompanied by more or 
less severe general symptoms and occasionally death-a 
train of phenomena to which the name "serum sickness" 
was later applied, since it w'as shown that it was the horse 
serum (foreign protein) that was the cause, and not the anti- 
toxin itself. In 1898 Bichet and Hericourt noticed that 
some of the dogs which they were attempting to immunize 
-against toxic eel serum not only were not immunized but 
suffered even more severely after the second injection. They 
obtained similar results with an extract of mussels which 
contain a toxin. Richet gave the nkme "anaphylaxis" 
{"no protection") to this phenomenon to distinguish it 
from immunity or prophylaxis (protection). 
All the above-mentioned observations led to no special 
investigations as to their cause. In 1903, Arthus noticed 
abscess formation, necrosis and sloughing following several 
injections of horse serum in immediately adjacent parts of 
the skin in rabbits ("Arthus' phenomenon"). Theobald 
Smith, in 1904, observed the death of guinea-pigs following 
properly spaced injections of horse serum. This subject 
was investigated by Otto and by Rosenau and Anderson in 
this country and about the same time von Pirquet and Schick 
were making a study of serum rashes mentioned above. The 
publications of these men led to a widespread study of the 
.subject of injections of foreign proteins. It is now' a well- 290 
ANAPHYLAXIS 
established fact that the injection into an animal of a foreign 
protein-vegetable, animal or bacterial, simple or complex- 
followed by a second injection after a proper length of time 
leads to a series of symptoms indicating poisoning, which 
may be so severe as to cause the death of the animal. Rich- 
et's term "anaphylaxis" has been applied to the condition 
of the animal following the first injection and indicates that 
it is in a condition of supersensitiveness for the protein in 
question. The animal is said to be "sensitized" for that 
protein.1 The sensitization is specific since an animal in- 
jected with white of chicken's egg reacts to a second injec- 
tion of chicken's egg only and not pigeon's egg or blood 
serum or any other protein. The specific poisonous sub- 
stance causing the symptoms has been called "anaphylo- 
toxin" though what it is, is still a matter of investigation. 
It is evident that some sort of an antibody results from the 
first protein injected and that it is specific for its own anti- 
gen. 
A period of ten days is usually the minimum time that 
must elapse between the first and second injections in guinea- 
pigs in order that a reaction may result, though a large pri- 
mary dose requires much longer. If the second injection is 
made within less time no effect follows, and after three or 
more injections at intervals of about one week the animal 
fails to react at all, it has become "immune" to the protein. 
Furthermore, after an animal has been sensitized by one 
injection and has reacted to a second, then, if it does not 
die from the reaction, it fails to react to subsequent injec- 
tions. In this latter case it is said to be " antianaphylactic. " 
It must be remembered that proteins do not normally get 
into the circulation except by way of the alimentary tract. 
Here all proteins that are absorbed are first broken down 
to their constituent amino-acids, absorbed as such and 
these are built up into the proteins characteristic of the 
animal's blood. Hence when protein as such gets into the 
blood it is a foreign substance to be disposed of. The blood 
1The term "allergie" was introduced by Von Pirquet to designate the 
state of the animal's being sensitized and "allergic" as the adjective derived 
therefrom. It does not seem to the author that there is any advantage gained 
by the introduction of these terms. ANAPHYLAXIS 
291 
contains proteolytic enzymes for certain proteins normally. 
It is also true that the body cells possess the property of 
digesting the proteins of the blood and building them up 
again into those which are characteristic of the cell. Hence 
it appears rational to assume that the foreign proteins act 
as stimuli to certain cells to produce more of the enzymes 
necessary to decompose them, so that they may be either 
built up into cell structure or eliminated as waste. If in 
this process of splitting up of protein a poison were pro- 
duced, then the phenomena of "anaphylaxis" could be 
better understood. As a matter of fact Vaughan and his 
co-workers have shown that by artificially splitting up pro- 
teins from many different sources-animal, vegetable, path- 
ogenic and saprophytic bacteria-a poison is produced which 
appears to be the same in all cases and which causes the 
symptoms characteristic of anaphylaxis. On the basis of 
these facts it is seen that anaphylaxis is simply another 
variety of immunity. The specific antibody in this case is 
an enzyme which decomposes the protein instead of precipi- 
tating it. The enzyme must be specific for the protein 
since these differ in constitution. Vaughan even goes so 
far as to say that the poison is really the central ring com- 
mon to all proteins and that they differ only in the lateral 
groups or side chains attached to this central nucleus. The 
action of the enzyme in this connection would be to split 
off the side chains, and since these are the specific parts of 
the protein, the enzyme must be specific for each protein. 
The pepsin of the gastric juice and the trypsin of the pan- 
creas split the native proteins only to peptones. As is well 
known, these when injected in sufficient quantity give rise 
to poisonous symptoms, and will also give rise to anaphy- 
laxis under properly spaced injections. They do not poison 
normally because they are split by the intestinal erepsin to 
amino-acids and absorbed as such. Whether Vaughan's 
theory of protein structure is the true one or not remains to 
be demonstrated. It is not essential to the theory of ana- 
phylaxis above outlined, i. e., a phenomenon due to the 
action of specific antibodies which are enzymes. On physio- 
logical grounds this appears the most rational of the few 292 
ANAPHYLAXIS 
explanations of anaphylaxis that have been offered and was 
taught by the author before he had read Vaughan's theory 
along the same lines. 
On the basis of the author's theory the phenomena of 
protein immunity and antianaphylaxis may be explained in 
the following way which the author has not seen presented. 
The enzymes necessary to decompose the injected protein 
are present in certain cells and are formed in larger amount 
by those cells to meet the increased demand due to injection 
of an excess of protein. They are retained in the cell for a 
time at least. If a second dose of protein is given before the 
enzymes are excreted from the cells as waste, this is digested 
within the cells in the normal manner. If a third dose is 
given, the cells adapt themselves to this increased intra- 
cellular digestion and it thus becomes normal to them. 
Hence the immunity is due to this increased intracellular 
digestion. 
On the other hand, if the second injection is delayed long 
enough, then the excess enzyme, but not all, is excreted from 
the cells and meets the second dose of protein in the blood 
stream and rapidly decomposes it there, so that more or less 
intoxication from the split products results. This uses up 
excess enzyme, hence subsequent injections are not digested 
in the blood stream but within the cells as before. So that 
"antianaphylaxis" is dependent on the exhaustion of the 
excess enzyme in the blood, and the condition is funda- 
mentally the same as protein immunity, i. e., due to intra- 
cellular digestion in each case. 
As has been indicated "serum sickness" and sudden death 
following serum injections are probably due to a sensitiza- 
tion of the individual to the proteins of the horse in some 
unknown way. Probably hay fever urticarial rashes and 
idiosyncrasies following the ingestion of certain foods-straw- 
berries eggs, oysters, etc., are anaphylactic phenomena. 
In medical practice the reaction is used as a means of 
diagnosis in certain diseases, such as the tuberculin test in 
tuberculosis, the mallein test in glanders. The individual 
or animal with tuberculosis becomes sensitized to certain 
proteins of the tubercle bacillus and when these proteins in ANAPHYLAXIS 
293 
the form of tuberculin are introduced into the body a reac- 
tion results, local or general, according to the method of 
introduction. The practical facts in connection with the 
tuberculin test are also in harmony with the author's theory 
of anaphylaxis as above outlined. Milder cases of tubercu- 
losis give more vigorous reactions because the intracellular 
enzymes are not used up rapidly enough since the products 
of the bacillus are secreted slowly in such cases. Hence 
excess of enzyme is free in the blood and the injection of 
the tuberculin meets it there and a vigorous reaction results. 
In old, far-advanced cases, no reaction occurs, because the 
enzymes are all used in decomposing the large amount of 
tuberculous protein constantly present in the blood. The 
fact that an animal which has once reacted fails to do so 
until several months afterward likewise depends on the fact 
that the excess enzyme is used in the reaction and time must 
elapse for a further excess to accumulate. 
The anaphylactic reaction has been made use of in the 
identification of various types of proteins and is of very 
great value since the reaction is so delicate, particularly 
when guinea-pigs are used as test animals. Wells has 
detected the 0.000,001 g. of protein by this test. It is evi- 
dent that the test is applicable in medico-legal cases and in 
food examination and has been so used. 294 
SUMMARY OF IMMUNITY 
A Tabulation of Antigens and Antibodies as at 
Present Recognized. 
Class of 
Antigen 
Antibody 
Action of Antibody 
Receptor 
Toxin 
Antitoxin 
Combines with toxin and 
hence prevents toxin 
from uniting with a 
cell and injuring it, 
i. e., neutralizes toxin. 
I. 
Enzyme 
Antienzyme 
Combines with enzyme 
and thus prevents en- 
zyme from uniting 
with anything else and 
showing its action, i.e., 
neutralizes enzyme. 
I. 
Solution of 
protein 
Precipitin 
Unites with its antigen 
and causes its precipi- 
tation from solution. 
II. 
Solution of 
protein 
? 
Causes phenomenon of 
anaphylaxis (?) 
(?) 
Suspension of 
cells 
Agglutinin 
Unites with its antigen 
causes its clumping to- 
gether and settling out 
of suspension. 
II. 
Suspension of 
cells 
Opsonin 
Unites with its antigen 
and makes the cells 
more easily taken up 
by phagocytes. 
H (?) 
Suspension of 
cells 
Amboceptor 
Unites with its antigen 
and also with comple- 
ment which latter then 
dissolves the antigen. 
III. 
Precipitin 
Antiprecipitin 
Neutralizes precipitin. 
I. 
Agglutinin 
Antiagglutinin 
Neutralizes agglutinin. 
I. 
Opsonin 
Antiopsonin 
Neutralizes opsonin. 
I. 
Amboceptor 
Antiamboceptor 
(two kinds) 
Neutralizes amboceptor. 
I. 
Complement 
Anticomplement 
Neutralizes complement. 
I. ANAPHYLAXIS 
295 
Summary of Immunity as Applied to Protection 
from Disease. 
