ANTHONY VAN LEEUWENHOEK 
Who first saw bacteria THE FUNDAMENTALS 
OF 
BACTERIOLOGY 
I 
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 165 ENGRAVINGS AND 6 PLATES 
LEA & FEBIGER 
PHILADELPHIA AND NEW YORK 
1917 COPYHIGHT 
LEA & FEBIGER 
1917 TO 
GRACE HAMILTON MORREY 
AMERICAN PIANIST PREFACE. 
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 
General 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 class-room periods per 
week. Each student is compelled to take two laboratory 
periods of three hours per week along with the class work. 
The outline 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 Bacteri- 
ology, covering the standard text-books as well as works of 
reference and the leading periodicals dealing with the sub- 
ject. Thus the latest information has been incorporated. 
No attempt has been made to give detailed references in a 
work of this character. VI 
PREFACE 
The photomicrographs are original except where otherwise 
indicated and are all of a magnification of one thousand 
diameters where no statement to the contrary appears. 
These photographs were made with a Bausch & Lomb Pro- 
jection Microscope fitted with a home-made camera box. 
Direct current 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 centigrade. 
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, N. 
Chicago, Ill.; Mr. C. B. Hoover, Superintendent of Sewage 
Disposal Plant, Columbus, O.; Mr. C. P. Hoover, Super- 
intendent of Water Filtration Plant, Columbus, O.; The 
Hydraulic Press Manufacturing Company, Alt. Gilead, ().; 
Loew Manufacturing Company, Cleveland, O.; Metric 
Metal Works, Erie, Pa.; Sprague Canning Machine Company, 
Chicago, Ill.; U. S. Marine Hospital Service.; Wallace and 
Tiernan Company, 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. 
Columbus, 1917. 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 34 
PART I. 
MORPHOLOGY. 
CHAPTER II. 
Cell Structures--Cell Wall-Protoplasm-Plasmolysis-Plasmop- 
tysis-Nucleus-Vacuoles-Capsules-Metachromatic Granules 
-Flagella-Spores 39 
CHAPTER III. 
Cell Forms-Coccus-Bacillus-Spirillum 49 
CHAPTER IV. 
Cell Groupings 52 
CHAPTER V. 
Classification-Migula's 56 VIII 
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 
Vibration 61 
CHAPTER VII. 
Chemical Environment-Reaction of Medium-Chemical Com- 
position 70 
I 
CHAPTER VIII. 
Chemical Environment (Continued)-General Food Relation- 
ships-Metabolism 75 
CHAPTER IX. 
Physiological Activities-Fermentation of Carbohydrates-Split- 
ting of Fats 82 
CHAPTER X. 
Physiological Activities (Continued)-Putrefaction of Proteins- 
Cycles of Nitrogen, Carbon, Sulphur, Phosphorus .... 91 
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 98 
CHAPTER XII. 
Physiological Activities (Continued)-Production of Enzymes- 
Discussion on Enzymes-Toxins-Causation of Disease . . 109 CONTENTS 
IX 
CHAPTER XIII. 
Disinfection - Sterilization - Disinfectants - Physical Agents- 
Pasteurization 118 
CHAPTER XIV. 
Disinfection and Sterilization (Continued)-Chemical Agents . 144 
CHAPTER XV. 
Disinfection and Sterilization (Continued)-Choice of Agent- 
Practical Sterilization and Disinfection 151 
PART III. 
THE STUDY OF BACTERIA. 
CHAPTER XVI. 
Culture Media-Broth, Gelatin, Agar, Potatoes, Milk, Blood 
Serum 157 
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 168 
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 178 
CHAPTER XIX. 
Study of the Morphology of Bacteria-Bacteriological Microscope 
-Hanging Drop Slides-Staining-Gram's Method-Spores- 
Acid-fast Bacilli-Capsules-Flagella 184 X 
CONTENTS 
CHAPTER XX. 
Study of the Physiology of Bacteria-Temperature-Incubators- 
Thermal Death Point-Oxygen Relationships-Study of Physi- 
ological Activities-Appearance of Growth on Culture Media . 196 
CHAPTER XXL 
Animal Inoculation-Material for Bacteriological Examination . 210 
PART IV. 
GENERAL PATHOGENIC BACTERIOLOGY. 
CHAPTER XXII. 
Introduction - Infection - Acute Infection - Chronic Infection 
-Specific-Non-specific-Koch's Postulates-Virulence-Sus- 
ceptibility 213 
CHAPTER XXIII. 
Pathogenic Bacteria Outside the Body-As Saprophytes-As 
Facultative Saprophytes-Latent-Carriers 218 
CHAPTER XXIV. 
Channels of Infection-Skin-Mucosae-Respiratory Tract-Ali- 
mentary Tract-Dissemination in the Body-Paths of Elimi- 
nation-Specificity of Location 222 
CHAPTER XXV. 
Immuni ty-Natural-Artificial-Active-Passive-Production of 
Immunity-Vaccine-Antiserum 228 
CHAPTER XXVI. 
Theories of Immunity - Pasteur - Chauveau - Baumgartner - 
Metchnikoff-Ehrlich-Principles of Ehrlich's Theory . . . 234 CONTENTS 
XI 
CHAPTER XXVII. 
Ehrlich's Theory (Continued)-Receptors of the First Order- 
Antitoxin-Antienzyme-Preparation of Antitoxins-Units . 238 
CHAPTER XXVIII. 
Ehrlich's Theory (Continued)-Receptors of the Second Order- 
Agglutinins-Agglutination Reaction-Preci pitins-Precipitin 
Test 242 
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 248 
CHAPTER XXX. 
Phagocytosis-Opsonins-Opsonic Index-Bacterial Vaccines- 
Preparation of-Use of-Aggressins 256 
I 
CHAPTER XXXI. 
Anaphylaxis-Author's Theory-Tuberculin Test-Summary . 264 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 
writh Koch's proof that Bacterium anthracis is the cause 
of anthrax in 1876. Nevertheless this discovery of Koch's 
was preceded by numerous observations and experiments 
which led up to it. Some of this work was done in attempt- 
ing to disprove the old "spontaneous generation" theory as 
to the origin of organisms; some in searching for the causes 
of disease and some in the study of fermentation and putre- 
faction. 
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. 
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 18 
HISTORICAL INTRODUCTION 
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. 
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 
succeeded in making a lens so powerfid that he could see 
in water and various infusions very minute living bodies 
never before observed. Leeuwenhoek contributed 112 papers 
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. 1). Rightly may he be styled 
"The Father of Bacteriology," if not of the microscope. He 
says in one paper: "With the greatest astonishment I 
observed that everywhere through the material which I SPONTANEOUS GENERATION 
19 
was examining were distributed animalcules of the most 
microscopic dimness which moved themselves about in a 
remarkably energetic way." Thus he considered these liv- 
ing 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 
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 
Fig. 1.-The first drawings of bacteria by Leeuwenhoek. The dotted line 
C-D indicates movement of the organism. 
ill 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. Uis critics raised 
the objections that heating the liquids destroyed their 
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 20 
HISTORICAL INTRODUCTION 
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 in Francois Appert, 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. 
Schultze, in 1836, attempted to meet the second objection to 
Spallanzani's experiment, i. e., the exclusion of air, by draw- 
ing air through his boiled infusions, first causing it to bubble 
Fig. 2.-Schultze's experiment. The set of bulbs next to the face con- 
tained KOH and the other set concentrated H2SO4. Air was drawn through 
at frequent intervals from May until August but no growth developed in 
the boiled infusion. 
through concentrated sulphuric acid to kill the "germs" 
(Fig. 2). His flasks fortunately showed no growths, but 
his critics claimed that the strong acid changed the proper- 
ties of the air so that it would not support life. Schwann, in 
1837, modified this experiment by drawing the air through 
a tube heated to destroy the living germs (Fig 3). His 
experiments were successful but the "spontaneous genera- 
tion" theorists raised the same objection, i. e., the change 
in the air by heating. Similar arguments were brought 
forward, also to the use of cotton plugs as filters by Schroeder SPONTANEOUS GENERATION 
21 
and Dusch in 1859 (Fig. 4). It remained for Chevreuil and 
Pasteur to overcome this objection in 1891 by the use of 
Fig. 3.-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. 4.-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 22 
HISTORICAL INTRODUCTION 
Fig. 5.-Pasteur's flask. 
Fig. 6.-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 
interior. 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 Monthly, April, 1877.) CAUSATION OF DISEASE 
23 
flasks with long necks drawn out to a point and bent over. 
These permitted a full access of air by diffusion but kept 
out living germs, since these cannot fly but are carried 
mechanically by air currents or fall of their own weight 
(Fig. 5). Hoffman, the year before (1860), had made similar 
experiments but these remained unnoticed. The Pasteur 
flasks convinced most scientists that "spontaneous genera- 
tion" has never been observed by man, though some few, 
notably Dr. Charlton Bastian, of England, vigorously sup- 
ported 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. 6). 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. 
CAUSATION OF DISEASE. 
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. Fracastorius (1484-1553), in 
a work published in 1546, elaborated a theory of "disease 
germs" and "direct and indirect contagion" very similar to 
modern views, though based on no direct pathological knowl- 
edge. 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 substances, 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 24 
HISTORICAL INTRODUCTION 
Fracastorius, his theory of a "living cause" for various dis- 
eases 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 held such views. 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 Naturcp (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." 
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 
1 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. CAUSATION OF DISEASE 
25 
only of the pus but also of the nasal discharges, saliva, 
urine, etc., of glandered horses. 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 bacterium in 
this case. 
In 1822 Gaspard showed the poisonous nature of material 
from infected wounds by injecting it into animals and caus- 
ing their death. Bearing on the "contagium vivum" theory 
was the rediscovery of the "itch mite'' (Sarcoptes 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 putre- 
faction did not occur in meat protected from contamination, 
and suggested that wound infection probably resulted from 
entrance 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. The fungous nature of 
Favus, a scalp disease, was recognized by Schonlein in 1839, 
and the organism was afterward called "Achorion schoen- 
leinii." 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") 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 instruments of 
that period did not enable Henle to prove his 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 26 
HISTORICAL INTRODUCTION 
of ringworm, is due to the fungus Trichophyton tonsurans. 
Klencke, in 1843, produeed generalized tuberculosis in a rab- 
bit by injecting tuberculous material into a vein in the ear, 
but did not carry his researches further. 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. 
Pollender, in 1849, and Davaine and Bayer, 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 Ankylosto- 
mum duodenale was shown by Griesinger 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, Tenia solium, in 1852, 
Cohn, an infectious disease of flies due to a parasitic fungus 
{Empusa muscoe) in 1855, and Zenker the Trichinella spiralis 
in trichinosis of pork ("measly pork") in 1860. The organ- 
isms 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 men- 
tioned, showed that anthrax could be transmitted from 
animal to animal by inoculation of blood, but only if the 
blood contained the minute rods which he believed to be the 
cause. In 1865 Villemin repeatedly caused tuberculosis in 
rabbits by subcutaneous injection of tuberculous material 
and showed that this disease must be infectious also. In 
this 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 Erance. SIR JOSEPH LISTER ROBERT KOCH CAUSATION OF DISEASE 
27 
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 
Sinrochceta obermeieri. Losch ascribed tropical dysentery 
to an ameba, named by him Amoeba coli, in 1875. Finally, 
Koch, in 1876, isolated the anthrax bacillus by means of gelatin 
plates (first used by Emil Chr. Hansen in his studies on yeast), 
worked out the life history of the organism and reproduced 
the disease by the injection of pure cultures and recovered 
the organism from the inoculated animals, thus establish- 
ing 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 find- 
ings, 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 brassicce) to be the cause of a disease in cabbage 
plants, the first proved instance of a unicellular animal caus- 
ing 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 condi- 
tion, 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- 28 
HISTORICAL INTRODUCTION 
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 solid cultures for 
isolation and study, 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 trypanosomiases 
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 dis- 
covery of diphtheria antitoxin by Behring, and independently 
by Roux, in 1890, a discovery which opened up the wide 
field of immunity which is so persistently cultivated at the 
present time. 
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 
and 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 LOUIS PASTEUR PUTREFACTION AND FERMENTATION 
29 
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 appli- 
cation 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 hydrochloric 
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" 
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 Hesseling, 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 butyric 
fermentations. Kette, in 1869, asserted the probability of 30 
HISTORICAL INTRODUCTION 
Pasteur's researches furnishing a scientific basis for many 
processes of change in the soil. In 1873 Schlosing 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. 
Ail 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, 
Reaumur, Hill and others. Muller, of Copenhagen, in 1786 
published the first attempt at classification, a most impor- 
tant 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 geol- 
ogist, who took great interest in natural history in general, 
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 classification on a mod- 
ern basis and added greatly to the knowledge of morphology STUDY OF FORMS 
31 
and natural history of bacteria. He described spore forma- 
tion and the development of spores into active bacteria, 
and showed the close relationships as well as differences 
between the bacteria and the lower algae. 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. 