The discussion of "immunity problems" in the preceding 
chapters serves to show that protection from disease either 
as a condition natural to the animal or as an acquired state 
is dependent on certain properties of its body cells or fluids, 
or both. The actual factors so far as at present known may 
be summarized as follows: 
1. Antitoxins which neutralize true toxins; shown to exist 
for very few diseases. 
2. Cytolytic substances which destroy the invading organ- 
ism: in reality two substances; amboceptor, which is speci- 
fic, and complement, the real dissolving enzyme. 
3. Phagocytosis or the destruction of the invading organ- 
isms within the leukocytes. 
4. Opsonins which render the bacteria more readily taken 
up by the phagocytes. 
5. Enzymes other than complement possibly play a part 
in the destruction of some pathogenic organisms or their 
products. This remains to be more definitely established. 
6. It is possible that in natural immunity there might 
be no receptors in the body cells to take up the organisms 
or their products, or the receptors might be present in 
certain cells but of a very low chemical affinity, so that com- 
bination does not occur. It is even highly probable that 
many substances formed by invading organisms which might 
injure specialized cells, such as those of glandular, nervous 
or muscle tissue, have a more rapid rate of reaction with, or 
a stronger affinity for, lower unspecialized cells, such as con- 
nective and lymphoid tissue, and unite with these so that 
their effects are not noticed. 
The importance of these different factors varies in differ- 
ent diseases and need not be considered in this connection. 
The question " which of the body cells are engaged in the 
production of antibodies" is not uncommonly asked. On 
physiological grounds it would not seem reasonable that the 
highly specialized tissues above mentioned could take up 
this work, even though they are the ones which suffer the 296 
SUMMARY OF IMMUNITY 
greatest injury in disease. Hence it is to be expected that 
the lower or unspecialized cells are the source, and it has 
been shown that the antibodies are produced by the phago- 
cytes (though not entirely as Metchnikoff maintained), by 
lymphoid tissue generally, by the bone marrow and also by 
connective-tissue cells, though in varying degrees. 
Since immunity depends on the activity of the body cells 
it is evident that one of the very best methods for avoiding 
infectious diseases is to keep these cells up to their highest 
state of efficiency, to keep in "good health." Hence good 
health means not only freedom from disease but also protection, 
against disease. List of Laboratory Exercises Given in Connection 
with the Class Work Included in this Text-book. 
Exercise 1. Cleaning glassware. 
Exercise 2. Preparation of broth medium from meat 
juice. 
Exercise 3. Preparation of gelatin medium from broth. 
Exercise 4. Preparation of agar medium from broth. 
Exercise 5. Potato tubes. 
Exercise 6. Potato plates. 
Exercise 7. Plain milk tubes. 
Exercise 8. Litmus milk tubes. 
Exercise 9. Sugar broth media. 
Exercise 10. Blood-serum tubes. 
Exercise 11. Inoculation of tubes. Action on complex 
proteins. 
Exercise 12. Production of gas from carbohydrates. 
Exercise 13. Production of indol. 
Exercise 14. Reduction of nitrates. 
Exercise 15. Chromogenesis: Illustrates nicely the varia- 
tion with environment. 
Exercise 16. Enzyme production. 
Exercise 17. Making of plate cultures; isolation in pure 
culture. 
Exercise 18. Stain making and staining. 
Exercise 19. Cell forms and cell groupings. 
Exercise 20. Hanging drop slides. 
Exercise 21. Staining of spores. 
Exercise 22. Staining of acid-fast bacteria. 
Exercise 23. Staining of capsules. 
Exercise 24. Staining of metachromatic granules. 
Exercise 25. Staining of flagella. 
Exercise 26. Study of individual species. 
Exercise 27. Determination of thermal death-point. 
Exercise 28. Action of disinfectants on bacteria. 
Exercise 29. Action of sunlight on bacteria. INDEX 
A 
Abbe, 17 
condenser, 200 
microscope, improvements in, 30, 
36 
Abilgaard, 26 
Abrin, 262 
Absorption of free nitrogen, 117 
tests, 267 
Accidental carriers, 241 
structures, 43 , 
Acetic acid, 99 
bacteria, carbon oxidation, 114 
fermentation, 32 
Acetobacter acidi oxalici, 83 
xylinum, 83 
Achorion schwnleinii, 27, 34 
Acid, acetic, 99 
fermentation, 32 
agglutination, 266 
amino, relation to green plants, 
119 
butyric, 99 
fermentation, 32, 99 
carbolic, first used, 29 
disinfectant action of, 159 
fast bacteria, fat content, 84 
staining of, 209 
fermentation, 93 
Bulgarian fermented milk, 98 
ensilage, 98 
industrial uses, 97 
lactic acid, 96 
sauerkraut, 98 
hydrochloric, 246 
production of, 110 
soils, 81 
Acquired immunity, 251, 252 
Actinomyces bovis, 30, 36 
Actinomycosis, cause of, 30, 36 
path of entrance of, 244 
Actions, reducing, 113 
Activating enzymes, 125 
Active immunity, definition of, 
251, 252 
Active immunity, production of, 
252 
Activities of bacteria, importance 
of, 31 
overproduction, of cells, 258 
physiological, definition of, 87 
in identification, 216 
Acute coryza, 244 
disease, 233 
Adulteration of food, anaphylactic 
test in, 293 
complement-fixation test in, 
279 
immunity reactions in, 255 
precipitin test in, 269 
Aerobes, facultative, 76 
strict, 76 
Aerobic, 76, 215 
Agar, composition of, 179 
gelatinizing temperature, 179 
medium, preparation of, 179 
melting point of, 179 
plating in, 188 
sterilization of, 180 
Agent, chemical, for disinfection, 
156-163 
choice of, for disinfection, 164 
physical, for disinfection, 131 
Agglutinating group, 266 
Agglutination, acid, 266 
diagnostic value of, 266 
in identification of bacteria, 266 
macroscopic, 265 
microscopic, 265 
phenomenon, 265 
Agglutinin, 265 
absorption test for, 267 
action of, 266 
anti-, 270 
antigenic action of, 270 
bacterial, 265 
chief, 267 
co-, 267 
function of, 266 
normal, 266 
partial, 267 INDEX 
299 
Agglutinin relation to precipitins, 
269 
specificity of, 267 
theory of formation, 265 
use of, 266 
Agglutinogen, 266 
Agglutinoid, 270 
Aggressins, 288 
Air, bacteria in, 71 
filtration of, 153 
"germ-free," 153 
Albumin in bacteria., 84 
Alcohol as antiseptic, 160 
as disinfectant, 160 
Alcoholase, 125 
Alcoholic fermentation, 31, 100 
Alexin, 271, 273 
Algae, relation to bacteria, 37 
Alimentary tract as path of en- 
trance, 246 
Alkalies as disinfectants, 158 
Allergic, 290 
Allergie, 290 
Amboceptor, 273 
anti-, 275 
co-, 274 
in cobra, 275 
formation of, 273 
hemolytic, 278 
partial, 274 
in rattle snake, 275 
specificity of, 274 
theory of formation, 273 
Amboceptorogen, 274 
Amebic dysentery, 29, 35 
Ameboid cells, 247 
colonies, 224 
Amino-acids, relation to green 
plants, 119 
Ammonia, structural formula, 103 
Ammoniacal fermentation, 32 
Amoeba coli, 29, 35 
Amphitrichic, 46 
Amylase, 124 
Anaerobes, 76 
cultivation, methods of, 188 
principles underlying, 188 
facultative, 76 
isolation of, 190 
relation to elements, 86 
strict, 76 
Anaerobic, 76, 215 
acid, butyric, 99 
Anaerobic acid fermentation, 98 
bacteria, first discovered, 32 
fermentation of polysaccharides, 
95 
Analysis of ash, 82 
chemical, of tubercle bacilli, 85 
Anaphylactic, anti-, 290 
phenomena, 292 
reaction, uses of, 293 
Anaphylatoxin, 290 
Anaphylaxis, 289 
anti-, 292 
antibodies in, 291 
theory of, 290, 291, 292 
Anaximander, 18 
Anderson, 289 
Anderson and McClintic, phenol 
coefficient, 165 