1776. Spallanzani, boiled and sealed infusions. 
1786. Muller, first classified "animalcules." 
1787. Wollstein, glanders pus infectious. 
1797. Viborg, transmitted glanders repeatedly. 
1807. Prevost, grain rust, Puccinia graminis. 
1810. Appert, directions for "canning." 
1822. Gaspard, infectiousness of material from wounds. 
1834. Renucci, itch-itch mite (Sarcoptes scabiei). 
1836. Schultze, air through acid to kill "germs." 
1837. Chevreuil and Pasteur, protected meat did not 
putrefy; suggested wound infection due to entrance 
of germs from without. 
1837. Caignard-Latour, Schwann, alcoholic fermentation 
-yeast. 
1837. Schwann, air through heated tubes to kill germs. 
1837. Bassi, muscardine of silkworms, Botrytis bassiana. 32 
HISTORICAL INTRODUCTION 
1838. Boehm, cholera, saw organisms in stools (not the 
cause). 
1838. Ehrenberg, study of forms. 
1839. Schbnlein, 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. 
1845. Liebert, a potato rot, Peronospora infestans. 
1846. Eichstedt, Pityriasis versicolor, Microsporon furfur. 
1849. Leidy, Joseph (American geologist), considered 
"vibrios" to be plants. 
1849. Pollender, Anthrax, saw rods in blood. 
1850. Davaine and Payer, Anthrax , saw rods in blood. 
1851. Griesinger, Egyptian chlorosis, Ankylostoma duo- 
denale. 
1851. Bilharz, Bilharzia disease, Schistosomum hematobium. 
1852. Kiickenmeister, tapeworm, Toenia solium. 
1852. Perty, saw spores in bacteria. 
1854. Cohn, classed bacteria as plants. 
1855. Cohn, Disease of Flies, Empusa muscm. 
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. 
1865. Villemin, repeatedly transmitted tuberculosis to 
rabbits. 
1865. Lister, introduced antisepsis in surgery. 
1866. Rindfleisch, Pyemia, organisms in the pus. 
1866. Von Hesseling, 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. STUDY OF FORMS 
33 
1871. Klebs, Pyemia, organisms in the pus. 
1872. Bollinger, spores in anthrax. 
1872-75. Cohn, definite classifieation. 
1873. Obermeier, Recurrent fever, Spirochaeta obermeieri. 
1873. Schlosing and Miinz, nitrification due to organisms. 
1875. Losch, Amebic dysentery, Amoeba coli. 
1875-76. Tyndall, germs in the air. 
1876. Robert Koch, Anthrax, Bacterium anthracis. 
1877. Bollinger, Actinomycosis, Actinomyces bovis (Strep- 
tothrix bovis). 
1877. Weigert, used anilin dyes for staining. 
1877. Woronin, Cabbage disease, Plasmodiophora brassicoe. 
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 con- 
nected with the fission yeasts and the yeasts and torulce. 
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 P0S1TI ON-RE LA TI ON SHI PS 
35 
thread bacteria do show true branching and rudimen- 
tary fruiting bodies (Streptothrix) and appear to be a link 
connecting the true bacteria and the molds. 
Fig. 7.-A thread of blue-green 
algae. 
Fig. 8.-A thread of a small blue- 
green algae. 
Fig. 9.-A thread of bacteria. Com- 
pare with Figs. 7 and 8. 
Fig. 10.-A chain of spherical blue- 
green algae. 
Fig. 11.-A chain of spherical 
bacteria. 
Fig. 12.-A pair of spherical blue- 
green algae. 36 
POSITION-RELA T ION SHIPS 
Further, the chemical composition of bacteria is more like 
that of other fungous plants than of any of the forms 
classed as animals. 
Fig. 13.-Spherical bacteria. 
Several pairs are shown. 
Fig. 14.-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. 15.-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 
1 Myxomycetes excepted, and they are probably to be regarded as animals 
-Mycetozoa. POSITION-RELA TIONSHIPS 
37 
are characteristically storers of energy while animals are 
liberators of it. Some bacteria which have the power of 
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 fortheir 
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 (g). 
The size varies widely among different kinds but is fairly 
constant in the same kind. The smallest described form is 
said to be only 0.18/z long by O.OOju 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 
40/x in length, but the vast majority are from 1 y to 4^ or 5ju 
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. 16), 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 40 
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 refractive. This is chiefly due 
to the presence of a cell wall, but is not a proof of its 
existence. 
Fig. 16.-Cells of bacteria show- 
ing plasmolysis. The cell substance 
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. 17.-Vacuoles in the bac- 
terial cell. The lighter areas are 
vacuoles. 
Nucleus.-A nucleus as such is not present in bacterial 
cells, except in a few large rare forms. Nuclein, the char- 
acteristic chemical substance in nuclei, which when aggre- 
gated forms the nucleus, is scattered throughout the cell con- 
tents and thus intimately mingled with the protoplasm, and 
cannot be differentiated by staining as in most cells. This 
close association of nuclein and protoplasm may explain 
the rapid rate of division of bacteria (Chapter VIII, p. 80). 
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. CAPSULE 
41 
Vacuoles.-Vacuoles appear as clear spaces in the proto- 
plasm when the organism is examined in the living condition 
or when stained very slightly (Fig. 17). During life these are 
filled with liquid or gaseous material which is sometimes 
waste, sometimes reserve food, sometimes digestive fluids. 
Students are apt to confuse vacuoles with spores (p. 44). 
Staining is the surest way to differentiate (Chapter XIX, 
p. 192). 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. 18). It is usually 
gelatinous, so that bacteria which form capsules frequently 
Fig. 18.-Bacteria seen within 
capsules. 
Fig. 19.-Metachromatic gran- 
ules in bacteria. The dark round 
spots are the granules. The cells 
of the bacteria are scarcely visible. 
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 is a poor term and 
misleading (zoon = 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 
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 42 
CELL STRUCTURES 
serve as an additional protection to the organism, doubtless 
more by selective absorption than mechanically. 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 capsule. 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. 19). They 
vary widely in chemical composition. Some of them are 
glycogen, some fat droplets. Others are so-called "granu- 
lose" closely related to starch but probably not true starch. 
Others are probably nuclein. Of many the chemical com- 
position is unknown. They are also called " Babes-Frnst cor- 
puscles" 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 granules"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. 20) (juoros = alone, single; 
rpcxos = 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 FLAGELLUM 
43 
bundle of flagella at one end and are called lophotrichic (Fig. 
21) (Xo</>os = tuft). Sometimes at approaching division the 
flagella may be at both ends and are then amphitrichic (Fig. 
22) (an<f)L = both). It is probable that this condition does not 
Fig. 20.-A bacterium showing a 
single flagellum at the end-mono- 
trichic. 
Fig. 21.-A bacterium showing 
a bundle of four flagella at the 
end-lopho trichic. 
persist long, but represents the development of flagella at 
one end of each of a pair resulting from division of an organ- 
ism which has flagella at one end only. In many bacteria 
Fig. 22.-A bacterium showing 
flagella at each end-amphitrichic. 
Fig. 23.-A bacterium showing 
flagella all around-peritrichic. 
the flagella arise from all parts of the surface of the cell. 
Such bacteria are peritrichic (Fig. 23) (irepL = around). The 
position and even the number of the flagella are very con- 
stant for each kind and are of decided value in identification. 44 
CELL STRUCTURES 
Flagella are too fine and delieate 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. 194). 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 act SPORES 
45 
somewhat like the chromosome of higher cells as a "center of 
attraction" so that the protoplasm gradually collects around 
it. The spot disappears or is enclosed in the collected proto- 
plasm. 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 developed 
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 contents and the 
old cell wall disappear or dissolve and the spore becomes 
"free" (Fig. 24). 
Fig. 24.-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 ah 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. 25). It may be from the middle of one 
side which gives equatorial germination (Fig. 26). 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, 46 
CELL STRUCTURES 
though sometimes spherical. A spore may be formed in the 
middle of the organism without (Fig. 27) or with (Fig. 28) 
Fig. 25.-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. 26.-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. 
a change in size of the cell around it. If the diameter 
through the cell is increased, then the cell with the con- 
tained spore becomes spindle-shaped. Such a cell is termed 
Fig. 27.-Spores in the middle of 
the rod without enlargement of the 
rod. The lighter areas in the rods 
are spores. 
Fig. 28.-Spores in the middle 
of the rod with enlargement of the 
rod around them. The lighter areas 
in the rods are spores. 
a "clostridium " Sometimes the spore develops in the end 
of the cell either without (Fig. 29) or with enlarging it SPORES 
47 
(Fig. 30). In a few forms the spore is placed at the end of 
the rod and shows a marked enlargement. This is spoken 
of as the " plectridium" or more commonly the "drumstick 
spore" (Fig. 31). The position and shape of the spore are 
Fig. 29.-Spores at the end of the 
rod with no enlargement of the rod 
around them. The lighter areas in 
the rods are spores. 
Fig. 30.-Spores at the end of the 
rod with enlargement of the rod, 
A, A, A, A. 
constant for each kind of bacteria. In one or two instances 
only, two spores have been observed in a single organism. 
The fact that the protoplasm is denser and the spore cap- 
sule thicker (the percentage of water in each is decidedly 
Fig. 31.-Drumstick spores at the end of the rod. 
less than in the growing cell) gives the spore the property 
of much greater resistance to all destructive agencies than 
the active bacterium has. For example, all actively growing 
cells are destroyed by boiling in a very few minutes, while 48 
CELL STRUCTURES 
some spores require several hours' boiling. The same rela- 
tion holds with regard to drying, the action of chemicals, 
light, etc. This resistance explains why it happens that 
food materials boiled and sealed in cans to prevent the 
entrance of organisms 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 leg 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 
protozoa-the ameba for example. Possibly the spore 
represents a very rudimentary beginning of a reproductive 
function such as is gradually evolved in the higher thread 
bacteria, 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 ATI). CHAPTER III. 
CELL FORMS. 
Though there is apparently a wide variation in the shapes 
of different bacterial cells, yet 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. 32). The coccus may be 
large, that is, from L5ju to 2ju in diameter. The term 
macrococcus is sometimes applied to these large cocci. If 
the coccus is less than 1 y 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 typi- 
fied by a section of a lead-pencil. The name bacillus, plural 
bacilli, is applied to this type (Fig. 33). The bacillus may 
be short (Fig. 34), 1 y or less in length, or long, up to 40)u 
in rare cases. Most bacilli are from 2y to 5y or Qy long. 
The ends of the rod are usually rounded, occasionally scpiare 
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. 35). 
1 The pronunciation of this word according to English standards is 
k6k-si; the continental pronunciation is k6k-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.) 50 
CELL FORMS 
The third cell form is the spiral, typified by a section of a 
cork-screw and named spirillum, plural spirilla (Fig. 36). A 
very short spiral consisting of only a portion of a turn is some- 
times called vibrio (Fig. 37). Vibrios when seen under the 
Fig. 32.-Cocci. 
Fig. 33.-Bacilli. 
microscope look like short curved rods. The distinction 
between the two can be made only by examining the organ- 
ism alive and moving in a liquid. The vibrio shows a char- 
acteristic spiral twisting motion. Very long flexible spirals are 
Fig. 34.-Short bacilli. 
Fig. 35.-Curved bacilli. Only 
the one in the center of the field is 
in focus. The others curve out of 
focus. 
usually named spirochetes (Fig. 38). The spiroehetes 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 CELL FORMS 
51 
pointed, bulged, club-shaped or even slightly branched. 
These peculiar and bizarre forms practically always occur 
when some of the necessary conditions for normal growth, 
Fig. 36.-Spirilla. 
Fig. 37.-Vibrio forms of spirilla. 
Compare with Fig. 35. 
discussed in Chapters VI and VII, are not fulfilled. They 
are best regarded as involution or degeneration forms for this 
reason (Fig. 39). In a very few cases it is not possible to 
obtain the organism without these forms (the diphtheria 
Fig. 38.-Spirochetes. 
Fig. 39.-Involution forms. The 
organisms are tapering and branched 
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. 40.-Streptospirillum grouping. 
Fig. 41.-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 (SlttXos = double); of 
three or more, a streptospirillum (arpeirTos = necklace, chain) 
(Fig. 40). 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. 41), bacilli in groups of two, and streptobacillus 
(Fig. 42), bacilli in chains are frequently used. Still the terms 
thread, filament, or chain are more common for streptobacillus. CELL GROUPINGS 
53 
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 tico planes only or in all three planes. 
Fig. 42.-Streptobacillus grouping. 
Fig. 43.-Typical diplococcus 
grouping. Note that the individual 
cocci are flattened on the appos- 
ing 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. 44.-Long streptococcus 
grouping. 