Andry, 25, 33 
Anilin dyes, as antiseptic, 162 
as disinfectants, 162 
introduction of, 30 
as stains, 204 
Weigert, 36 
fuchsin, 205 
gentian violet, 205 
water, 205 
Animal carriers, 239 
inoculation, uses of, 227 
Animalcules, 19, 33 
Animals, disinfection of, 170 
experimental, 227 
food relationships of, 39 
Ankylostoma duodenale, discovery 
of, 27, 34 
Egyptian chlorosis, cause of, 
28, 35 
hookworm disease, cause of, 
28 
Anthrax, 17, 28, 35 
bacterium a facultative sapro- 
phyte, 238 
isolation of, 29 
due to a bacterium, 29 
in human beings, 238 
path of entrance, 243 
intestine, 246 
stomach, 246 
persistence due to spores, 251 
produced by exhaustion, 251 
protective inoculation in, 30 
spores, 29, 35 
transmission by flies, 242 300 
INDEX 
Anthrax vaccine, 254 
Anti-agglutinins, 270 
aggressins, 288 
amboceptors, 275 
antisera in snake poisoning, 
275 
anaphylactic, 290 
anaphylaxis due to intracellular 
digestion, 292 
protein immunity compared 
to, 292 
bacterial immunity, 254, 255 
bodies, 259 
place of production, 295 
tabulation of, 294 
body, action, 260 
chemical composition, 260 
formation of, 128, 260 
complement, 274 
complementophil amboceptor, 
275 
cytophil amboceptor, 275 
diphtheritic serum, 263 
enzyme, 122, 262 
function of, 262 
Antigen, 259 
chemical composition of, 260 
in complement-fixation, 277 
syphilitic, 277, 279 
in Wassermann test, 279 
Antigens, fats and fatty acids as, 
260 
in preparation of vaccine, 285 
tabulation of, 294 
Antipollenin, 263 
Antiprecipitins, 270 
Antisepsis, 131 
Lister, introduced, 35 
primitive, 25 
Antiseptic, 131 
action of anilin dyes, 162 
carbolic acid as, 159 
cold as, 148 
Antisera in snake poisoning, 275 
Antisnake venoms, 275 
Antitetanic serum, 263 
Antitoxic immunity, 254, 255 
Antitoxin, 261 
collection of, 263 
diphtheria, 30, 252 
preparation of, 263 
standard, 264 
tetanus, 252 
Antitoxins, 261-264 
as factors in immunity, 295 
preservative in, 263 
specific, 261 
Antivenin, 263 
Apes, 227 
Apparatus of Barber, 196 
Appearance of growth on culture 
media, 217 
Appert, 20, 31, 34 
Aqueous gentian violet, 205 
Arborescent growth, 221 
Aristotle, 18 
Aromatic compounds, production 
of, 104, 111 
Arrak, 100 
Arsenate, reduction of, 114 
Arsenite, oxidation of, 115 
Arthtjs, 289 
phenomenon, 289 
Articles, unwashable, disinfection 
of, 169 
washable, disinfection of, 169 
Artificial immunity, 251, 252 
Ase, termination of name of en- 
zyme, 124 
Asepsis, 131 
Aseptic, 131 
Ash, analysis of, 82 
Asiatic cholera, 27, 34, 73, 238, 239, 
246, 248, 249 
Attenuated, 253 
Autoclave, air pressure sterilizer, 138 
pressure sterilizer, 138 
Autogenous vaccines, 284 
in epidemic, 241 
Autoinfection, 234 
Autolysis, 149 
self-digestion, 126 
Autotrophic, 86 
Available nitrogen, loss of, 113 
Azotobacter, 118 
B 
Babes-Ernst corpuscles, 45 
Bacilli, butter, 209 
colon, 248 
grass, 209 
size and shape of, 52 
tubercle, chemical analysis of, 85 
Bacillus, 52, 60, 62 
anthracis, 17, 36 
spore staining, 209 INDEX 
301 
Bacillus of blue milk, 31 
Ducrey's, 245 
subtilis, 77, 83 
spore staining, 209 
Bacteria, absorption of N by, 117 
acid fast, 84, 209 
adaptability, range of, 90 
advantage of motility to, 45 
aids in isolation of, 197 
anaerobic, 32 
cause of disease in animals, 30 
of souring of milk, 32 
cell groupings of, 55 
chains of, 38 
chemical composition of, 39, 81 
elements in, 82 
classed as fungi, 37 
as plants, 33, 35 
definition of, 40 
development of, 90 
distribution of, 71 
energy relationships, 39 
environmental conditions for 
growth, 72 
first classification of, 34 
drawings of, 20 
seen, 19, 33 
food relationships of, 39 
injurious, 72 
isolation of, 194 
measurement of, 40, 203 
metabolism of, 86 
methods of study of, 171 
morphology of, 41 
motile, 45 
nitric, 114 
nitrous, 114 
nucleus of, 42 
occurrence, 71 
pathogenic, outside the body, 
237 
phosphorescent, 111, 112 
position of, 37 
rate of division, 43 
of motion, 45 
relation to algse, 33, 37 
to elements, 86 
to gas and oil, 95 
to phosphate rock, 115 
to protozoa, 40 
to soil fertility, 120 
to sulphur deposits, 116 
to yeasts and torulse, 37 
Bacteria, reproduction of, 37, 55 
root tubercle, 86, 87 
size of, 37, 40 
soil, chief function of, 119 
source of N, 102 
speed of, 45 
spiral, 53 
staining of, 204-212 
sulphur, 63 
thermophil, 75, 77 
universal distribution of, 90 
in vinegar-making, 99 
Bacteriace^®, 62, 66, 70 
Bacterial agglutinin, 265 
vaccines, 282 
preparation of, 283, 284 
Bacterin, 253 
Bacteriocidin, 272 
Bacteriological culture tubes, 184 
examination, material for, 228 
microscope, 200 
Bacteriology, pathogenic, defini- 
tion of, 231 
reasons for study of, 217 
as a science, 17, 32 
Bacteriolysin, 272 
Bacteriopurpurin, 62, 63, 112 
Bacteriotropin, 281 
Bacterium abortus, agglutinin of, 
265 
coli in autoinfection, 234 
gas formation by, 95 
oxygen limits for, 77 
pneumonia through intestinal 
route, 246 
in preparation of sugar broths, 
176 
definition of, 62, 67, 70 
enteriditis, cause of food poison- 
ing, 104 
fluorescens, oxygen limits, 77 
typhosum, 73 
agglutinin, 265 
in phenol coefficient method, 
166 
pneumonia through intestinal 
route, 246 
Ballon pipette, 193 
Balsam, mounting in, 207 
Barber, 253 
apparatus, 196 
Barnyards, disinfection of, 167 
Baskets, wire, 184 302 
INDEX 
Bassi, 27 
silkworm disease, 34 
Bastian, 24 
Baumgartner, 256 
Beaded growth, 221 
Bed-bugs, 241 
Beds, contact, 116 
hot, 117 
Beer, pasteurization of, 141, 144, 
145 
Beggiatoa, 63 
BeggiatoacetE, 63 
Behring, 30 
Belfanti, 271 
Berg, 27, 34 
Berkefeld filter, 154 
Bichloride of mercury as disin- 
fectant, 158 
Bilharz, 28, 35 
Bilharzia disease, 28, 35 
Biochemical reactions, definition 
of, 87 
Biological relationships, immunity 
reactions, 255, 270 
Bipolar germination of spore, 48 
Bismarck brown, 209, 212 
Black-leg, 51, 73, 238, 243, 248, 251 
vaccine, 254 
Bleaching powder as disinfectant, 
158 
Blood, collection of, 228 
cytolytic power of, 272 
detection of, 269 
serum, liquid, sterilization of, 
182 
Loeffler's, 182 
medium, preparation of, 182, 
183 
sterilization of, 182 
vessels in dissemination of or- 
ganisms, 247 
Blue milk, bacterial cause of, 34 
fermentation of, 31, 34 
Boehm, 27, 34 
Boiling as disinfectant, 133 
Boils, 237, 240, 243 
Bollinger, 29, 30, 35, 36 
Bonnet, 20, 33 
Bordet, 271 
Botrytis bassiana, 27, 34 
Bottles, staining of, 206 
Bougies, 154 
Bouillon, 173 
Boyer, 260 
Bread, salt rising, 95, 97 
Bronchopneumonia, 233, 246 
Broth, appearance of growth in, 
218 
extract of, 176 
glycerine, 176 
medium, 173 
nitrate, 177 
sterilization of, 174 
sugar, 176 
Brownian movement, 47, 203 
Brushes, disinfection of, 169 
Bubonic plague, 239 
Buchner, 271 
Budding of yeasts, 37 
Bulgarian fermented milk, 98 
Burning as disinfectant, 132 
Burying as disinfectant, 154 
Butschli, 41, 43 
Butter, 97 
bacilli, staining of, 209 
rancidity of, 101 
Butyric acid fermentation, 32, 99 
Buzzards, 241 
C 
Cabbage disease due to protozoa, 
36 
Cadaverin, 104 
Caignard-Latour, 31, 34 
Calcium hypochlorite as disinfect- 
ant, 158 
oxide as disinfectant, 158 
Candles, filter, 153, 154 
Canned goods, food poisoning by, 
104 
spoilage of, 51 
Canning, introduced, 21, 34 
principles involved, 133 
Capsule, 44, 45 
of spore, 48 
staining of, 210 
Carbohydrates in bacterial cell, 84 
fermentation of, 93-101 
Carbol-fuchsin, 206 
Carbolic acid as antiseptic, 159 
as disinfectant, 159 
first used, 29 
Carbol-xylol, 209 
Carbon cycle, 107 
dioxide, 108 INDEX 
303 
Carbon, function of, in bacteria, 88, 
101 
oxidation of, 114 
in proteins, liberation of, 105 
source of, 88 
uses of, 88, 101 
Carboni, 271 
Cardano, 18 
Carrier problem, solution of, 240 
Carriers, 239 
accidental, 241 
carrion eating animals as, 241 
control of, 240 
intermediate hosts as, 242 
protective measures against, 242 
universal, 240 
of unknown organisms, 239 
Cars, stock, disinfection of, 170 
Catalase, 125 
Catalytic agents, function of, 123 
Catalyzer, 123 
Cattle, 227 
Causation of disease, 24, 128 
Cell, constituents of, 84 
contents of, 41, 83 
forms of, 58, 59 
staining for, 212 
typical, 52 
groupings, 55, 58, 59 
staining for, 195 
metabolism, 90 
structures of, 41 
wall, 41, 59 
composition of, 83 
Cells, chemical stimuli of, 257 
overproduction activity of, 258 
specific chemical stimuli of, 258 
Cellular theory of immunity, 256, 
280 
Cellulose, definition of, 83 
occurrence of, 83 
Chain, 56 
Channels of infection, 243 
alimentary tract, 246 
conjunctive, 244 
external auditory meatus, 244 
genitalia, 245 
intestines, 246 
lungs, 245 
milk glands, 244 
mouth cavity, 244 
mucosae, 244 
nasal cavity. 244 
Channels of infection, pharynx, 245 
skin, 243 
stomach, 246 
tonsils, 245 
Chaos, 25 
Characteristic groupings, 58 
Characteristics of enzymes, 121 
of toxins, 126 
Charrin, 265 
Chart, descriptive, 217 
Chauveau, 256 
Cheese, eyes in, 96 
failures, 110 
Limburger, 101 
odor of, 99 
poisoning, 104 
ripening of, 35 
Chemical composition of bacteria, 
39, 81, 85 
elements in bacteria, 82 
disinfectants, action of, 156-163 
stimuli, 257-260 
theory, fundamentals of, 256 
Chemotherapy, 249, 255 
Chevreuil, 21, 27, 31, 34 
Chicken cholera, 30 
pox, 239, 246 
Chief agglutinin, 267 
cell, 267 
Chitin, 72 
ChlamydobacteriacEu®, 63 
Chlamydothrix, 63 
Chloride of lime as disinfectant, 158 
Chlorine as disinfectant, 157 
Chloroform as antiseptic, 162 
as disinfectant, 162 
Chlorophyl, 37, 112 
Chlorosis, Egyptian, 27, 35 
Cholera, Asiatic, carriers of, 239 
organisms in, 27, 34 
facultative saprophytes, 238 
path of elimination of, 248- 
of entrance of, 246 
relation to moisture, 73 
specific location of, 249 
hog, 242, 248, 252 
Cholesterins as cell constituents, 84 
Chromogenesis, 112 
Chromoparic, 112 
Chromophoric, 112 
Chronic disease, 232 
Chronological table, 33-36 
Chymosin, 124 304 
INDEX 
Circulation of carbon, 107 
of nitrogen, 107 
of phosphorus, 107 
of sulphur, 108 
Classification, advantage of, 59 
early, 33, 35, 59 
Migula's, 62-63 
S. A. B., 63-70 
Cleaning of slides, 207 
Clearing of sections, 209 
Closed space disinfection, 161 
Clostridium, 49 
botulinum, 87, 104, 128, 238, 261 
pasteurianum, 118 
tetani, 128, 209, 233, 261, 263 
Clothing, disinfection of, 170 
Coagglutinins, 267 
Coagulases, 124 
Coagulating enzymes, 124 
Coagulation temperature of pro- 
teins, 51 
Coal, spontaneous heating of, 88 
Coamboceptors, 274 
Cobra, 275 
Coccacej®, 62, 66, 68 
Coccus, appearance of, on divid- 
ing, 57 
cell form of, 52 
groupings of, 52, 56, 57 
division of, 52 
Coenzymes, 122 
Cohn. 28, 33, 35, 59 
Cold as antiseptic, 148 
incubator, 215 
storage, 148 
Colds, due to universal carriers, 
240 
path of entrance of, 244 
vaccines in, 241 
Colonies, characteristit s of plate, 
223-226 
definition of, 173 
Color production, 112 
Colorimetric method of standardi- 
zation, 175 
Combustion, spontaneous, 116 
Commensal, 87 
Commercial preparation of lactic 
acid, 99 
products, why keep, 131 
vaccines, 285 
Communicable disease, 232 
Complement, 273 
Complement deviation test, 277 
effect of temperature on, 274 
fixation test, 276-279 
lecithin as, 274 
relation to toxins and enzymes, 
273 
source of, 277 
Complementoid, 274 
Complementophil haptophore, 273 
Complements, nature of, 274 
Composition, chemical, 81-85 
related to fungi, 39 
relation to food, 81 
Concentration of antitoxin, 264 
Condenser, 200 
Conditions for growth, general, 72 
maximum, 72 
minimum, 72 
optimum, 72 
spore formation, 51 
Congenital immunity, 251, 252 
Conjunctiva as path of entrance, 
244 
Constant temperature apparatus, 
213 
Contact beds, 116 
Contagion, direct and indirect, 34 
Contagious abortion, agglutination 
test, 268 
complement-fixation text, 277 
path of elimination, 248 
of entrance, 245 
Contagium, definition of, 232 
vivum theory, 25, 28, 33 
Contamination of food by carriers, 
241 
Continuous pasteurization, 141 
Contrast stains, 205 
Convalescents, control of, 239-240 
Cornalia, 29 
Corpuscles, Babes-Ernst, 45 
red, in complement-fixation test, 
278, 279 
malaria, etc., in, 249 
Corrosive sublimate as disinfect- 
ant, 158 
Corynebacterium diphtheria;, 64, 69, 
128, 233, 234, 261, 263 
Coryza, acute, 244 
Cotton plugs, 21, 184 
Coughing, 248 
Crateriform liquefaction, 221 
Cream ripening, 97 J INDEX 
305 
Creite, 271 
Crenothrix, 61 
Creolin as disinfectant, 160 
Cresols as disinfectants, 159 
Culture, definition of, 171 
medium, definition of, 171 
essentials of, 172 
inoculation of, 186, 192 
kinds of, 172 
liquid, 172, 173 
methods of using, 184 
optimum moisture for, 73 
plating of, 188 
reaction of, 81, 216 
selective, 198, 199 
solid, 172, 173 
standardization of, 174, 175 
synthetic, 183 
titration of, 174 
use of, 173 
tubes, description of, 184 
Cultures, anaerobic, 188-192 
from internal organs, 229 
mass, 188 
plate, 188 
potato, 186 
puncture, 185 
pure, definition of, 171 
isolation of, 194-199 
slant, 186 
slope, 186 
stab, 185 
Curled edge, 225 
Cutaneous inoculation, 228 
Cycle, carbon, 107 
nitrogen, 107 
phosphorus, 107 
sulphur, 108 
Cystitis, 234 
Cytolysin, 272 
Cytolysins, 271-279 
Cytolytic, 272 
power of blood, 272 
serums, failure of, 275 
substances in immunity, 295 
Cytophil group, 273 
Cytoplasm, 41 
Cytotoxic, 272 
D 
Dallera, 289 
Dark field illumination, 204 
Davaine, 28, 35 
Death-point, thermal, 75 
determination of, 215 
Decomposition, how caused, 108 
importance of, 108 
of urea, 106 
Deep culture tubes, 190-191 ' 
Degeneration forms, 54 
Delousing method in typhus, 242 
De Martin, 35 
Denitrification, 114 
Deodorant, 131 
Descriptive chart, 217 
Diagnosis, agglutination test in, 
265-267 
anaphylaxis in, 292 
complement-fixation test in, 277 
immunity reactions in, 255 
material for bacteriological, 228- 
22.9. 
precipitin test in, 269 
Diastase, 124 
Diffusion of food through cell wall, 
41 
Digestion of proteins, 102 
Dilution method of isolation, 194 
plates, 194, 195 
Dimethylamine, structural form- 
ula, 103 
Diphtheria antitoxin, 30, 263, 264 
bacilli, granules in, 45 
involution forms, 54 
carriers, 239 
location of, 245, 249 
path of entrance, 245 
toxin, M. L. D., 264 
Diplobacillus, 55 
Diplococcus, 56 
Diplococcus, 66, 69 
Diplospirillum, 55 
Discharges, 228 
intestinal, 248 
nasal, 248 
urethral, 248 
vaginal, 248 
Discontinuous sterilization, 133. 
Disease, acute, 233 
of animals to man, 232 
Bilharzia, 28, 35 
cabbage, 30, 35 
causation of, 24, 128 
communicable, 232 
contagious, 34, 232 
of flies, 28, 35 306 
INDEX 
Disease, germ, 25, 27, 33 
hookworm, 28, 35 
infectious, 232, 240 
Johne's, 246, 248 
non-specific, 233 
protozoal, eradication, 242 
transmission, 242 
silkworm, 27, 29, 34, 35 
skin, 243 
specific^ 27, 30, 233 
transmission of, 26, 232 
Dishes, Petri, 181 
Disinfectant, 131 
action of anilin dyes, 162 
closed space, 161 
dry heat as, 133 
moist heat as, 132, 133 
standardization of, 165 
steam as, 132, 133 
Disinfectants, chemical, action of. 