Fig. 45.-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- 
diplococeus grouping (Fig. 43), or in chains-streptococcus 
grouping (Figs. 44 and 45). When the grouping is in diplococci, 54 
CELL GROUPINGS 
the individual cocci most commonly appear as hemispheres 
with the plane surfaces apposed (Fig. 43). Sometimes they ap- 
pear as spheres, and occasionally are even somewhat elongated. 
The individuals in a streptococcus grouping are most com- 
monly 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. 44 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 together, 
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 algee 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 necessarily a multiple 
of four and the resultant flat mass has an irregular, uneven 
outline. This grouping is termed staphylococcus (<jTacl>vXo$ = 
a bunch of grapes) (Fig. 46). It is the most common group- 
ing 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. 47). 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-jdane division. 
If there are no sarcina, but tetrads or staphylococci (rarely 
merismopedia), then the division is in two planes. If none 
of the foregoing is observed but only diplo- or strepto- CELL GROUPINGS 
55 
cocci, 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. 46.-Staphylococcus group- 
ing. The large flat masses are 
staphylococcus grouping. Diplo- 
coccus grouping, tetrads and short 
streptococci are also evident. 
Fig. 47.-Saroma 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. 
streptococcus-in chains. 
tetracoccus, tetrads-in 4's. 
staphylococcus-irregular flat masses. 
sarcina-regular, solid packets, multiples 
of 8. 
coccus-round or spherical. 
bacillus-rod-shaped or 
cylindrical. 
diplobacillus-in 2's. 
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 fixed through the International Congress 
of Zoologists and of Botanists respectively. Unfortunately, 
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 
recapitulate, 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 it 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 CLASSIFICATION 
57 
activities. One of these systems has been adopted by the 
laboratory section of the American Public Health Associa- 
tion and by the Society of American Bacteriologists and is 
Fig. 48.-Illustrates the genus 
Streptococcus. Typical chains, no 
staphylococcus grouping, no sarcina 
grouping, no flagella. 
Fig. 49.-Illustrates the genus 
Micrococcus. Diplococcus, tetrads, 
short chains and staphylococcus; 
no sarcina, no flagella. 
practically the standard in this country. It is that of the 
German Bacteriologist Migula and is the only one which 
will be considered here and used throughout this book. 
Fig. 50.-Illustrates the genus 
Sarcina. Sarcina grouping, no 
flagella. 
Fig. 51.-Illustrates the genus 
Bacillus. A bacillus with peritri- 
chic flagella. (Student prepara- 
tion.) 
Personally, the author would prefer some slight modifications, 
but as he is not a botanist, will not presume to make them. 
Since practically the entire discussion in this book is con- 58 
CLASSIFICATION 
Fig. 52.-Illustrates the genus 
Pseudomonas. A bacillus with flag- 
ella at the end only. 
Fig. 53.-Illustrates the genus 
Microspira. It is (though the 
photograph does not prove it) a 
short spiral with one flagellum at 
the end. 
Fig. 54.-Illustrates the genus 
Spirillum. Spiral bacteria with 
more than three, in this case four, 
flagella at the end. 
Fig. 55.-Illustrates the genus 
Spiroch®ta. 
Fig. 56.-Illustrates the genus 
Chlamydothrix. Fine threads with a 
delicate sheath. 
Fig. 57.-Illustrates the genus 
Crenothrix. The thickness of the 
cell walls is due to deposits of iron 
hydroxide. (After Lafar.) CLASSIFICATION 
59 
cerned with the first three families, the generic character- 
istics in these only will be given. The full classification as 
Fig. 58.-Illustrates the genus Beggiatoa. The filament A is so full of 
sulphur granules that the individual cells are not visible. B has fewer 
sulphur granules. In C the granules are nearly absent and the separate 
cells of the filament are seen. (After Winogradsky, from Lafar.) 
well as a thorough discussion of this subject is given in 
Lafar's Handbuch, whence the following is adopted: 
ORDER I. EUBACTERIA. 
Cells without nuclei, free from sulphur granules and from 
bacteriopurpurin (p. 100); colorless, or slightly colored. 
1. Family: Coccace^e (Zopf) Migula, all cocci. 
Genus 1. Streptococcus Billroth: division in one plane only 
(Fig. 48). 
" 2. Micrococcus (Hallier) Cohn: division in two 
planes only (Fig. 49). 
" 3. Sarcina Goodsir: division in three planes only 
• (Fig. 50). 
N on-flagellated, 
non-motile 
Flagellated, 
motile 
" 4. Pianococcus Migula: division in two planes only. 
" 5. Planosarcina Migula: division in three planes only. 
2. Family: Bacteriace.e Migula, all bacilli. 
Genus 1. Bacterium (Ehrenberg) Migula: no flagella; 
non-motile. 
" 2. Bacillus (Cohn) Migula: flagella peritrichic 
(Fig. 51). 
" 3. Pseudomonas Migula: flagella at the end: mono- 
trichic, lophotrichic, amphitrichic (Fig. 52). 60 
CLASSIFICATION 
3. Family: Spirillaceje Migula, all spirilla. 
Genus 1. Spirosoma Migula: non-flagellated; non-motile. 
" 2. Microspira (Schroter) Migula: flagella one to 
three at the end (Fig. 53). 
" 3. Spirillum (Ehrenberg) Migula: flagella more than 
three at the end (Fig. 54). 
Cells stiff 
Cell flexible 
" 4. Spirochceta Ehrenberg: motile; no flagella (Fig. 
55). 
4. Family: Chlamydobacteriace.®. 
Cells cylindrical in long threads, and surrounded by a 
sheath. Reproduction also by gonidia formed from an 
entire cell. 
Genus 1. Chlamydothrix Migula (Fig. 56). 
" 2. Crenothrix Cohn (Fig. 57). 
" 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 Beggiatoacete. 
Genus 1. Thiothrix Winogradsky. 
" 2. Beggiatoa Trevisan. Of interest since it is with- 
out a sheath, is motile, but without flagella 
(Fig. 58). 
2. Family RhodobacteriacejE. 
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. 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 62 
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; temperature; 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 
injurious 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 
63 
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 Bacillus typhosus (of 
typhoid fever) and Microspira comma (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 64 
GENERAL CONDITIONS OF 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 freez- 
ing-point of water, since reproduction by division has been 
observed in the water from melting sea-ice at a temperature 
of - 1.5°. Thus bacteria as a class have a range for existence 
of about 373° ( - 273° to +100°) and for development 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; 
1 With 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 
65 
but is very much lower when grown in the absence of oxygen. 
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 tem- 
perature at which bacteria are killed is a matter of great 
practical importance in many ways and numerous deter- 
minations of this have been made with a great many organ- 
isms 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, the 
mere statements are worthless. It may be stated that all 
young, actively growing (non-spore-containing) disease-pro- 
ducing bacteria, when exposed in watery liquids and in small 
quantities are killed at a temperature of 60° within half an 
hour. It is evident that this fact has very little practical 
application, since the conditions stated are rarely, if ever, 
fulfilled except in laboratory experiments. (See Sterilization 
and Pasteurization, 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 66 
GENERAL CONDITIONS EOli 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 
light, 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 
expresssed 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 0, 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 
67 
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 com- 
mon "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 
destroyed. 
Parodko has determined the oxygen limits for five com- 
mon organisms as follows: 
Minimum. 
Maximum. Vol. Mm. 
In atmospheres. Mm. Hg. per cent. Hg. 
Bacillus fluorescens . 1.94 to 2.51 1474 to 1908 0.00016=0.0012 
Sarcina lutea . . . 2.51to3.18 1908 to 2417 0.00015=0.0011 
Bacillus vulgaris . . 3.63 to 4.35 2749 to 3306 0 0 
Bacillus coli . . . 4.09 to 4.84 3108 to 3478 0 0 
Bacillus 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. 64) 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- 68 
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. 
ELECTRICITY. 
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 
1 It 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 
anaerobic bacteria shows the fallacy of such beliefs. MECHANICAL VIBRATION 
69 
formed (ozone, chlorine, acids, bases, etc.), the same result 
will follow (see Ozone, pp. 67, 138 and 145). 
RADIATIONS. 
Rontgen or x-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. 
MECHANICAL VIBRATION. 
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. 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). 
(See Disinfection and Disinfectants, Chapter XIII.) 
INJURIOUS CHEMICAL SUBSTANCES. 
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 constituents vary remarkably with the 
kind and amount of food material available-a rich protein 
CHEMICAL COMPOSITION. CHEMICAL COMPOSITION 
71 
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 to 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 PaOs . . 18.14 " to 73.94 per cent. 
Cl 2.29 
K as K2O . . 11.1 " to 25.59 
Ca as CaO . . 12.64 " to 14.0 " 
Mg as MgO . . 0.7 " to 11.55 
Fe as FezOs . . 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 wall and protoplasm, including 72 
CHEMICAL ENVIRONMENT 
the nuclein. These differ markedly in chemical composition. 
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; Bacterium tuberculosis, Ilam- 
merschlag, Dreyfuss, Nishimura; Bacillus subtilis, Drey- 
fuss; Bacterium xylinum, Brown; Bacterium, acidi oxalici, 
Banning and a few others.) It is certainly not an important 
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 associated 
with these substances are compounds containing nitrogen. 
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 
part 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. 
1 "By cellulose is understood a carbohydrate of the general formula 
(CeHioOs) not soluble in water, alcohol, ether, or dilute acids but soluble 
in an ammoniacal solution of copper oxide. It gives with iodine and sul- 
phuric acid a blue color and with iodine zinc chloride a violet and yields 
dextrose on hydrolysis."-H. Fischer. CHEMICAL COMPOSITION 
73 
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 recent 
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 
properties. 
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 endotoxins 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 glycogen. 
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 
all bacteria, still illustrates the high percentage of protein 74 
CHEMICAL ENVIRONMENT 
compounds which undoubtedly occurs in most, as well as 
showing the large amount of fatty substance in a typical 
" acid-fast" organism: 
In the dried 
organisms 
8.5 per cent, tuberculinic acid 
24.5 " nucleoprotamin 
23.0 " neucleoprotein 
8.3 " proteinoid 
26.5 " fat and wax 
9.2 " ash 
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 inorganic, 
the term autotrophic is applied to the organism, and hetero- 
trophic if the compound is organic. It is very probable that 
anaerobes, exclusive of a few nitrogen absorbers, are meta- 
trophic for all the elements they utilize. With the excep- 
tion 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 
1 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 kinds of bacteria so far as known are proto-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. 76 
CHEMICAL ENVIRONMENT 
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 also 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 require 
no further explanation. 
The changes which the above-mentioned types of food 
material undergo in the various anabolic and katabolic pro- 
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 "biochemical reactions." 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 
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 meat-poisoning in man (Bacillus botulinus) and certain 
fungi found on spoiled fodder or grain. GENERAL FOOD RELATIONSHIPS-METABOLISM 
77 
essential for the growth of every bacterium, two of them 
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 carbon 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 group is said to be able to oxidize marsh gas, CII4, 
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 also high partly for the same 
reason that carbon is, i. e., respiration. Oxygen is alos 
one of the constituents of protoplasm, and combined with 
hydrogen 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, 78 
CHEMICAL ENVIRONMENT 
through a shifting or rearrangement of the atoms in the 
compounds used as food the oxidation of carbon is brought 
about. Enzymes are probably responsible for this action. 
Carbon dioxide is produced by anaerobes as well as 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 to supply this element in the 
protoplasmic structure. In some of the sulphur bacteria it 
is a source of energy, since either free sulphur 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. GENERAL FOOD RELATIONSHIPS-MET ABOLISM 
79 
Whether there is a speeial class of phosphorus bacteria 
remains to be discovered. That phosphorus is oxidized dur- 
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 
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 materials 
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 determine. 
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. 80 
CHEMICAL ENVIRONMENT 
The most rapid rate so far observed is a division in eighteen 
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, in diameter, 
its volume =0.0000000000005 c.c. 
| hour = 2 
1 hour = 4 
2 hours = 16 
4 hours = 256 
5 hours = 1024 = 103 + 
15 hours = 1,000,000,000 = 10s = 0.5 c.c. 
35 hours = 1021 + = 500.0 cu.m. 
About 65 hours = 2 x 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 following 
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 = 2na 
2 " " S = 22na 
3 " " S = 23na 
With two-plane or three-plane division, assuming that each 
organism attains full size, as was assumed in the first calcu- 
lation, the "mass as large as the earth" would be attained 
in about 32 hours and 22 hours respectively. 
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." GENERAL FOOD RELATIONSHIPS-METABOLISM 
81 
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- 
tact in all parts of the mass which favors rapid chemical 
action. The following calculation illustrates this: 
The volume of a coccus Iju in diameter is 0.5236 x 10 13 c.c. 
The surface of a coccus 1/x in diameter is tt x IO-8 sq. cm. 
It is not uncommon to find in milk on the point of sour- 
ing 1,000,000,000 bacteria per c.c. 
Assuming these to be cocci of 1/z diameter the volume of 
these bacteria in a liter is only 0.05 c.c. 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. (NH4)2SO4 and 0.0000000007 mg. 