156-163 
first experiment in, 21 
factors affecting, 164-165 
Disinfection, agents in, 131-163 
by boiling, 132, 133 
by burning, 132 
by burying, 154 
definition of, 130 
first chemical, 34 
hot air, 21, 133 
physical agents, 131-155 
practical, 166-170 
precautions in, 170 
puerperal fever, 28, 34 
steam, 134-138 
surgeon's hands, 28 
Dissemination of organisms, 247 
Distaso, 42, 43 
Distilling sour mash, 98 
Division, planes of, 55-58 
rate of, 43, 91 
Dobell, 43 
Dorset, 84 
Dosage of vaccines, 286 
Dose, minimum lethal, 264 
standard test, 264 
Douglas, 42, 43, 280 
Dourine, 245, 248 
Drumstick spore, 49 
Dry heat, 21, 133 
Drying, 131, 132 
Dubini, 27, 34 
Ducrey's bacillus, 245 
Dunham's peptone, 177 
Durham, 265 
Dyes, anilin, as antiseptics, 162 
introduction of, 30 
as stains, 204 
Dysenteries, 242, 246, 248, 249 
Dysentery, amebic, 29, 35 
tropical, 29 
E 
Ectoplasm, 41 
Edema, malignant, 237, 243 
Edge of colony, 225 
Effuse colony, 224 
Egg sensitization, 292 
Ehrenberg, 33, 34 
Ehrlich, 256, 276 
Ehrlich's theory, 256-260 
Eichstedt, 28, 34 
Electric milk purifier, 152 
Electricity, 79, 150 
Elements in bacteria, 82, 86, 88, 89 
Elimination of organisms, 248 
Empusa muscce, 28, 29, 35 
Emulsin, 122 
Endo-enzymes, 126 
Endogenous infection, 235 
Endoplasm, 41 
Endotoxins, 128, 276 
Energy relationships, 39 
transformations, 8&-90 
Ensilage, 98 
Enteritis, 233 
Entire edge, 225 
Entrance of organisms, 243-246, 
247 
Environmental conditions, 72, 130, 
213 
Enzymes, 84, 121-126 
in anaphylaxis, 291 
in immunity, 295 
Enzymoid, 262 
Epidemics, 241 
Epitheliolysin, 272 
Eosin, 204 
Equatorial spore 49 
germination of, 48, 49 
Eradication of disease, 236, 242 
Erysipelas, hog, 248 
Erythrobadllus prodigiosus, 66, 68, 
70, 77, 113 
Essential structures, 41 INDEX 
307 
Essentials of a culture medium, 172 
Esters, 84, 110 
Ether as disinfectant, 162 
Eubacteria, 62 
Exanthemata, 248 
Exhaustion factor in immunity, 
251 
theory of immunity 256 
Existence, conditions for, 72 
Exo-enzymes, 126 
Exogenous infection, 235 
Exotoxins, 128 
Experiment, Pasteur's, 21 
Schroeder and Dusch's, 22 
Schultze's, 21 
Schwann's, 22 
Tyndall's, 24 
Experimental animals, 227 
External auditory meatus, 244 
genitalia, 245 
Extracellular enzymes, 126 
Extract broth, 176 
Eyes in cheese, 96, 97 
F 
Factors affecting disinfectants, 
164, 165 
immunity, 250, 251 
in immunity to disease, 295 
Facultative, 215 
aerobes, 76 
anaerobes, 76, 192 
parasites, 87 
saprophytes, 238 
Failure of cytolytic serums, 275 
of vaccines, 286 
Fat colors, 112 
splitting enzymes, 124 
Father of bacteriology, 19 
of microscope, 19 
Fats as antigens, 260 
occurrence of, 84 
rancidity of, 101 
in sewage disposal, 101 
splitting of, 101 
Favus, 27, 34, 243 
Feces, bacteria in, 72 
Feeding, as inoculating method, 
228 
Feinberg, 43 
Ferment, organized, 126 
unorganized, 126 
Fermentation, 31, 93 
acid, 93, 96 
acetic, 32, 99 
butyric, 32, 99 
alcoholic, 31, 34, 100 
ammoniacal, 32 
bacterial, 32 
blue milk, 31, 34 
of carbohydrates, 93-101 
definition of, 93 
gaseous, 93, 94, 96 
tubes, 184, 190 
yeast, 34, 99 
Fermented milk, Bulgarian, 98 
Fever, due to invisible organisms, 
25 
Malta, 268 
recurrent, 29, 35 
Rocky Mountain spotted, 242 
scarlet, 246, 248 
Texas, 232, 233, 242 
typhoid, 232, 248 
typhus, 242 
yellow, 242 
Fibrin ferment, 124 
Filament, 56 
Filiform growth, 221 
Film, fixing of, 207 
preparation of, 207 
Filter, Berkefeld, 154 
candles, 153-154 
Mandler, 154 
Pasteur-Chamberland, 154 
sprinkling, 115, 116 
Filterable virus, 234 
Filtration, 152-154 
First order, receptors of, 261, 262 
Fischer, 42, 45 
Fixation test, complement, 276 
Fixed virus, 253 
Fixing of film, 207 
Flagella, 45-47 
staining of, 210 
Flash process of pasteurization, 145 
Fleas, 241 
Flexner, 276 
Flies, 28, 35, 241 
Flugge, 271 
Fodor, von, 271 
Food adulteration, complement- 
fixation test in, 279 
immunity reactions in, 255 
precipitin test in, 269 308 
INDEX 
Food contamination by carriers, 
241 
poisoning, 87, 104, 238 
requirement compared with 
man, 92 
uses of, 86 
Foot-and-mouth disease, 244, 248 
Forage poisoning, 87 
Foreignbody pneumonia, 245 
Formaldehyde as disinfectant, 160 
Formalin, 160 
Formol, 160 
Forms, cell, 52-54 
degeneration, 54 
growth, 55 
involution, 54 
study of, 32-34 
Fox fire, 111 
Foxes, 241 
Fracastorius, 25, 33 
Free acid, 175 
receptors, 259 
spores, 48 
Fruiting organs, 37 
Fuchs, 31, 34 
Fuchsin, 205 
anilin, 205 
carbol, 206 
Fungi, bacteria as, 37 
Funnel-shaped liquefaction, 221 
G 
Gabbet's blue, 206 
method of staining, 209 
Gall-bladder, 248 
Galvanotaxis, 79 
Gas formation in cheese, 96, 97 
natural, 95 
production of, 110 
Gaseous fermentation, 93-95 
Gaspard, 26, 34 
Gelatin, advantage of, 178 
composition of, 179 
cultures, first used, 30, 36 
liquefaction of, 103 
medium, 177 
plating of, 188 
standardization of, 178 
sterilization of, 178 
Gemmation, 37 
General conditions for growth, 72 
infections, vaccines in, 286 
Generation, spontaneous, 17-24 
Generic names introduced, 33 
Genitals, 245 
Gentian violet, selective action of, 
162 
stain, 205 
Germ, free air, 153 
theory of disease, 25 
German measles, 233 
Germination of spore, 48 
Germs, 33 
in air, 24, 35 
Gescheidel, 271 
Giemsa stain, 43 
Glanders, 26, 233, 238, 244, 248, 
249, 268, 277 
Glands, mammary, 248 
salivary, 248 
Gleichen, 32 
Globulin in bacteria, 84 
Glycerine broth, 176 
Glycerinized potato, 172 
Glycogen as cell constituent, 84 
Goats, 227 
Gonidia, 63 
Gonococcus, 245 
Gonorrhea, 248, 249 
Good health, 296 
Grain rust, 26, 34 
Gram positive organisms, 162, 208 
negative organisms, 162, 208 
Gram's method of staining, 208 
solution, 208 
Granular edge, 225 
Granules, metachromatic, 212 
Neisser's, 45 
polar, 45 
Granulose in bacteria, 84 
Grape juice, pasteurization of, 141 
Grass bacilli, 209 
Green plants, N nutrition of, 118 
Griesinger, 27, 28, 35 
Group, agglutinating, 266 
haptophore, 261, 262, 266, 270, 
273 
precipitating, 270 
toxophore, 261, 262 
zymophore, 262, 270, 273 
Groupings, cell, 55-58 
Growth, appearance in media, 217 
Gruber, 265, 268 
Gruby, 28, 34 
Gum-like substance in bacteria, 83 H 
Haeckel, 280 
Hanging drop slide, 203 
Haptophore, complementophil, 273 
cytophil, 273 
group, 261, 262, 266, 270, 273 
Harness, disinfection of, 169 
Hay fever, 263, 292 
Health, 296 
Heat as disinfectant, 132-144 
due to oxidation, 112 
production of, 116 
Heated serum, 271, 277, 278, 279 
Heating of manure, 116 
Hellmich, 84 
Helmont, Van, 18 
Hemagglutinin, 265 
Hemicellulose, 83 
Hemolysin, 272 
Hemolytic amboceptor, 278 
Hemorrhagic septicemia, 246 
Henle, 27, 34, 233 
Hericourt, 289 
Herpes tonsurans, 28, 34 
Hesseling, von, 32 
Heterologous sera, 276 
Heterotrophic, 86 
Hill, 33 
Hilton, 27 
Hoffman, 24 
Hog cholera, 231, 242, 248, 252,253 
erysipelas, 248 
Holders, 143 
Holmes, 28 34 
Homologous sera, 276 
Hookworm disease, 28, 34 
Horses, 227, 263 
Host, 87 
Hot beds, 117 
Hunger in immunity, 251 
Hydrochloric acid, 246 
Hydrogen, function of, 98 
ion concentration standardiza- 
tion, 175, 176 
ox dation of, 114 
peroxide, 162 
sulphide, 115 
Hydrophobia, 249 
Hydrostatic pressure, 79 
Hygienic laboratory, 165 
Hypochlorites, 157, 158 
INDEX 
309 
I 
Ice cream poisoning, 104 
Identification of bacteria, 216, 217 
in blood, 269 
in meat, 269 
in milk, 269 
Immersion oil, 201 
Immunity, 236, 250-296 
acquired, 251, 252 
active, 251, 252-255 
antibacterial, 254, 255 
antitoxic, 254, 255 
artificial, 251, 252 
classification of, 251 
congenital, 251 
factors in, 295 
modifying, 250 
inherited, 251, 252 
natural, 295 
passive, 251, 252, 253 
to protein, 290 
reactions, value, 255 
relative, 250 
summary of, 295 
theories of, 256 
Inactivate, 272 
Incubation period, 26, 232 
Incubator, 213 
cold, 215 
room, 213 
Index, chronological, 31 
opsonic, 281 
phagocytic, 281 
Indicator, 278 
Indol, 104 
Infection, 232 
auto, 234 
channels of, 243 
endogenous, 235 
exogenous, 235 
mixed, 234 
primary, 234 
secondary, 234 
wound, 17, 25, 26, 27, 30, 34, 36,, 
233, 234, 240, 243, 248 
Infectious diseases, 232 
control of, 240 
Infective organisms, specificity of. 
location, 249 
Infestation, 232 
Infested, 232 
Influenza, 239, 241, 246 310 
INDEX 
Infusoria, 33 
Inhalation, 228 
Inherited immunity, 251, 252 
Inoculation of animals, 227 
uses of, 227 
of cultures, 186, 188 
definition of, 192 
methods of, 227 
needles, 192 
Inoculations, first protective, 30 
of smallpox, 24 
Insects, 241, 242 
Instruments, sterilization, 136, 167 
Intracardiac, 228 
Intracellular enzyme, 166 
Invasion, 232 
Invertase, 124 
Involution forms, 53, 212 
Iodine, 157 
Iron bacteria, 86 
function of, 89 
Irregular forms, 53 
Isolation of anaerobes, 190 
of pure cultures, aids in, 197-199 
methods, 194-196 
Itch mite, 27, 34 
J 
Jablqt, 32 
Jack-o-lantern, 105 
Jar, Novy, 192 
Jenner, 26, 34, 253 
Johne's disease, 248 
K 
Kette, 32, 35 
Kidneys, 248 
Kinase, 125 
Kircher, 18, 25, 33 
Klebs, 29, 35 
Klencke, 28, 34 
Koch, 17, 27, 29, 30, 33, 36 
Koch's postulates, 233 
Kraus, 268 
Kruse, 254 
Kuchenmeister, 28, 35 
L 
Lab, 124 
Lachrymal canal, 244 
Lactacidase, 125 
Lactic acid bacteria, 97 
fermentation, 96-99 
Lancisi, 25, 33 
Landois, 271 
Latour, 31, 34 
Laveran, 25, 30 
Lecithin as antigen, 279 
as cell constituent, 84 
as complement, 274 
Leeuwenhoek, 19, 32, 33 
Legumes, 118 
Leidy, 27, 33, 34, 35 
Le Moignac, 284 
Leprosy, 233, 244, 249 
Lesser, 32 
Lethal dose, 264 
Leukocytes, washing of, 281 
Lice as carriers, 241 
Liebert, 28, 34 
Light, action on bacteria, 75 
as disinfectant, 148 
production of, 111 
Linnaeus, 25 
Lipase, 124 
Lipochromes, 113 
Lipoids as antigen, 274 
Lipovaccines, 284 
Liquefaction of gelatin, 221 
of protein, 103 
Liquid blood serum, 182 
manure, disinfection of, 169 
media, 172 
Liquids, sterilization of, 153 
Lister, 29, 30, 35 
Litmus milk, 177 
Living bacteria, examination of, 
201 
cause theory, f8, 33 
Localized infections, vaccines in, 
286 
Location of organisms, specificity 
of, 249 
Lockjaw, 231, 233 
Loeffler s blood serum, 182 
blue, 206 
Loop needles, 193 
Lophotrichic, <6 
Losch, 29, 35 
Lungs, 245, 249 
Lye washes as disinfectants, 159 
Lymph channels in dissemination, 
247 
Lysol as disinfectant, 160 M 
McClintock, 165 
McCoy 160 
Macrococcus, 52 
Macroscopic agglutination, 265 
Malaria, 25, 30, 32, 242 
Malarial parasite, 30, 249 
Malignant edema, 237, 243 
Mallease reaction, 269 
Mallein test, 292 
Malta fever, 268 
Mammary glands, 248 
Mandler filter, 154 
Manure, liquid, disinfection of, 169 
heating of, 40 
Mar gar opus annulatus, 242 
Martin, 32 
Mass cultures, 188 
Massart, 42 
Maximum conditions, 72, 73, 74, 76 
Measles, 246, 248, 250 
German, 233, 239 
Measly pork, 28 
Measurement of bacteria, 203 
special unit of, 40 
Meat broth, 173 
identification of, 269 
juice, 173 
poisoning, 104 
Mechanical vibration, 80 
Medico-legal examination, 269, 
279, 293 
Medium. See Culture medium 
Meningitis, 239, 244 
Meningococcus, 244 
Mercuric chloride, 158 
Merismopedia, 57 
Metabiosis, 103 
Metabolism, 86-91 
Metachromatic granules, 44, 45, 
59, 212 
Metastases, 235 
Metatrophic, 86 
Metchnikoff, 256, 280 
Methods of inoculation of animals, 
227 
of cultures, 186-188 
of obtaining pure cultures, 194 
Methylamine, 103 
Methylene blue, 205, 206 
Mice, white, 227 
Microbiology, 231 
INDEX 
Micrococcus, 52, 60, 62, 66, 68, 69, 
245 
Micrometer, 203 
Micromillimeter, 40 
Micron, 40 
Microorganisms, 32 
Microscope, improvements in, 30, 
36 
invention, 19 
Leeuwenhoek's, 19 
use of, 200 
Miscospira, 61, 63 
Miscrosporon furfur, 28, 34 
Middle ear, 241 
Migula's classification, 62 
Milk, blue, 31, 34 
Bulgarian, 98 
digestion of, 102 
flavors in, 110 
glands, 244 
identification of, 269 
litmus, 177 
pasteurization of, 141, 144-147 
as path of elimination, 248 
preparation of, 177 
purifier, electric, 152 
souring of, 32 
sterilization of, 177 
tuberculous, 248 
Minimum conditions, 72, 73, 74, 77 
lethal dose, 264 
Mirror, use of, 200 
Mixed infection, 234 
vaccine, 285 
Mixotrophic, 86 
M. L. D., 264 
Mohler, 167 
Moist heat, 133 
Moisture, 73 
Mold colonies, 226 
Molds in alcoholic fermentation, 
100 
in relation to bacteria, 37, 39 
Molecular respiration, 88, 89 
Monas, 33 
Monkeys, 227 
Monotrichic, 45 
Montague, 24 
Mordants, 204, 211 
Morphology, 41-58 
in identification, 171, 212 
Mosquitoes and malaria, 25, 242 
Motile bacteria, 45 
311 312 
INDEX 
Motion of bacteria, 47 
Brownian, 47, 203 
Mounting in balsam, 207 
Mouth cavity, 244 
Mu, 40 
Mucosae as channels of infection, 
244 
MfiLLER, 33, 34, 59 
Mumps, 239 
Municipal disinfection, 170 
Muntz, 32, 35 
Muscardine, 34 
Mycelia, 39, 226 
Mycobacteriaceoe, 64 
Mycobacterium, 64, 69 
of Johne's disease, 2.9 
leprae, 209 
smegmatis, 209 
tuberculosis, 83, 176, 209 
Mycoproteid, 83 
Mycorrhiza, 119 
Myxomycetes, 38 
N 
Nageli, 29, 35 
Nasal cavity, 244 
discharges, 248 
Natural gas, 95 
immunity, 251, 252, 296 
Needham, 20, 33 
Needles, inoculation, 192 
Negative complement-fixation test, 
278 
phase, 287 
Neisser's granules, 45 
stain, 212 
Nencki, 83 
Nephrolysin, 272 
Neufeld, 281 
Neurin, 104 
Neurotoxin, 272 
Neuvel, 43 
Nichrome wire, 193 
Nitrate broth, 177 
Nitrates in soil, 115 
Nitric bacteria, 114 
Nitrification, 32, 35 
Nitrite, oxidation of, 114 
Nitrogen, absorption of, 117 
in bacterial cell, 89 
circulation, 109 
cycle, 107 
Nitrogen fertilizers, 120 
liberation, 104 
nutrition of green plants, 118 
use of, 103 
Nitrous bacteria, 114 
Non-pathogenic, 87 
Non-specific disease, 233 
Normal agglutinins, 266 
serum, 272 
Nosema bombycis, 29, 35 
Novy, 183 
jar, 192 
Noxious retention theory, 255 
Nuclein, 42, 43 
Nucleoprotein, 43 
Nucleus, 42, 43 
Nutrition of green plants, 118 
Nuttal, 271 
O 
Obermeier, 29, 35 
Objective, oil immersion, 200, 201 
Oblique germination of spore, 48 
Occurrence of bacteria, 71 
Official classification, 59 
Oidium albicans, 27, 34 
Oil bath, 167 
essential for clearing, 209 
immersion objective, 200, 201 
relation of bacteria to, 116 
Omodei, 27 
Opsonic index, 281, 282, 287 
method, 282 
power, 287 
Opsonin, 281 
Opsonins, 281, 282, 295 
Optimum conditions, 72, 73, 74 
Order, receptors of first, 261-264 
of second, 265-270, 281 
of third, 271-279 
Organic acids, 84, 110 
catalyzers, 123 
Organisms, dissemination of, in 
body, 247 
filterable, 234 
pathogenic, elimination of, 248 
entrance of, 243-246, 247 
specific relation to tissue, 249 
ultramicroscopic, 234 
Organized ferments, 126 
Osmotic pressure, 78, 149, 216 
I Otitis media, 244 INDEX 
313 
Otto, 289 
Overproduction theory, 257, 258 
Owen, 27, 34 
Oxidation, 114, 115 
Oxidizing enzymes, 125 
Oxygen, compressed, 77 
as disinfectant, 156 
function of, 88 
nascent, 77 
relationships, 215, 220 
requirement, 88 
source, 76, 77 
Oyster sensitization, 292 
Oznam, 26 
Ozone, 77, 150, 157 
P 
Pancreas. 248 
Papillate, 221 
Paget, 27, 34 
Paraffin oil, 190 
Parasite, 87 
facultative, 87 
strict, 87 
Parodko, 77 
Partial agglutinin, 267 
amboceptor, 274 
Passive immunity, 251, 252 
Pasteur, 17, 21, 29, 30, 31, 32, 35, 
253, 256, 283 
flask, 21, 23, 24, 193 
treatment of rabies, 253 
Pasteur-Chamberland filter, 154 
Pasteurization, 139-147 
continuous, 141 
flash process, 145 
Pathogenic, 87 
bacteria, definition of, 231 
outside the body, 237 
bacteriology, scope of, 235 
organisms, destroyed by boiling, 
133 
elimination of, 248 
entrance of, 243-247 
Paths of elimination, 248 
of entrance, 243-247 
Peacock, 26 
Pebrine, 29, 35 
Pedesis, 47 
Peptone solution, Dunham's, 177 
Period of incubation, 26, 232 
Peritonitis, 234 
Peritrichic, 46 
Peronospora infestans, 28, 34 
Perty, 33, 35 
Pet animals, 241 
Petri dishes, 181, 188 
Petroleum, 95 
Pfeiffer, 271 
Pfeiffer's phenomenon, 271 
Pfeifferella mallei, 65, 69, 265 
Phagocytes, 247 
Phagocytic index, 281 
Phagocytosis, 243, 280-288, 295 