(NH4)2HPO4. The bacteria numbered about 1000 per c.c. 
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). CHAPTER IX. 
PHYSIOLOGICAL ACTIVITIES. 
The objects in view in the discussion of the "physiological 
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 careful study 
of these "activities" that individual kinds of bacteria are 
identified. It should always be borne in mind that the bac- 
teria, in bringing about these changes which are so charac- 
teristic in many instances, are simply engaged in their own 
life struggle, in securing the elements which they need for 
growth, in liberating energy for vital processes, or occa- 
sionally 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 of acid. The former is called "gaseous fermentation" 
the latter "acid fermentation." The gases commonly pro- 
1 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 constituents 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. FERMENTATION OF CARBOHYDRATES 
83 
duced are carbon dioxide (CO2) hydrogen and marsh gas 
(CH4). 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. 59.-Cylinder to show 
the formation of gas by bacteria. 
The gaufee shows 265 pounds. 
It went beyond 500 pounds. 
Fig. 60.-A burning natural gas 
well at night. From a photograph 
colored. 84 
PHYSIOLOGICAL ACTIVITIES 
bacteria which produce disease, though even here some CH4 
is present. The common Bacillus 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 
CII4 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. 
rfhe author has conducted a number of experiments to 
demonstrate this action as follows: Material taken from the 
bottom 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. 59). 
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 analysis by 
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. 60 and 61). 
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 Hour used in the process (Fig. 62). 
1 See "Oil and Gas in Ohio," Bownocker: Geological Survey of Ohio, Fourth 
Series, Bull. I, pp. 313-314. PERMENTATION OF CARBOHYDRATES 
85 
Another is in the formation of the "holes" or "eyes" so 
characteristic of Swiss and other types of cheese (Fig. 63). 
A great many organic acids are formed during the "acid 
fermentation" of carbohydrates by bacteria. Each kind of 
Fig. 61.-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. 86 
PHYSIOLOGICAL ACTIVITIES 
Ihe "lactic acid bacteria" form a very large and important 
group and are indispensable in many commercial processes. 
Fig. 62.-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. 64). 
Fig. 63.-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, and a FERMENTATION OF CARBOHYDRATES 
87 
similar change occurs in the popular "Bulgarian fermented 
milk." The reaction is usually represented by the equation: 
Milk-sugar. Lactic acid. 
C12H22O11 + H2O + (bacteria) = dCsHeOs 
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 is 
Fig. 64.-A cream ripener. In this apparatus cream is "ripened," i. e., 
undergoes lactic acid fermentation, preparatory to making it into butter. 
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. 65) 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 88 
PHYSIOLOGICAL ACTIVITIES 
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 bacteria. 
The sugar is first converted into alcohol by yeasts, then the 
Fig. 65.-Filling a silo on the University farm. 
alcohol is oxidized to acid by the bacteria (Fig. 66). The 
reaction may be represented as follows: 
Dextrose. Ethyl alcohol. Acetic acid. 
C6H12O6 = 2C2H5OH + 2CO2 
C2H6OH + O2 + (bacteria) = CH3COOH + H2O 
Butyric acid is generally produced where fermentation of 
carbohydrate 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 90). FERMENTATION OF CARBOHYDRATES 
89 
As has been indicated alcohol is a common accom- 
paniment 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. 66.-A vinegar ripener. The tank shown opened at the side is filled 
with a special type of beech shavings which thus provide a very large surface. 
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- 90 
PHYSIOLOGICAL ACTIVITIES 
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 attacked, 
as is illustrated by the difficulties due to accumulation of 
fats in certain types of sewage-disposal works. The chemi- 
cal change is represented by the equation: 
Fat. Glycerin. Fatty acid. 
C3H5 (C„H2B.iO2)3 + 3 H2O = C3H5 (OH), 4- 3 (C„H2n O2). CHAPTER X. 
PHYSIOLOGICAL ACTIVITIES (Continued). 
PUTREFACTION OF PROTEINS. 
The word "putrefaction" is now restricted to the action of 
bacteria on the complex nitrogen-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 
(cairpos = 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 undergo 
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 proteins in 
the natural state or in solution, but also in the coagulated 
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 coagu- 
lated blood serum is acted on. In the latter case the serum 92 
PHYSIOLOGICAL ACTIVITIES 
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 saprophitic 
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 complex 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 /CH3 
N^H; s^mP^es^ Pt°maine is , methylamine, in 
which one H is replaced by methyl, a gaseous ptomaine; 
/CH3 
with two hydrogens replaced by methyl, N^-CH3) dimethyl- 
amine, a gas at ordinary temperature also; and trimethyl- 
/CH3 
amine, N^-CH3, a liquid. All three of these occur in her- PUTREFACTION OF PROTEINS 
93 
ring brine and are responsible for the charaeteristic odor 
of this material. Putrescin and cadaverin-tetramethylene 
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 C5H13NO or C2H3N(CH3)3OH 
= trimethyl-oxyethyl 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 poisoning," "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 Bacillus enteritidis 
group. Occasionally a case of poisoning by the true toxin 
(see Chapter XII) of Bacillus botulinus occurs. 
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 
ammonia. 
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), 94 
PHYSIOLOGICAL ACTIVITIES 
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, or 
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 free H. 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 
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 
1 It 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 conditions and likewise phosphine. These escape into the air 
and the "spontaneous combustion" of the phosphine ignites the marsh gas. PUTREFACTION OF PROTEINS 
95 
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, CO2 and H2O with a variety of organic acids as inter- 
mediate products in some cases. The strong odor of 
ammonia 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 
ammonia 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. 
The three physiological activities already discussed explain 
how bacteria break down the chief complex, energy-rich 
substances-carbohydrates, fats and proteins which consti- 
tute 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 96 
PHYSIOLOGICAL ACTIVITIES 
Phosphates in the soil 
Fig. 67.-Diagram to illustrate the circulation of phosphorus through the 
agency of bacteria. 
in' 
the air 
Fig. 68.--Diagram to illustrate the circulation of carbon through the 
agency of bacteria. 
Nitrates in soil 
Fig. 69.-Diagram to illustrate the circulation of nitrogen through the 
agency of bacteria. PUTREFACTION OF PROTEINS 
97 
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 further to be noted 
that not only is all this dead organic matter removed, but 
it is converted into forms which are again available for 
plant growth. Carbon dioxide forms the source of the car- 
bon 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 
Sulphates in the soil 
Fig. 70.-Diagram to illustrate the circulation of sulphur through the 
agency of bacteria. 
nitrates which is the form in which nitrogen 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 available. Hence bacteria are 
indispensable in nature, especially in keeping up the circu- 
lation 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 Jbe 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. 102). 
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. 
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, II, CII4, N, NH3, II2S, gaseous mercaptans, gaseous 
ptomaines, volatile fatty acids, ethereal salts or esters and 
others, both of pleasant and of foul odor, but of unknown 
composition. 
GAS PRODUCTION. 
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 
99 
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. 71.-Culture of phosphorescent bacteria in an Ehrlenmeyer flash 
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, 100 
PHYSIOLOGICAL ACTIVITIES 
hence they are frequently found on various sea foods, espe- 
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 appreciable 
quantities of heat. The light produced may be sufficient 
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. 71). None of the phos- 
phorescent 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 0 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 REDUCING ACTIONS 
101 
which they grow. Others are not soluble in water but are 
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 influ- 
enced 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 sulphates, 
for others); by the temperature (many bacteria cease to 
produce color at all if grown at body temperature, 37°- 
Bacillus prodigiosus-or if grown for a longer time at tem- 
peratures a few degrees higher). 
REDUCING ACTIONS. 
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 
bacteria 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 fertility. 
The building up of protein from these mineral salts by bac- 
teria in the intestines of herbivorous animals has been sug- 102 
PHYSIOLOGICAL ACTIVITIES 
gested by Armsby as a considerable source of nitrogenous 
food, and this suggestion appears possible. 
The liberation of nitrogen from nitrates or nitrites, either 
as free nitrogen or as ammonia, is spoken of as "denitrifica- 
tion," though this term was formerly applied to such libera- 
tions from compounds of nitrogen generally even from 
proteins. 
Certain bacteria may also reduce sulphates and other 
sulphur compounds to H2S, a phenomenon frequently 
observed in sewage and likewise of importance in the soil. 
It is possible that phosphates may be similarly reduced. 
Further and more careful study of the reducing actions of 
bacteria is needed. 
OXIDATION. 
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 
('H4 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" purposes. 
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 CH3CH2OII+O2 = 
CH3COOH+H2O (see Fig. 66). 
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, OXIDATION 
103 
and the other, the nitric bacteria, oxidizes the nitrous to 
nitric acid, giving nitrates with bases. A striking peculiarity 
of these two classes of organisms is that they may live entirely 
on inorganic food materials, are proto-autotrophic, proto- 
trophic for oxygen (aerobic) and autotrophic for the other 
elements. Their carbon is derived from CO2 or carbonates. 
The importance of such organisms in keeping up the supply 
of nitrates in the soil can scarcely be overestimated. 
Fig. 72.-Sprinkling filters of the Columbus sewage-disposal plant- 
devices which provide a good supply of oxygen for the bacteria that 
oxidize the organic matter in the sewage. 
The oxidation of the II2S, 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. 104 
PHYSIOLOGICAL ACTIVITIES 
The oxidation of H2S to free S may be an explanation of 
the origin of the great deposits of sulphur which occur in 
Louisiana and along the Gulf coast. These deposits occur 
in the same general regions in which natural gas and oil 
occur. The sulphur might have been derived from the same 
organic material carried down by the Mississippi which 
yielded the oil and gas. 
A purposeful utilization of the oxidizing power of bacteria 
is in "contact beds" and "sprinkling filters" in sewage dis- 
posal works. In these instances the sewage is thoroughly 
mixed with air and brought in contact with large amounts 
of porous material so as to expose an extensive surface for 
oxidation (Fig. 72). 
Ftg. 73.-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 ABSORPTION OF FREE NITROGEN 
105 
which are in a state of very fine division due to the disin- 
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. 73). 
ABSORPTION OF FREE NITROGEN. 
This is likewise one of the most important practical 
activities of certain types of bacteria present in the soil. 
Fig. 74.-Root tubercles on soy bean. X f. 
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. 74) spoken of as "root tubercles" which are at 
certain stages filled with bacteria. When conditions are 
favorable, these bacteria live in symbiotic relationship with 106 
PHYSIOLOGICAL ACTIVITIES 
the plant tissues, receiving carbonaceous and other food 
material from them and in return furnishing nitrogenous 
material to the plant. This nitrogenous material is built up 
from free nitrogen absorbed from the air by the bacteria. 
The utilization of this peculiar property 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 insofar as the nitrogen is concerned. The technical 
name of these bacteria is Pseudomonas radicicola. 
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. 75.-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. 75), while 
another is the anaerobic Bacillus (Clostridium') pastorianus. 
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. ABSORPTION OF FREE NITROGEN 
107 
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 
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 grow- 
ing in the tubercles. That nitrogen circulates throughout 
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 probably 
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. 96), 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 108 
PHYSIOLOGICAL ACTIVITIES 
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 
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. Jt 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 110 
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 7^ 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 
111 
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 in 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 en- 
zymes and where two modifications exist it is usually the 112 
PHYSIOLOGICAL ACTIVITIES 
dextrorotatory 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 1 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 commonly 
called amylase1 (starch-splitting enzyme); invertase,1 which 
splits cane-sugar into dextrose and levulose: C12H22O11 + 
II2O = C6Hi2O6 + C6H12O6. Lipase1 or a fat-splitting 
enzyme, which decomposes fat into glycerin and fatty acid: 
Fat Glycerin Fatty acid 
C3H5(OCnH2n.1O)3 + 3H2O = C3H5(OH)3 + 3CnH2nO2. 
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 
113 
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 or 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; C6Hi2O6 = 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 denitrifying 
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 body. In the former case the 114 
PHYSIOLOGICAL ACTIVITIES 
enzymes 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 applied 
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 
115 
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 
tetanus toxin and kept at 20° shows no indication of poison, 
but if the temperature is raised to 37,° symptoms of poisoning 
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 
larger 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 
(Ricinus 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 116 
PHYSIOLOGICAL ACTIVITIES 
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. 
Bacillus tetani or lockjaw bacillus, Bacterium diphtheria or 
the diphtheria bacillus, Bacillus botulinus or a bacillus caus- 
ing a type of meat poisoning, Pseudomonas pyocyanea or the 
blue pus bacillus are the most important. Other pathogenic 
bacteria do not secrete their toxins under the above condi- 
tions, but only give them up when the cell is disintegrated 
either within or outside the body. For the reason that 
endotoxins are therefore difficult to obtain their character- 
istics have not been much studied. The description of 
toxins as above given is intended to apply to the exotoxins 
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; (6) 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.) CULTURAL CHARACTERISTICS 
117 
The reaction of bacteria to various stains is dependent on 
their physicochemical structure and hence is a result of 
physiological processes, but is best discussed separately 
(Chapter XIX). 
STAINING. 
CULTURAL CHARACTERISTICS. 
The same is true of the appearance and growth on different 
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 metluxls 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 destruc- 
tion of "infective" organisms, that is, organisms producing 
disease in man or animals. A wider knowledge of bacteri- 
ology 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 labor- 
atory, instruments, glassware, apparatus, etc., are "steril- 
ized" while desks, walls and floors are "disinfected." The PHYSICAL AGENTS 
119 
surgeon "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 "sterile." 
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 disinfectant 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- 
sidered as an antiseptic rather than as a disinfectant process. 
While it is true that the complete removal of water would 120 
DISINFECTION-STERILIZ A TION-DISINFECT A NTS 
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 are 
killed by drying, many more, including the spore formers, 
are not. lienee 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 
Fig. 76.-A small laboratory hot-air sterilizer. 
water or steam). Very frequently in veterinary practice, 
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 
organ when it is desired to obtain bacteria from the interior 
free from contamination with surface organisms. PHYSICAL AGENTS 
121 
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. 76). 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 certain 
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," 
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 subjected to streaming steam 
for half an hour on each of three successive days. In can- 
ning 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 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 122 
DISINFECTION-ST ERI LIZ A TION-DISINFEC TA N TS 
destructive to bacteria than continuous heating for the same 
length of time, but the development of the spores is the most 
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 intermittent heating and cooling is of the same impor- 
tance as the development of the spores in this case. (Better 
results are secured with such substances by collecting them 
aseptically in the first place.) 
Fig. 77.-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 
be substituted. It is just as efficient and frequently more 
easily applied. The principle of the numerous forms of 
"steam sterilizers" (Fig. 77) is essentially the same. There PHYSICAL AGENTS 
123 
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 
Fig. 78.-Vertical gas-heated 
laboratory autoclave. 
Fig. 79.-Horizontal gas-heated labora- 
tory autoclave. 124 
DISINFECTION-STERILIZATION-DISINFECTANTS 
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. 80.-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 
be obtained from sizes as small as one or two gallons up to 
huge structures which will take one or two truckloads of PHYSICAL AGENTS 
125 
material (Figs. 78-90). 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. 81.-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 
in an autoclave of the type shown in Fig. 79 it required ten 
minutes for 500 c.c. of water at 15 pounds' pressure to reach 126 
DISINFECTION-STERI LIZ A TION-DISINFECTANTS 
a temperature of 100°, starting at room temperatures, 20° to 
25°. Autoclaves may be used as simple steam sterilizers by 
Fig. 82.-Horizontal steam chest used in canning. Height, 32 inches; 
width, 28 inches; length, 10 feet. 
leaving the eseape 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. 80 which are supplied with steam 
from the University central heating plant. A very great saving of time is 
thus secured. PHYSICAL AGENTS 
127 
A process closely akin to sterilization by heat is pasteur- 
ization. This means the heating of material at a tempera- 
Fig. 83.-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 128 
DISINFECTION-STERILIZATION-DISINFECTANTS 
vary but are essentially two in principle. 1. The material in 
small quantities in suitable containers (bottles) is placed in 
Fig. 84.-A battery of cylindrical process kettles in actual use in a canning factory. 
the apparatus; the temperature is raised to 60° to 65° and 
maintained for twenty to thirty minutes and then the whole PHYSICAL AGENTS 
129 
is cooled (beer, wine, grape juice, bottled milk) (Figs. 91, 92, 
and 93). 
Fig. 85.-A steam chamber used in government disinfection work. Size, 
4 feet 4 inches x 5 feet 4 inches x 9 feet. 
Fig. 86.-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 raised 130 
DISINFECTION-STERILIZATION-DISINFECTANTS 
Fig. 87.-Portable steam chamber used in government disinfection work. 
Fig. 88.-Steam chambers^on deck of the U. S. quarantine station barge 
" Defender." PHYSICAL AGENTS 
131 
to 60° and by "retarders" is kept at this temperature for 
twenty to thirty minutes (Figs. 94 to 97). In another type the 
Fig. 89.-Steam chambers in hold of U. S. quarantine station barge "Pro- 
tector." Disinfected space. 
Fig. 90.-Municipal disinfecting station, Washington, P. C, Fig. 91.--A pasteurizer for milk in bottles. 
Fig. 92.-A pasteurizer for grape juice, cider, etc,, in bottles, PHYSICAL AGENTS 
133 
temperature is raised to as high as 85° for a few seconds only, 
" flash process" (Fig. 98), and then the material is rapidly 
Fig. 93.-A pasteurizer for beer in bottles. 
Fig. 94.-A continuous milk pasteurizer. 134 
DISINFECTION-STERILIZATION-DISINFECT ANTS 
cooled. It is certain that all pathogenic microorganisms, except 
the very few spore formers in that stage, are killed by proper 
Fig. 95.-A pasteurizer for cream to be used in making ice-cream. 
pasteurization. The process is largely employed in the 
fermentation and dairy industries. Fig. 96.-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. 97.-A continuous pasteurizing plant in operation. Similar to Fig. 
96, but larger. Capacity, 12,000 pounds per hour. A, pasteurizer; B, seven 
compartment holder; C, D, coolers. 136 
DISINFECTION-STERILIZATION-DISINFECT ANTS 
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. 98.-A "flash process" pasteurizing outfit, with holder. A, flash 
pasteurizer; B, holder; C, cooler. 
4. Light.-It has been stated (p. 65) 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 
137 
oxidation, but it should not be forgotten that a very slight 
protection will prevent the action of light (Figs. 99 and 100). 
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. 99.-Effect of light on bacteria. X 7/10. 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. 
Outside 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," 138 
DISINFECTIONS TERI LIZ A TION-DISINFEC TA N TS 
p. 114) 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. 100.-Effect of light on bacteria. X 7/10. This plate was treated 
exactly as the plate in Fig. 99, 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. 99. (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 AGENTS 
139 
Fig. 101.-An electric milk purifier (pasteurizer). The milk flowing 
from cup to cup completes the circuit when the current is on. The effect 
is certainly a heat effect. Sparking occurs at the lips of the cups. 
Fig. 102.--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. 140 
DISINFECTION-STERILIZ A TION-DISINFECTANTS 
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. 103.-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 
1 The author has tested an "electric milk purifier" (Fig. 101) 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 
141 
hold back the bacteria. It is used on a large scale in the 
purification of water for sanitary or manufacturing reasons 
(Fig. 102). Air is also rendered "germ free" in some surgical 
operating rooms, " serum laboratories" and breweries by filtra- 
tion. In the laboratory it is a very common method of steriliz- 
ing liquids which would be injured by any other process. The 
Fig. 104.-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. 
apparatus consists of a porous cylinder with proper devices 
for causing the liquid to pass through either by suction 
(Fig. 103), 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. 104). The two 
main types of porous cylinders ("filter candles," "bougies") 142 
DISINFECTION-STERILIZATION-DISINFECTANTS 
are the Pasteur-Chamberland (Fig. 105) and the Berkefeld. 
The former are made of unglazed porcelain of different 
degrees of fineness, the latter of diatomaceous earth (Fig. 
106). The designs of complete 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. 105.-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 way that the surface 
soil is not contaminated either directly or by material brought 
up from below by digging or burrowing animals, insects, PHYSICAL AGENTS 
143 
worms, or movement of ground water to the surface. Also 
that the underground water supply which is drawn upon 
for use by men or animals is not also contaminated. Fre- 
Fig. 106.-Berkefeld filter candles about one-half natural size. 
quently infected material, carcasses of animals, etc., are 
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 (seep. 146). 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. 
ELEMENTS. 
Oxygen.-Oxygen as it occurs in the air is probably not 
injurious to living bacteria but aids them with the excep- ELEMENTS 
145 
tion of the anaerobes. In the nascent state especially as 
liberated from ozone (O3) hydrogen peroxide (H2O2) and 
hypochlorites (Ca(C10)2) it is strongly bactericidal. 
Fig. 107.-Apparatus for sterilizing water with liquid chlorine. 
Chlorine.-Chlorine is actively disinfectant and is coming 
into use for sterilizing water on a large scale in municipal 
plants (Fig. 107). Iodine finds extended use in aseptic sur- 
gical operations and antiseptic dressings. Bromine, mer- 
cury, silver, gold, nickel, zinc and copper are markedly 
germicidal in the elemental state but are not practical. 146 
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 (HgCh), 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 HgCl2 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. 108). 
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 alkalis are, 
of course, destructive to bacteria, but their corrosive effect ORGANIC COMPOUNDS 
147 
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. 108.-Tanks for bichloride of mercury, government quarantine 
disinfecting plant. 
ORGANIC COMPOUNDS. 
Carbolic Acid or Phenol.-Carbolic acid or phenol (C6H5OH) 
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 148 
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. 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 of Bacterium anthracis, 
from Bacillus coli and from Micrococcus pyogenes aureus in 
all cases even when the organisms had been exposed twenty- 
four hours in 5 per cent, solutions. Erom these results and 
from the wide variations reported in the literature it is clear 
that soap solutions alone cannot be depended on as dis- 
infectants. Medicated soaps do not appear to offer any 
advantages in this respect. 
Formaldehyde.-Eormaldehyde (HCIIO) 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- 
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 ORGANIC COMPOUNDS 
149 
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, Commis- 
sioner 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 
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 dilutions 
as high as 1 to 2000 of the gas. It is very irritant to mucous 
membranes of most individuals. 150 
DISINFECTION AND STERILIZATION 
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 list 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- 
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). 
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, especially 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 pene- 
trated, though old growths may be weakened by the accu- 
mulation 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. 
Metallic salts, especially bichloride of mercury, are precipi- 
tated by albuminous substances and if employed at all must 
CHOICE OF AGENT. 152 
DISINFECTION AND STERILIZATION 
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 rea- 
son 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. 
PRACTICAL STERILIZATION AND DISINFECTION. 
The methods for sterilizing in the laboratory have been 
discussed and will be referred to again in the next chapter. 
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. PRACTICAL STERILIZATION AND DISINFECTION 
153 
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. Surgeon's 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 prevents 
rusting. 
Rooms, offices and all spaces which may be readily made 
practically gas-tight are best disinfected by means of for- 
maldehyde by any of the methods above described (Figs. 
109 and 110). 
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, 
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 
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 154 
DISINFECTION AND STERILIZATION 
which a deep crater is made for the placement of from 1 
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 
Fig. 109. - Formaldehyde 
generator used in city work for 
room disinfection. 
Fig. 110. - Government formalde- 
hyde generator. 
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. PRACTICAL STERILIZATION AND DISINFECTION 
155 
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. 
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. 
Fig. 111.-Chamber used in government work for formaldehyde disinfec- 
tion. The small cylinder at the side is the generator. 
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 disinfectant 
solutions as soon as soiled, and then boiled or steamed. 
Unwashable articles-burn all possible. Use formaldehyde 
gas method in a closed receptacle (Fig. 111). 
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. 156 
DISINFECTION AND STERILIZATION 
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. 112). 
Fig. 112.-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. 
THE STUDY OF BACTERIA. 
CHAPTER XVI. 
CULTURE MEDIA. 
The study of bacteria may be taken up for the disci- 
plinary and pedagogic value of the study of a science; with 
the purely scientific idea of extending the limits of knowl- 
edge; 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 "culture" and the "soil" or material 
on which they are grown is called a "culture medium." 
Insofar 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 158 
CULTURE MEDIA 
organism as discussed in the chapters on Physiological 
Activities of Bacteria. 
The thorough study of a bacterium involves, therefore: 
its isolation in pure culture, its study with the microscope 
to determine morphological features and special staining 
reactions; growth on culture media for determining its 
physiological activities, as well as the morphological charac- 
teristics of the growths themselves; animal inoculations and 
special serum reactions. Since isolation in pure culture 
requires material for growing the organism, the first subject 
to be considered is culture media. 
A culture medium for a given bacterium must not only 
contain the elements necessary for its food, insofar as these 
may not be derived from the air, but must contain these 
elements in a form readily available to the bacterium. 
Further, the medium must not be too dry in order to furnish 
sufficient moisture for growth and to prevent too high a con- 
centration of the different ingredients. The reaction must 
be carefully adjusted to suit the particular organism dealt 
with. There must be no injurious substances present. The 
above are the chief points to be borne in mind in preparing 
culture media. 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 
composition 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 
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 study- 
ing some few of the physiological activities and especially 
for determining characteristic appearances of the isolated 
growths of bacteria. These isolated growths of bacteria on MEAT BROTH 
159 
solid media are technically spoken of as "colonies," 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 is used as a medium itself, 
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 dextrose 
(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 thor- 
oughly 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 magnesium, 
both of which must be filtered off and the water loss restored 
by adding the proper amount of distilled water. The boil- 
ing is done at this point because the medium must later 
be heated to sterilize it and it is best to get rid of the 
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 concentra- 
tion of mineral ingredients lost by the boiling. The chlorine 
is also an essential element. The reaction is now deter- 
1 The 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. 160 
CULTURE MEDIA 
mined best by titration with twentieth-normal sodium 
hydroxide solution with phenolphthalein as indicator. The 
amount of normal alkali or acid as found by titration is now 
added to the medium to make the desired end reaction (most 
commonly 1 to 1.5 per cent, acid to phenolphthalein) and the 
medium is again thoroughly boiled and filtered boiling hot. 