theory, 256 
Pharynx, 245 
Phase, negative, 287 ' 
positive, 287 
Phenol coefficient, 165, 166 
as disinfectant, 159 
production of, 104, 111 
Phenolphthalein, 174 
Phenomenon, anaphylactic, 292 
Arthus', 289 
Pfeiffer's, 271 
Phosphate reduction, 114 
rock, 115 
Phosphorescence, 111 
Phosphorus cycle, 108 
in proteins, 105 
uses of, 89 
Photogenesis, 111 
Physical agents for disinfection, 
131-155 
Physiological activities, 93-129 
definition of, 87 
in identification, 216 
Physiology of bacteria, 71-171 
Phytotoxins, 127, 128 
Pickling, 98 
Pigeons, 227 
Pigments, 84, 112, 113 
Pimples, 234, 240, 243 
Pinoy, 284 
Pipettes for inoculation, 193 
Piroplasma bigeminum, 233, 242, 
249 
Piroplasmoses, 242, 249 
Pirquet, von, 289 
Pityriasis versicolor, 28, 34 
Plague, 246 
Planes of division, 56, 57 
Pianococcus, 62 
Planosarcina, 62 
Plants and animals, 39 314 
INDEX 
Plasmodiophora bassicoe, 30, 36 
Plasmolysis, 41, 42, 78 
Plasmoptysis, 42, 78 
Plate colonies, study of, 224-226 
cultures, 180, 188, 191 
Plates, dilution, 194, 195 
gelatin first used, 30, 36 
Platinum needles, 193 
Plectridium, 49 
Plenciz, 26, 31 
Plugs, cotton, 21, 184 
Pneumococcus, 240, 245 
Pneumonia, 240, 245, 246, 248 
vaccination against, 241 
Poisoning, cheese, 104 
food, 87, 104, 238 
ice cream, 104 
meat, 104 
Polar germination, 48, 49 
granules, 45 
Poliomyelitis, 244 
POLLENDER, 28, 35 
Polysaccharides, fermentation of, 
95 
Polyvalent vaccine, 285 
Pork, measly, 28 
Position of bacteria, 37 
of flagella, 45, 46 
of spore, 49 
Positive phase, 287 
test, 278 
Postulates, Koch's, 233 
Potato, acidity of, 182 
glycerinized, 182 
media, 180-182 
rot, 28, 34 
Power, opsonic, 287 
Practical sterilization and disin- 
fection, 166-170 
Pragmidiothrix, 63 
Precipitinogen, 269 
Precipitinoid, 270 
Precipitins, 268-270 
anti-, 270 
Preparation of antitoxin, 263 
of bacterial vaccines, 283, 284 
of film, 207 
Preservation of slides, 207, 208 
Preservative, alcohol as, 160 
in vaccine, 284 
Pressure, hydrostatic, 79 
osmotic, 78, 149 
oxygen, 76, 77 
Pressure, steam, 136 
sterilization, 136-139 
Prevention of disease, 235, 236, 
253, 255, 283 
Preventive vaccination, colds, 241 
pneumonia, 241 
rabies, 253 
smallpox, 26, 34, 253 
vaccines, autogenous, 285 
stock, 285 
Prevost, 26 
Primary infection, 234 
Process kettle, 137 
Pro-enzyme, 121 
Prophylaxis, 289 
Protamine in bacteria, 84 
Protease, 124 
Protective inoculation, first, 30 
Protein in bacteria, 84 
coagulation temperature, 51 
composition of, 102 
decomposition of, 105 
differentiation of, 255 
foreign, 289 
identification of, 293 
immunity, 290 
putrefaction of, 102-109 
split products of, 291 
splitting of, 106 
structure of, 291 
synthesis of, 113 
Proteus vulgaris, 67, 70, 77 
Protoautotrophic, 115 
Protoplasm, 41, 59 
Prototrophic, 86 
Protozoa, cause of disease, 30 
cell wall in 41 
in intermediate hosts, 242 
relation to bacteria, 40 
specificity of localization, 249 
Protozoal diseases, transmission of, 
242 
Pseud omonadacete, 65, 70 
Pseudomonas pyocyanea, 62, 65, 
70, 128, 265 
Ptomaines, 103, 104 
Puccinia graminis, 26, 34 
Puerperal fever, 28, 34 
Punctiform colonies, 223 
Puncture cultures, 185 
Pure culture, 171, 194-199 
Purification of streams, 73 
of water, 150 INDEX 
315 
Purin bases in bacteria, 84 
Pus cocci, 73 
infectious, 26 
organisms in, 35 
Putrefaction, 27, 31, 33 
definition of, 102 
of proteins, 102-109 
end products of, 103 
in soil, 106 
Putrescin, 104 
Q 
Quarantine, 239 
disinfection, 170 
Quicklime as a disinfectant, 155, 
158 
Quinsy, 245 
R 
Rabbits, 227 
Rabies, bacteriological examina- 
tion in, 229 
Pasteur treatment of, 253 
path of elimination in, 248 
transmission of, 239 
specificity of localization in, 249 
Rabiger's method of staining, 210 
Radiations, 79 
Radium, 79 
Rancidity of butter, 101 
Rashes, serum, 289 
urticarial, 292 
Rate of division, 43, 91 
of movement, 45 
Rats, 227, 241 
Rayer, 28, 35 
Reaction of medium, 81, 174, 175, 
216 
Reactions, biochemical, 87 
immunity, 255, 269, 279, 292, 
293 
surface, 91 
Reaumur, 33 
Receptors, 257, 258, 259, 261-280 
as factors in immunity, 295 
of first order, 262 
free, 259, 261, 262 
of second order, 265 
tabulation of, 294 
of third order, 273 
Recurrent fever, 29, 35 
Red corpuscles, 249, 278, 279 
Redi, 19 
Reducing actions, 112, 113 
enzymes, 125 
Refrigeration as antiseptic, 148 
Reinke, 80 
Relapses, 235 
Relationships of bacteria, 37-40 
biological, 255, 270 
Rennet, 124 
Renucci, 27, 34 
Reproduction, 37, 63, 90 
Resistance to disease, 241, 250 
of spores, 50 
Respiratory function, 88 
tract, 246 
Retarders, 143 
Rheumatism, 245 
Rhizobium leguminosarum, 65, 68 
69, 118 
Rhizoid colonies, 222, 223 
Rhizopus nigricans, 226 
Rhodobacteriace®, 63 
Rhodococcus, 66, 69 
Richet, 289 
Ricin, 262 
Rideal, 165 
Rideal-Walker method, 165 
Rimpau, 281 
Rindfleisch, 29, 35 
Ringworm, 28 
Ripening of cheese, 32 
of cream, 97 
Robin, 262 
Rock, phosphate, 115 
Rocky Mountain spotted fever, 
242 
Rogers, 265 
Rbntgen rays, 79 
Room temperature, 213 
Rooms, disinfection of, 167 
incubator, 213 
Root tubercle bacteria, 86, 87, 108 
tubercles, 117 
Rosenau 289, 
Rot, potato, 28, 34 
Round worm, 232 
Roup, 244 
Roux, 30 
Rubbing as inoculation, 195 
Rust, grain, 26, 34 
Ruzicka, 42 316 
INDEX 
S 
Saccate liquefaction, 222 
Safranin, 205 
Saliva, 248 
Salivary glands, 248 
Sake, 100 
Salt-rising bread, 95 
Saprogenic, 102 
Saprophilic, 103 
Saprophyte, 87, 238 
Sarcina, 57, 58, 60, 66, 68, 69 
lutea, 77 
ventriculi, 83 
Sarcoptes scabiei, 27, 34 
Sauerkraut, 98 
Scarlet fever, 246, 248, 250 
Scavengers, bacteria as, 108 
Schick, 289 
Schistosomum hematobium, 28, 35 
Schlosing, 32, 35 
Schonlein, 27, 34 
Schroeder and Dusch, 21 
Schultze, 21, 34 
Schwann, 21, 31, 34 
Sea, bacteria in, 71, 111 
Sealing air-tight, 20 
Secondary infection, 234 
Sections, staining of, 209 
Selective media, 198, 199 
Self-limited, 233 
Semmelweiss, 28, 35 
Sensitization, 290 
Sensitized animal, 290 
bacteria, 254 
vaccine, 254 
Septicemias, hemorrhagic, 246 
Sero-bacterins, 254 
Serum, antidiphtheritic, 263 
antitetanic, 263 
heated, 271, 277, 279 
rashes, 289 
sickness, 289, 292 
simultaneous method, 253 
therapy, 253 
Serums, cytolytic, failure of, 275 
Sewage disposal, 101, 116 
sulphate, reduction in, 114 
Shape of spore, 48 
Sickness, serum, 289, 292 
Side-chain theory, 256, 258 
Silkworm disease, 27, 29, 34, 35 
Size of bacteria, 37, 40 
Skatol, 104 
Skin, channel of infection, 243 
diseases, 243 
glanders, 248 
lesions, 228 
pocket, 227 
Slant cultures, 186 
Slide, cleaning of, 207 
hanging drop, 203 
staining on, 207 
Slope cultures, 186 
Sludge tanks, 116 
Small intestine, 249 
Smallpox, 24, 26, 34, 239, 246, 248 
babies, 252 
vaccines, 253 
Smith, 289 
tubes, 184 
Snake poisons, 263, 275 
venoms, 128 
Sneezing, 248 
Soap, 160 
medicated, 160 
Society of American Bacteriolo- 
gists, classification, 63 
descriptive chart, 217 
key, 68 
Sodium hypochlorite, 158 
Soil, acid, 81 
bacteria, 119 
bacteriology, 35 
enrichment, 117 
fertility, 120 
organisms, 74 
Solid media, 172, 173 
Solution, Gram's, 208 ■ 
stock, 205 
Sore throat, 240, 241 
Sound, 80 
Sour mash, 98 
Source of complement, 277 
Souring, 98 
Spallanzani, 20, 31, 34 
Species determination, 59, 60 
Specific amboceptor, 274, 278, 279 
antibody, 291 
chemical stimuli, 257, 258, 259 
disease, 27, 30, 233 
Specificity of agglutinins, 267 
of amboceptor, 274 
of location, 249 
of opsonins, 281 
Spermotoxin, 272 INDEX 
317 
Spherical form, 52 
Spherotilus, 63 
Spirillacece, 63, 65 
Spirilloses, 241, 242 
Spirillum, 53, 54, 55, 61, 63, 66, 
68, 69 
rubrum, 113 
Spirochaeta, 61 
obermeieri, 29, 35 
Spirochetes, 53, 242 
Spirosoma, 63 
Splenic fever, 28 