The titration and boiling ordinarily cause a precipitate to 
form which is largely phosphates 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 discon- 
tinuously in streaming 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. 
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 c.c. 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 Bacterium 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 (Bacillus 
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 GELATIN CULTURE MEDIUM 
161 
Bacillus coli. This mixture is then boiled to kill the Bacillus 
coli, retitrated 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 autoclave 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- 
ciaLpurposes 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 hot 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 
to 0.S per cent. acid. The milk should be sterilized discon- 
tinuously to avoid splitting up the lactose as well as action 
on the casein and calcium 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 0 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 162 
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 charac- 
teristic in appearance, as is the growth of many on gelatin 
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, 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 gela- 
tin solution, cooled to below 60°, the white of an egg for 
each 1000 c.c., 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 absorbent 
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 pro- 
teose and peptone and it will not gelatinize on cooling. It 
may be heated in the autoclave for ten to fifteen minutes at 
10 pounds' pressure and sometimes not be hydrolyzed, but AGAR MEDIUM 
163 
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 sterilized in 
these, since, if put into flasks these must be sterilized, 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 gelatinizes at 
about 38° and remains solid until again heated to near boil- 
ing. 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 patho- 
genic 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 
broth. The agar solution should be kept hot from the begin- 164 
CULTURE MEDIA 
ning to the end exeept the cooling down to below (>0°, 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 temperature 
does not injure the agar. 
Potato Media.-Potatoes furnish a natural culture medium 
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. 
117). These are prepared as follows: Large tubes are used. 
With a cork-borer of a size to fit the tubes used, cylinders 
about one and one-half inches long are cut from fairly large, 
healthy potatoes. The skin is cut off square from the ends 
and each cylinder 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 oxi- 
dizing 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 sterilized 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." 
Now glass dishes of the general form shown in Fig. 113, 
called "Petri dishes," or plates are used for practically all POTATO MEDIA 
165 
plate culture work. For "potato plates" slices from pota- 
toes are cut as large and as thick as the relative sizes of 
potato and dish permit (Fig. 114). The slices should be thin 
enough not to touch the lid and thick enough to be firm. 
Fig. 113.-Petri dish with the lid partly raised. X 5. 
Fig. 114.-A potato plate. X J. 
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. 
It sometimes happens that the natural acidity of potatoes 166 
CULTURE MEDIA 
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 securing 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 
an apparatus for coagulating the serum by heat. A copper 
water bath with a tightly closed air compartment or the hori- 
zontal autoclave (Fig. 80) is sufficient 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 solid- 
ified 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 suggested 
dialyzing the serum to free it from salts and thus prevent 
its coagulation when heated. Whether the removal of the BLOOD SERUM MEDIA 
167 
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. CHAPTER XVII. 
METHODS OF USING CULTURE MEDIA. 
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. 117), the other 15 cm. long by 13 mm. (Nos. 
1 to 7, Fig. 117). 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 readily rust (Figs. 115 and 116). 
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. 118 so that the gas may be collected in the 
closed arm of the tube, measured (Fig. 119) and tested if 
desired. METHODS OE USING CULTURE MEDIA 
169 
One method of using gelatin and also agar is as " puncture" 
or "stab" cultures. The tubes (the narrower tubes are to be 
Fig. 115.-Round wire basket. 
Fig. 116.-Square wire basket. 
Fig. 117.-Culture tubes with media in them. X j. 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; 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. 170 
METHODS OF USING CULTURE MEDIA 
preferred for most "stab" cultures) are filled one-third full 
of the medium while it is still fluid, plugged, sterilized and 
allowed to cool in the vertical position. The medium 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. 117, Nos. 4 and 5). 
Fig. 118.-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, sloping 
surface on which the bacteria are inoculated (Fig. 117, 
Nos. 6 and 7). 
Potato tubes are likewise used for "slant" or "slope" 
cultures (Fig. 117, No. 8). Potatoes as "plate cultures" 
have been referred to. Agar and gelatin are very largely METHODS OF USING CULTURE MEDIA 
171 
Fig. 119.-Method of estimating percentage of gas in a fermentation tube 
by means of the "gasometer." The reading is 45 per cent. 
Fig. 120.-A toxin flask showing a large surface growth. 172 
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 in to 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. 117). 
For using large quantities of medium, Florence flasks, 
Ehrlenmeyer flasks, special toxin flasks (Fig. 120) or various 
other devices (Vaughan and Novy's "mass cultures," Figs. 
121 and 122) 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. METHODS OF USING CULTURE MEDIA 
173 
/I. "Vignal tubes" of the style shown (Fig. 123) are made 
from glass tubes of about 6 to 8 mm. outside diameter, 
Fig. 121.-Tank with raised lids. (Vaughan.) 
Fig. 122.-Tank with lids lowered. (Vaughan.) 
Figs. 121 and 122.-Vaughan and Novy's mass culture apparatus. 174 
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. 123. 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 tube 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. " I er mentation 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- METHODS OF USING CULTURE MEDIA 
175 
ized. Inoculation is made to the bottom and anaerobes 
grow well (Fig. 124). 
1). For slope or plate, or any type of surface cultures the 
Xovy jar (Fig. 125) is the most practical device. It is good 
Fig. 124.-Deep tubes showing anaerobic growth. 1, shows a few small 
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 176 
METHODS OF USING CULTURE MEDIA 
upset and mixed with the alkali when it is desired to do so. 
The cover is now clamped in position with all joints well 
vaselined. Then the outlet tube is connected with a suc- 
tion 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 replaced. 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. 125.-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. 126). 
It is used most frequently to inoculate all forms of solid METHODS OF USING CULTURE MEDIA 
177 
media. The platipum 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. 127) 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-fourth 
Fig. 126.-Straight needle. 
Fig. 127.-Straight and loop needles. 
Fig. 128.-Pasteur flask-"ballon pipette." 
of an inch on the side of the end portion of the rod. The 
low cost, less than one-twentieth of platinum, justifies 
its use. 
Sterile graduated pipettes varying in capacity from 1 c.c., 
graduated in hundredths, upward, permit the transfer of 
definite amounts of liquids. Large quantities are conven- 
iently transferred by means of Pasteur flasks (Fig. 128). 
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. 129 
to 132). The chief uncertainty with this method is that 
occasionally two kinds of bacteria stick together so closely ISOLATION OF BACTERIA IN PURE CULTURE 
179 
that even the separate colonies contain both organisms. 
This is not common, however. The plate colonies frequently 
Fig. 129.-Dilution plates. X fo- 1, shows the first dilution, the colo- 
nies are so numerous and small that they are invisible (compare Fig. 130); 
2, shows fewer and hence larger colonies, but too crowded to isolate (compare 
Fig- 131); 3, shows the colonies larger and well separated, so that it is easy 
to isolate from them (compare Fig. 132). 
develop from groups of bacteria which were not separated, 
but as these are of the same kind the culture is essentially 
pure. 
Fig. 130.-A portion of plate 1 in Fig. 129 as seen under the low-power 
objective. X 100. Very small, closely crowded colonies. 
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- 
ified in a Petri dish with a sterile heavy platinum needle, 180 
ISOLATION OF BACTERIA IN PURE CULTURE 
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 
streaks on a slope tube or plate of medium with a needle 
inserted but once into the material to be plated. On the first 
streak a large part of the bacteria are rubbed off and a 
continuous growth results, but usually on the last of a series 
only isolated colonies appear, which are presumably pure. 
The ideal method for securing pure cultures is to be abso- 
lutely certain that the culture starts from a single organism. 
Fig. 131.-From the thinnest part 
of plate 2, Fig. 129, as seen under the 
low-power objective. X 100. .Colo- 
nies much larger than on plate 1, 
but still crowded. 
Fig. 132.-The smallest colony 
on plate 3, Fig. 129, 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. 133 and 134). 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, ISOLATION OF BACTERIA IN PURE CULTURE 
181 
Professor W. A. Starin of the author's department, a 
former student of Professor Barber, has done some excellent 
work with this apparatus. 
Fig. 133.-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, October20,1908, vol. 5, No.4, p.381.) 182 
ISOLATION OF BACTERIA IN PURE CULTURE 
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 
mixture, such as ability to form spores, different resistance 
to antiseptics, special food requirements, and pathogenic 
Fig. 134.-Photograph of microscope with Barber's isolation apparatus 
set up to use. 
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) 
and then plated out. Or (6) an antiseptic which restrains 
the growth of some organisms and not others may be placed ISOLATION OF BACTERIA IN PURE CULTURE 
183 
in the culture media (carbolic acid, various anilin dyes, 
excess acid, or alkali, ox bile, etc.), when the more resistant 
organisms grow on the final plates, the others not. (c) 
Special food substances (various carbohydrates) from which 
the organism desired forms special products (acids, alde- 
hydes) that may be shown on the plates by various indica- 
tors, 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 (b) and (c) is much used 
in the separation of the organisms of the "intestinal group" 
in human practice, (d) The inoculation of a susceptible ani- 
mal 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-third 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. 
The function of immersion oil is to prevent the dispersion HANGING DROP SLIDE 
185 
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. 135). A ray of light (J //) 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 
Fig. 135.-Diagram of use of immersion oil. 
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. 136). 
HANGING DROP SLIDE. 
It is sometimes necessary to examine living bacteria and 
for this purpose the device known as the "hanging drop Fig. 136.-Diagram of paths of rays in microscope. HANGING DROP SLIDE 
187 
slide" is used (Fig. 137). 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 material 
from a solid culture suspended in broth or physiological 
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 ins diaphragm of the condenser must be 
nearly closed, so that the light coming through consists mainly 
Fig. 137.-Hanging drop slide. 
of parallel vertical rays, otherwise the transparent 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 " Brown- 
ian 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 calibrated 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 bacteria as light, electricity, 
heat, etc., in the study of "tropisms" and "taxes" has been 
investigated on various modifications of the above-described 
hanging drop slide. 
Cell forms and cell groupings may be studied in the same 188 
STUDY OF INDIVIDUAL BACTERIA-STAINING 
way but these features are best determined on stained 
preparations. 
"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 elementary student, 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 imperfectly 
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 adsorption 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 substances 
are called mordants. The principal mordants used are alka- 
lies, 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 anil in dyes. These dyes, as is well STAINING 
189 
known, are of nearly every conceivable color and shade but 
relatively very few are used in bacteriological work. The 
elementary 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 of which the following are the most 
common: 
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 filter. 
3. Anilin Fuchsin. 
Saturated alcoholic solution of fuchsin ..... 1 part 
Anilin water (see below) 10 parts 
Mix and filter. 
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 container. 
Anilin Water.-Anilin water is made by putting 3 or 4 c.c. 
of anilin "oil'' in a 120 c.c. flask, adding 100 c.c. of distilled 
water, shaking vigorously for a minute or so and filtering 
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 parts 
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. 190 
STUDY OF INDIVIDUAL BACTERIA-STAINING 
6. Gabbet's (methylene) blue (solution). 
Dry methylene blue 4 parts 
Concentrated ELSOi 25 parts 
Distilled water 75 parts 
Dissolve the dry dye in the acid and add the solution to the dis- 
tilled water and filter. 
Staining solutions are conveniently kept in square drop- 
ping bottles inserted in a block as shown in Fig. 138. This 
form of holder necessitates the use of one hand only in secur- 
ing the stain and dropping it on the preparation. 
Fig. 138.-Author's staining set. Square bottles are set in square holes in 
the block. The capacity of each bottle is 30 c.c. 
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: STAINING 
191 
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, handkerchief, 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 thor- 
oughly 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 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 192 
STUDY OF INDIVIDUAL BACTERIA-STAINING 
compound is insoluble in alcohol, and other bacteria do not 
contain this substance. Consequently when bacteria are 
stained by Gram's method (given below), those that con- 
tain 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 parts 
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 
minutes in the anilin gentian violet and be left about two 
minutes in the Gram 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 STAINING 
193 
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 
Bacterium anthracis, cultures on agar slants forty-eight hours 
in the 37° incubator are just right. For the spores of Bacillus 
tetani, the culture should be three days old, but may be as 
old as a week. 
Staining of "Acid-fast" Bacilli.-Bacterium tuberculosis, 
Bacterium of Johne's disease, "grass" and "butter bacilli," 
Bacterium lepra?., Bacterium smegmatis. 