Split products of proteins, 291 
Splitting enzymes, 124 
of fats, 101 
Spoilage of canned goods, 51, 78 
Spoiling of food, 91 
Spontaneous combustion, 105, 116 
generation, 17-24, 33, 34 
outbreaks of disease, 239 
Sporangia, 226 
Spore, 47-51 
anthrax, 29, 35 
capsule, 48 
germination, 48 
Spores, cause spoiling of canned 
goods, 51 
destroyed by boiling, 133 
first recognized, 33, 35 
light on, 75 
in pasteurization, 146 
resistance of, 50, 51 
staining of, 209 
two in bacterium, 50 
Sprinkling filters, 116 
Stab cultures, 185 
Stables, disinfection of, 167 
Stain, anilin fuchsin, 205 
gentian violet, 205 
aqueous gentian violet, 205 
Bismarck browm, 212 
carbol fuchsin, 206 
contrast, 205 
Gabbet's blue, 206 
Loeffler's blue, 206 
Neisser's, 212 
.Staining, 204-212 
acid-fast bacteria, 209 
bottles, 206 
capsules, 210 
cell forms, 212 
groupings, 212 
flagella, 210 
Staining, Gabbet's method, 209 
Gram's method, 208 
metachromatic granules, 212 
Neisser's method, 212 
, Rabiger's method, 210 
reasons for, 204 
sections, 209 
spores, 209 
Welch's method, 210 
Ziehl-Neelson, 210 
Standard antitoxin, 264 
methods, 217 
test dose, 264 
toxin, 264 
Standardization, colorimetric 
method, 175 
of culture media, 174 
> of disinfectants, 165 
H-ion method, 175 
of vaccines, 284 
Starin, 196 
Staphylococcus, 57, 58 
Staphylococcus, 66, 68, 69 
Steam at air pressure, 134 
sterilizers, 135 
I streaming, 135 
under pressure, 136 
Stegomyia, 242 
Sterile, 131 
Sterilization, 130 
in canning, 133 
discontinuous, 133 
by filtration, 21, 152 
first experiment by boiling (moist 
heat), 20 
by chemicals, 21 
by dry heat (hot air), 21 
by filtration, 21 
Sterilizers, pressure, 137 
Stimuli, chemical, 257, 258, 259 
Stock cars, 170 
solutions, 205 
vaccines, 285 
Stomach, 246 
Straight needles, 192 
Stratiform liquefaction, 222 
Strawberry poisoning, 292 
Streak methods of isolation, 196 
plates, 188 
Streptobacillus, 53, 56 
Streptococcus, 56, 60, 245 
Streptococcus, 60, 62, 66, 68, 69 318 
INDEX 
Streptospirillum, 55 
Streptothrix, 38 
Streptothrix bovis, 30, 36 
Strict aerobe, 76 
anaerobe, 76 
parasite, 87 
Structures, accidental, 43 
cell, 41 
essential, 41 
Subcutaneous inoculation, 227 
Subdural inoculation, 228 
Substrate, 123 
Successive existence, 103 
Sugar broth, 176, 177 
Sulphate reduction, 114 
Sulphur bacteria, 63, 86, 115 
deposits, 116 
function of, 89 
in proteins, 105 
Summary in immunity, 295 
Ehrlich's theory, 259 
Sunning, 148 
Surface reactions, 91, 92 
Surgical instruments, 167 
Susceptibility, 235 
Swine, 227 
Symbionts, 87, 103 
Symbiosis, 87 
Synthetic media, 172, 183 
Syphilitic antigen, 277, 279 
Syphilis, 233, 245, 248, 249 
Wassermann test, 277, 279 
T 
Tabulation of antigens and anti- 
bodies, 294 
Toenia solium, 28, 35 
Tapeworm, 28, 35, 232 
Taxes, 203 
Temperature conditions, 74 
effect on growth, 213 
factor in immunity, 251 
room, 213 
Test, complement deviation, 277 
fixation, 276, 279 
dose, 264 
for enzymes, 123 
Gruber-Widal, 268 
mallein, 292 
negative, 278 
positive, 278 
for toxins, 127 
Test, tuberculin, 292 
Wassermann, 277, 279 
Widal, 268 
Tpqfirlp 94Q 
Tetanus, 231, 238, 243, 249, 251f 
252 
antitoxin, 252 
toxin, 126 
Tetracoccus, 57 
Tetrad, 57 
Texas fever, 232, 233, 242 
Thaer, 31 
Theories of immunity, 256 
Theory, anaphylaxis (author's), 
290-292 
cellular, 256 
chemical, 256 
contagious disease, 34 
contagium vivum, 25, 28, 33 
Ehrlich's, 256-260 
exhaustion, 256 
germ, 25 
living cause, 33 
mosquito, 25 
noxious retention, 256 
overproduction, 257, 258 
phagocytosis, 256 
side-chain, 256 
spontaneous generation, 17 
unfavorable environment, 256 
Thermal death point, 75, 215 
Thermophil bacteria, 75, 77 
Thermoregulator, 213 
Thermostadt, 213 
Thiobacteria, 63 
Thiothrix, 63 
Thread, 56 
Thrombin, 124 
Thrush, 27, 34, 244 
Ticks, 241 
Tiedemann, 26 
Tinea, 28 
Tissue contrast stains, 205 
Titer, 268 
Titration, 174 
Tonsil, 245, 249 
Tonsillitis, 245 
Touissant, 283 
Toxin, diphtheria, 264 
effect of temperature, 262 
final test for, 127 
in food poisoning, 104 
molecule, 261, 262 INDEX 
319 
Toxin, standard, 264 
tetanus, 264 
Toxin-antitoxin method, 254 
Toxins and enzymes compared, 127 
as cell constituents, 84 
production of, 126-128 
of other organisms, 127 
specific localization, 249 
true, 128 
Toxoid, 262 
Toxophore group, 261, 262, 273 
Tract, alimentary, 246 
Transmission, accidental carriers 
in, 241 
agency of, 232 
of contagious diseases, 232 
of disease, 26, 28, 35, 239 
of glanders, 26, 34 
of protozoal diseases, 242 
of tuberculosis, 28, 29, 34, 35, 
238 
Transverse division, 54, 56 
Traub e, 271 
Treponema pallidum, 245 
Trichina, 27 
Trichina spiralis, 27, 34, 35 
Trichinosis, 28, 35 
Trichophyton, 243 
Trichophyton tonsurans, 28, 34 
Trimethylamine, 104 
Tropical dysentery, 29 
lands, 242 
Tropisms, 203 
True toxins, 128 
Trypanosomes, 242 
Trypanosomiases, 241, 243 
Tubercle bacteria, 85, 209 
Tuberculin reaction, 292, 293 
Tuberculosis, 73, 233, 238, 245, 
246, 248, 249 
due to bacteria, 30 
produced experimentally, 28, 34 
proved infectious, 29, 35 
Tuberculous milk, 248 
Tubes, culture, 184 
deep, 190 
fermentation, 184, 190 
Smith, 184 
Vignal, 189 
Two spores in a bacterium, 50 
Tyndall, 24 
Tyndallization, 133 
Tyndall's box, 23, 24, 35 
Typhoid bacilli, 73, 238 
bacillus, 45 
carriers, 239 
fever, 231, 233, 248, 265, 268 
transmission by flies, 242 
vaccine, 254 
Typhus, 242 
Typical cell forms, 52 
U 
Ultramicroscope, 204 
Ultramicroscopic organisms, 234 
Ultraviolet rays, 150 
Unfavorable environment theory, 
256 
Unit of antitoxin, 264 
of measurement, 40 
Universal carrier, 240 
Unorganized ferment, 126 
Unwashable articles, 169 
Urea, 106 
Urease, 125 
Urethral discharges, 248 
Urine, 72 
Urticarial rashes, 292 
V 
Vaccination in chicken cholera, 30 
negative phase in, 287 
in pneumonia, 241 
in smallpox, 26, 34, 253 
Vaccine, 253 
age of, 285 
anthrax, 254 
antigens for, 285 
autogenous, 285 
black-leg, 254 
derivation of, 253 
mixed, 285 
polyvalent, 285 
preservative in, 284 
sensitized, 254 
smallpox, 253 
Vaccines, bacterial, 283 
in colds, 241 
dosage of, 286 
in epidemics, 241 
failure of, 285-286 
in infections, 286 
preparation of, 283 
. standardization of, 284 320 
INDEX 
Vaccines, stock, 284 
theory of, 286 
use of, 283 
Vacuoles, 42, 43, 44, 59 
Vaginal discharges, 248 
Varo, 25 
Vaughan, 291 
Vaughan and Novy's mass cul- 
tures, 188 
Vegetable toxins, 127, 128 
Vegetables, forcing of, 117 
Vehicles, disinfection of, 169 
Venoms, antisnake, 275 
Viborg, 26, 34 
Vibration, mechanical, 80 
Vibrio, 33, 35, 53, 65, 68, 69 
cholerce, 66, 73 
Vignal tubes, 189 
Villemin, 29, 35 
Villous growth, 219, 221 
Vinegar, 99, 114 
Virulence, 235 
Virus, 234 
Vultures, 241 
W 
Walker, 165 
Wall, cell, 41 
composition of, 82, 83 
Warden, 260 
Washable articles, disinfection of, 
169 
Washing leukocytes, 281 
Wassermann test, 277 
Water, bacteria in, 73 
filtration of, 153 
purification of, 77, 150 
sterilization of, 157 
Webb, 253 
Weigert, 17, 30, 36, 42, 257, 258 
Welch's method of staining, 210 
Whooping cough, 246, 250 
Widal, 265 
test, 268 
Will o' the wisp, 105 
Wine, pasteurization of, 141 
Winogradsky, 32, 63, 86 
Wire baskets, 184 
nichrome, 193 
Wollstein, 26, 34 
Woronin, 30, 36 
Wound infections, 17, 25, 26, 27, 
30, 34, 36, 233, 234, 240, 243, 248 
Wright, 280 
X 
X-RAYS, 79 
Xylinum, acetobacter, 83 
Y 
Yeast, fermentation, 31, 34, 99, 
100, 114 
relation to bacteria, 37 
reproduction of, 37, 39 
Yellow fever, 242 
Z 
Zanz, 18 
Zenker, 27, 28, 35 
Zettnow, 43 
ZlEMANN, 43 
Ziehl-Neelson method of staining, 
210 
Ziehl's solution, 206 
Zoogloea, 44 
Zootoxins, 128 
Zymase, 125 
Zymogens, 121, 125 
Zymophore group, 273