Gabbet'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, 2 or 3 minutes. 
5. Wash, dry and examine. 
The sulphuric acid in Gabbet's blue removes the carbol- 
fuchsin from everything except the "acid-fast" bacteria, 
which remain red, and the blue stains the decolorized bacteria 
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 Loffler's blue 1 or 2 minutes. 
7. Wash, dry and examine. 
The results are the same as with Gabbet's method. 
13 194 
STUDY OF INDIVIDUAL BACTERIA-STAINING 
Staining of Capsules.-Rabiger's Method.-Films of the 
organism to show capsules should be freshly prepared, dried, 
but not fixed. The preparation is covered with a freshly 
prepared saturated solution of.gentian violet in formalin and 
this allowed to stain for from 30 to 90 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- 
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 6 to 
24 hours old is selected. A bit of the culture is removed and 
placed in a watch-glass of water. The bacteria are allowed 
to diffuse of themselves without stirring. After several min- 
utes a loop of this water is removed and spread on the slide 
with a single movement of the needle, dried and carefully 
fixed. The film is 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 mordant if necessary. After mordanting 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 STAINING 
195 
is apt to cause the film on the surface of the mordant to 
settle down on the slide and spoil the preparation. 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 min- 
utes. 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 
FeCl3 is more permanent than FeSO4 solution. 
Mordant sufficient for one student: 
5 per cent, solution of FeCls 20.0 c.c. 
25 per cent, solution of tannic acid 20.0 c.c. 
Anilin fuchsin 4.0 c.c. 
Normal NaOH . 1.5 c.c. 
The solution of FeCl3 is made up in the cold and must be 
perfectly clear. The tannic acid solution must be thor- 
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. 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 preparation of the mordant are essential. 
The stain to follow this mordant is anilin fuchsin. 
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 sur- 
face, on the slide and allow this to dry without spreading it 
out, then stain lightly with Loffler's blue. Such slides also 
show characteristic 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 determining 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 maintaining 
the temperature constant, a "thermoregulator" or "ther- 
mostat." For lower temperatures a cooling arrangement 
must be installed. For the great part of bacteriological work 
only two temperatures are used, 20° so-called "room tem- 
perature" (this applies to European "rooms" not to Amer- 
ican) and 37° or body temperature. Incubators for 37° of 
almost any size and style desired may be secured from sup- 
ply houses and need not be further described. Figs. 139 
and 140 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 
$60 to install. 
The styles of incubators for lower temperatures, 20° and 
below, are not so numerous nor so satisfactory. The author Fig. 139.-Small laboratory incubator, gas heated. 
Fig. 140.-Electric incubator. 198 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
has constructed a device which answers every purpose for a 
small class. The diagram, Fig. 141, 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. 141.-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. 5, 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. 
li. Oxygen relations-whether the organism is aerobic, 
anaerobic, or facultative is determined by inoculation in PHYSIOLOGICAL ACTIVITIES 
199 
gelatin or agar puncture or stab cultures and noting whether 
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, arrays, 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. 200 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
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 of bacteria, 
many 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. 
Colonies which are ordinarily round and regular may assume 
very diverse shapes, if there chance to be an excess of 
moisture 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 
adopted a standard "descriptive chart" for detailing the 
characteristics of a given organism which includes the terms 
to be used in describing the appearance on different culture 
media. This chart is inserted in this chapter. 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 pseudozoogle®. 
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 of fluids which contain pseudozoogle®, 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, colonies having the radiately filamentous appearance 
of mold colonies. 
NAPIFORM, liquefaction with the form of a turnip. 
NITROGEN REQUIREMENTS, the necessary nitrogenous food. 
This is determined by adding to nitrogen-free media the nitro- 
gen compound to be tested. 
OPALESCENT, resembling the color of an opal. 
OPTIMUM TEMPERATURE, temperature at which growth is most 
rapid. 
PELLICLE, in fluid bacterial growth forming either a continuous of 
an interrupted sheet over the fluid. 
PEPTONIZED, said of curds dissolved by trypsin. 
PERSISTENT, many weeks, or months. 
PLUMOSE, a fleecy or feathery growth. 
PSEUDOZOOGLEAi, 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 zoogle®. 
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 flocculence. 
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. 
ZOOGLE2E, firm gelatinous masses of bacteria, one of the most typical 
examples of which is the Streptococcus mesenterioides of sugar 
vats (Leuconostoc mesenterioides), the bacterial chains being 
surrounded by an enormously thickened, firm covering inside 
of which there may be one or many groups of the 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. AH 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 
(G) 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 
COa 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 Anaerobic (strict) 
1 Gelatin liquefied 
2 Gelatin not liquefied 
0.1 Acid and gas from dextrose 
0.2 Acid without gas from dextrose 
0.3 No acid from dextrose 
0.4 No growth with dextrose 
0.01 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 
0.00003 Blue chromogens 
0.00004 Green chromogens 
0.00005 Yellow' chromogens 
0.00006 Orange chromogens 
0.00007 Red chromogens 
0.00008 Brown chromogens 
0.00009 Pink chromogens 
0.00000 N on-chromogenics 
0.000001 Diastasic action on potato starch, strong 
0.000002 Diastasic action on potato starch, feeble 
0.000003 Diastasic action on potato starch, absent 
0.0000001 Acid and gas from glycerin 
0.0000002 Acid without gas from glycerin 
0.0000003 No acid from glycerin 
0.0000004 No growth with glycerin 
The genus according to the system of Migula is given its proper 
symbol which precedes the number thus: (7) 
Bacillus con (Esch.) Mig. becomes B. 222.111102 
Bacillus alcaligenes Petr. becomes B. 212.333102 
Pseudomonas campestkis (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 pneumoeoceL (see diplococcus of pneumonia). Source Date of Isolation Name Group No. (*) 
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 
0. Nutrient Broth. 
Surface growth, ring, pellicle, flocculent, membranous, 
none. 
Clouding slight, moderate, strong: transient, persistent: 
none: fluid turbid. 
Odor, absent, decided, resembling 
Sediment, compact, flocculent, granular, flaky, viscid 
on agitation, abundant, scant. 
7. Milk. 
Clearing without coagulation. 
Coagulation prompt, delayed, absent. 
Extrusion of whey begins in days. 
Coagulum slowly peptonized, rapidly peptonized. 
Peptonization begins on. .. .d, complete on... .d. 
Reaction, Id 2d 4d...., 10d...., 20d.... 
Consistency, slimy, viscid, unchanged. 
Medium browned, reddened, blued, greened. 
Lab ferment, present, absent. 
8. Litmus Milk. 
Acid, alkaline, acid then alkaline, no change. 
Prompt reduction, no reduction, partial slow reduction. 
9. Gelatin Colonies. 
Growth slow, rapid. 
Form, punctiform, round, irregular, ameboid, 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 
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. 
o. Toleration of Acids, great, medium, slight. 
Acids tested 
6. Toleration of NaOH, great, medium, slight. 
7. Optimum reaction for growth in bouillon, stated in 
terms of Fuller's scale 
8. Vitality on culture media, brief, moderate, long. 
9. Temperature relations. 
Thermal death-point (10) minutes' exposure in 
nutrient broth when this is adapted to growth of 
organism) C. 
Optimum temperature for growth ° C.; or 
best growth at 15° C., 20° C., 25° C., 30° C„ 37° C„ 
40° C„ 50° C., 60° C. 
Maximum temperature for growth °C. 
Minimum temperature for growth ° C. 
10. Killed readily by drying: resistant to drying. 
11. Per cent, killed by freezing (salt and crushed ice or 
liquid air) 
12. Sunlight: Exposure on ice in thinly sown agar plates; 
one-half plate covered (time 15 minutes), sensitive, 
not sensitive. 
Per cent, killed 
13. Acids produced 
14. Alkalies produced 
15. Alcohols 
16. Ferments, pepsin, trypsin, diastase, invertase, pectase, 
cytase, tyrosinase, oxidase, peroxidase, lipase, catalase, 
glucase, galactase, lab, etc 
17. Crystals formed: 
18. Effect of germicides: 
BRIEF CHARACTERIZATIOP 
I. 
Mark + or 0, and when two terms 
occur on a line erase the one which 
does not apply unless both apply. 
Diameter over l/i 
>< 
Chains, filaments 
0 
O 
Endospores 
ft 
c 
Capsules 
K 
Ph 
Zooglea, Pseudozooglea 
Ph 
o 
Motile 
S 
Involution forms 
Gram's stain 
Cloudy, turbid 
Ring 
Pellicle 
Sediment 
Shining 
Dull 
Wrinkled 
a 
Chromogenic 
1 
Round 
rURAL FEATI 
d 
Proteus-like 
ft 
Rhizoid 
o 
Filamentous 
Curled 
- 
Surface growth 
O 
Oia 
Needle growth 
Moderate, absent 
o 
aS 
Abundant 
b 
ft 
Discolored 
Starch destroyed 
Grows at 37° C. 
Grows in Cohn's sol. 
Grows in Uschinsky's sol. 
Gelatin (*) 
CO 
Blood-serum 
H 
ft J 
Casein 
i 
Acid curd 
1 
Rennet curd 
a 
Casein peptonized 
o 
JI 
Indol (5) 
K 
Hydrogen sulphide 
8 
Ammonia (5) 
2 
Nitrates reduced (6) 
A 
Fluorescent 
Luminous 
Animal pathogen, epizoon 
Plant pathogen, epiphyte 
- 
ION 
Soil 
Milk 
Fresh water 
5 
Salt water 
Sewage 
23 
0 
Iron 
bacterium 
Sulphur bacterium 
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) 
Agar I Chains (No. of elements) 
Hanging-block ] 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 
. [ Orientation (grouping) 
1 Chains (No. of elements) 
Hanging-block 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: other solvents 
Odor, absent, decided, resembling 
Consistency, slimy, butyrous, viscid, membranous, 
coriaceous, brittle. 
Medium, grayed, browned, reddened, blued, greened. 
3. Loeffler's Blood-serum. 
Stroke invisible, scanty, moderate, abundant. 
Form of growth, filiform, echinulate, beaded, spread- 
ing, plumose, arborescent, rhizoid. 
Elevation of growth, flat, effuse, raised, convex. 
Lustre, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verrucose. 
Chromogenesis (8) 
Medium grayed, browned, reddened, blued, greened. 
Liquefaction begins in d, complete in d, 
4. Agar Stab. 
Growth uniform, best at top, best at bottom: surface 
growth scanty, abundant: restricted, widespread. 
Line of puncture, filiform, beaded, papillate, villous, 
plumose, arborescent: liquefaction. 
Substance 
ethod used 
Minutes 
Temperature 
Killing quantity 
Amt. required to 
restrain growth 
III. PHYSICAL AND BIOCHEMICAL FEATURES. 
1. Fermentation-tubes con- 
taining peptone-water or 
sugar-free bouillon and 
asojjxaQ 
Saccharose 
Lactose 
Maltose 
uusaAio 
Mannit 
Gas production, in per cent. 
\co2/ 
- 
- 
- 
- 
Growth in closed arm 
Amount of acid produced Id. 
Amount of acid produced 2d. 
Amount of acid produced 3d. 
IV. PATHOGENICITY. 
1. Pathogenic to Animals. 
Insects, crustaceans, fishes, reptiles, birds, mice, rats, 
guinea-pigs, rabbits, dogs, cats, sheep, goats, cattle, 
horses, monkeys, man 
2. Pathogenic to Plants: 
3. Toxins, soluble, endotoxins. 
4. Non-toxin forming. 
5. Immunity bactericidal. 
6. Immunity non-bactericidal. 
7. Loss of virulence on culture-media: prompt, gradual, 
not observed in months. GROWTH ON DIFFERENT CULTURE MEDIA 
201 
Among the cultural appearances the following are of most 
importance: 
Fig. 142.-Broth cultures X L 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. 
Tn broth cultures the presence or absence of growth on 
the surface and the amount of the same. Whether the 202 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
broth is rendered cloudy or remains clear, and whether there 
is a deposit at the bottom or not (Fig. 142). 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. 143.-A fili- 
form stab or punc- 
ture culture. X 5. 
Fig. 144.-A beaded 
stab or puncture cul- 
ture. X j. 
Fig. 145.-A vill- 
ous stab or puncture 
culture. X 
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 GROWTH ON DIFFERENT CULTURE MEDIA 
203 
the organisms lose their motility. Xon-motile facultative 
anaerobes usually cloud the broth also, but settle out more 
rapidly than the motile ones. 
Fig. 146 
Fig. 147 
Fig. 148 
Fig. 149 
Fig. 146.-Crateriform liquefaction of gelatin. X j. 
Fig. 147.-Funnelform liquefaction of gelatin. X |. 
Fig. 148.-Saccate liquefaction of gelatin. X J. 
Fig. 149.--Stratiform liquefaction of gelatin. X |. 
Iii gelatin and agar punctures the oxygen relationship is 
shown by surface growth for aerobes, growth near the 
bottom of the puncture for anaerobes, ami a fairly uniform 
growth all along the line of inoculation for facultative 204 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
anaerobes. 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. 
Fig. 150.-Filiform 
slope culture. X 5. 
Fig. 151.-Filiform, 
slightly spreading, 
slope culture. X j. 
Fig. 152.-Beaded 
slope culture. X i 
Along the line of puncture the commonest types are 
filiform (Fig. 143), which indicates a uniform growth; beaded 
(Fig. 144), or small separate colonies; villous (Fig. 145), deli- 
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. GROWTH ON DIFFERENT CULTURE MEDIA 
205 
Further, in the gelatin puncture the liquefaction which 
occurs is frequently characteristic. It may be crateriform 
(Fig. 146), a shallow saucer at the surface; or funnel-shaped 
Fig. 153 
Fig. 154 
Fig. 155 
Fig. 156 
Fig. 153.-Effuse slope culture. X 
Fig. 154.-Rhizoid slope culture. X j. 
Fig. 155.-Rugose slope culture. X j. 
Fig. 156.-Verrucose slope culture. X | 
(Fig. 147); or it may be of uniform width all along the 
puncture, i. e., saccate (Fig. 148); or it may be stratiform, 
(Fig. 149), i. e., the liquefaction extends to the sides of the 
tube and proceeds uniformly downward, 206 
STUDY Ob' THE PHYSIOLOGY OF BACTERIA 
On agar, potato and blood serum slope tubes the amount 
of growth, its form and elevation, the character of the sur- 
Fig. 157.-Punctiform colonies on a plate. X 
158.-A rhizoid colony on a plate. Natural size. GROWTH ON DIFFERENT CULTURE MEDIA 
207 
face, and the consistency should be carefully noted, and in 
some few cases the character of the edge. Figures 150 to 156 
show some of the commoner types. 
Fig. 159.-Ameboid colonies on a plate. X J. 
Fig. 160.-Large effuse colony on a plate. The edge is lacerated. Inci- 
dentally the colony shows the rate of growth for six successive days. X 3. 208 
STUDY OF THE PHYSIOLOGY OF BACTERIA 
Fig. 162.-Colony with edge 
coarsely granular as seen under 
the low-power objective. X100. 
Fig. 161.-Colony with edge entire 
as seen under the low-power objective. 
X 100. 
Fig. 163.-Colony with edge 
curled as seen under the low-power 
objective. X 100. 
Fig. 164.-Colony with edge rhiz- 
oid as seen under the low-power 
objective. X 100. 
Fig. 165.-A small deep rhizoid colony as seen under the low-power objective. 
X 100. GROWTH ON DIFFERENT CULTURE MEDIA 
209 
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 
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. 157 to 165 show some of the commoner 
characteristics to be noted. 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 
animals 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 ANIMAL INOCULATION 
211 
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 subdural 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. 212 
ANIMAL INOCULATION 
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 on 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 platinum 
needle frequently give pure cultures of the organism sought. 
Slides may be made from such material and culture 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 
practice to pack material for examination in ice and use no 
preservatives. 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 insofar 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 cholera, 
swine, etc. 
Diseases which are due to unicellular pathogenic micro- 
organisms are called infections diseases, while if such diseases 214 
INTRODUCTION 
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, 
transmissible. 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. 
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- 
tion that characterizes acute infections. INTRODUCTION 
215 
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 Bacterium diphtheria, lockjaw due to 
Bacillus 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), bronchopneumonia, 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. 
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 
recognized infectious and contagious diseases 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 216 
INTRODUCTION 
frequently called "filterable." The term "virus" or "filter- 
able virus'' is likewise applied to these "ultra-microscopic" 
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 secondary generalized tuberculosis, or 
primary typhoid fever by a secondary pneumonia. Where 
several organisms seem to be associated simultaneously in 
causing the condition then the term mixed infection is used- 
in severe diphtheria, streptococci are commonly associated 
with the Bacterium diphtheric?. In many cases of hog- 
cholera, mixed infections in the lungs and in the intestines 
are common. 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 Bacillus 
coli normal to the intestine escapes into the peritoneal cavity, 
or passes into the bladder, a severe peritonitis or cystitis, 
respectively, is apt to result. "Boils" and "pimples" are 
frequently autoinfections. Such infections are also spoken 
of as endogenous to distinguish them from those due to 
the entrance of organisms from without-exogenous infec- 
tions. 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 
tubercle bacilli from lung to intestine, spleen, etc., the for- 
mation of abscesses in internal organs following a primary 
surface 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, may be fatal. 
On the other hand, the characteristic of the host which 
indicates its capacity for infection is called susceptibility. INTRODUCTION 
217 
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 in- 
vading organism and the susceptibility of the host. High 
virulence and great susceptibility mean a severe infection; 
low virulence and little susceptibility, a slight infection; 
while high virulence and little susceptibility or low viru- 
lence and great susceptibility might mean a moderate infec- 
tion varying in either direction. Other factors influencing 
the degree of infection are the number of organisms intro- 
duced, the point where they are introduced and various 
conditions. These will be discussed in another connection 
(Chapter 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 
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) Sick themselves. 
(6) Recovered from illness but carrying the organisms. 
(c) Never sick with the disease but carrying the 
organisms. 
(d) Serving as necessary intermediate hosts for cer- 
tain stages of the parasite-this applies to 
protozoal diseases only, as yet. 
I. (a) The bacilli of tetanus and malignant edema 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 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 organ- 
ism in its natural habitat. Bacillus botulinus, which causes 
a type of meat poisoning in man, does not even multiply 
in the body, but the disease symptoms are due to a soluble 
toxin which is produced during its growth outside the body. 
(6) 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 living 
a saprophytic existence outside the body for years. These 
can no more be attacked during their, saprophytic existence PATHOGENIC BACTERIA OUTSIDE THE BODY 
219 
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, however, 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. 
IL (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 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 
milk; bubonic plague from rats, etc. The mode of limiting 
this class of diseases is evidently to isolate the sick, dis- 
infect their discharges and their immediate surroundings, 
sterilize such products as must be handled or used, kill 
dangerous animals, and disinfect, bury properly, or destroy 
their carcasses. 
(6) This class of "carriers'' offers one of the most difficult 
problems in preventing infectious diseases. A perfectly 
healthy individual may give off dangerous organisms and 
infect others for years. Typhoid carriers have been known 
to do so for fifty-five years. Cholera, diphtheria, menin- 
gitis and other carriers are well known in human practice. 
The difficulty in detecting such individuals is obvious. 
Carriers among animals have not been so frequently demon- 
strated, but there is every reason for thinking that hog- 220 
PATHOGENIC EACTEPIA OUTSIDE THE PODY 
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. 
(c) 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 transmit 
the organisms through biting infected and non-infected ani- 
mals successively. The spirilloses and trypanosomiases 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. 
Typhoid transmission by Hies is well established in man. 
Why not hog-cholera from farm to farm by Hies, 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 Hy breed- 
ing are obvious protective measures. 
(d) 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 
most certain 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 (Margar opus annul abas'). 
Piroplasmoses in South Africa among cattle and horses, PATHOGENIC BACTERIA OUTSIDE THE BODY 
221 
and in other countries are transmitted in similar ways. Prob- 
ably many of the diseases due to spirochetes and trypano- 
somes 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 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. 
J. 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, Trychophyton 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 occa- 
sionally in animals are illustrations. Sharp-edged and freely 
bleeding wounds are less liable to be infected than contu- 
sions, 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 THE SKIN 
223 
to care for invading organisms (phagocytosis). Pathogenic 
organisms, especially pus cocci, frequently gain lodgment in 
the milk glands and cause local (mastitis) or general infection. 
B. 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 of even 
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. 
(c) The nasal cavity, on account of its anatomical structure 
retains pathogenic organisms which give rise to local infec- 
tions more frequently than other mucosae of its character. 
These may extend from here to middle ear, neighboring 
sinuses, or along the lymph spaces of the olfactory nerve 
into the cranial cavity (meningitis). Acute coryza ("colds" 
in man) is characteristic, (danders, occasionally, is primary 
in the nose, as is probably roup in chickens, leprosy in man. 
The meningococcus and the virus of poliomyelitis 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. 
(e) The tonsils afford a ready point of entrance for ever- 
present micrococci and streptococci whenever occasion offers 
(follicular tonsilitis, "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. 
(J) The pharynx is the seat of localized infection as in 
micrococcal, streptococcal and diphtherial "sore throat" in 
human beings, but both it and the esophagus are rarely 
infected in animals except as the result of injury. 224 
ENTRANCE OF PATHOGENIC ORGANISMS 
(</) 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 contagious 
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 
most readily through close association though not neces- 
sarily actual contact, the commonest path is through the 
lungs, which may or may not show lesions (smallpox, scar- 
let fever, measles, chicken-pox, whooping-cough, pneumonic 
plague in man, lobar and bronchopneumonias and influenza 
in man and animals, some cases of glanders and tubercu- 
losis). On the other hand, the fact that the Bacillus typhosus 
and Bacillus coll may cause pneumonia when they evi- 
dently have reached the lung from the intestinal tract, and 
the experimental evidence of lung tuberculosis above men- DISSEMINATION OF ORGANISMS 
225 
tinned show that this route cannot be excluded in inflam- 
mations of the lung. 
1). 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. 
(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 many 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 
stomach of ruminants practically all the time may explain 
the relative freedom of adult animals of this class from 
"choleras" and "dysenteries." 
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 simul- 
taneously. 226 
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 ultra-microscopic 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. LOCATION OF INFECTIVE ORGANISMS 
227 
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 apex (in man most commonly), 
"choleras" the small intestine, "dysenteries" the large 
intestine, influenza the lungs. In these cases the explana- 
tion is probably that the points attacked are the places 
where the organism is most commonly carried, with no 
specific relationship, since all of these organisms (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 frequently 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 piroplasms attack the 
red blood corpuscles, etc. 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 "chemotherapy" will ever profit 
from a knowledge of such chemical relationships remains 
to be developed. 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 incapable of manifesting them- 
selves, are unable to cause disease. The word has taken the 
place of the earlier term, resistance, and is the opposite of 
susceptibility. The term must be understood always in a 
relative sense, since no animal is immune to all pathogenic 
organisms, and conceivably not entirely so to anyone, since 
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 aniiftals; 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 tends 
to lower its resistance. Children appear more susceptible 
to scarlet fever, measles, whooping-cough, etc., than adults; 
young cattle more frequently have black-leg than older ones IMMUNITY 
229 
(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 anguish, 
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) By having the 
disease in utero. 
(b) By absorption 
of immune sub- 
stances from the 
mother. 
I. Natural 
A. Congenital 
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: 
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. 
(b) 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 
immunized animal. 230 
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 ("smallpox 
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). It is a familiar fact that with most 
infectious diseases recovery from one attack confers a more 
or less lasting immunity, though there are marked excep- 
tions. 
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 because 
the body cells of the animal immunized perform the real 
work of bringing about the immunity as will be discussed 
later. In passive immunity the blood serum of an actively 
immunized animal is introduced into a second animal, 
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 
231 
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 instances 
both the organism and an antiserum are used to cause both 
active and passive immunity (serum-simultaneous 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 (6) 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. 
(2) By drying the organism, as is done in the preparation of 
the vaccine for the Pasteur 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 impor- 
tant in this procedure as the drying, since it is doubtful if the 
"fixed virus" is pathogenic for man.) (3) The organism 
may be attenuated by growing at a temperature above the 
normal. This is the method used in preparing anthrax vac- 
cine 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 weakened. Black-leg 
vaccines are made by this method. (5) Chemicals are some- 
times added to attenuate the organisms, as was formerly 
done in the preparation of black-leg vaccine by Kruse's 232 
IMMUNITY 
method in Germany. This method is no longer used to any 
great extent. (6) Within the past few years the workers in 
the Pasteur Institute in Paris have been experimenting with 
vaccines 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). (7) Growing on artifi- 
cial culture media reduces the virulence of most organisms 
after a longer or shorter time. This method has been tried 
with many organisms 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 
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). ACTIVE IMMUNITY 
233 
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 adulterations 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 OE 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. Metchnikoff 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 PAUL EHRLICH THEORIES OF IMMUNITY 
235 
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 chemi- 
cal 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, chemical, etc. Chemical stimuli are 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 increase of its corresponding 
foodstuff. The cells of the body, or certain of them at 
least, seem to respond in a specific way when proteins or 
substances closely related to them are brought into direct 
contact with them, that is, without having been subjected 
to digestion in the alimentary tract, but injected directly 
into the 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, may be 236 
THEORIES OF IMMUNITY 
destroyed; if the stimulus is of proper amount then it excites 
the cell to* increased activity, and in the case of sped fie 
chemical stimuli the increased activity, as mentioned for 
the pancreas, shows itself in an increased production of what- 
ever is called forth by the chemical stimulus. In the case of 
the proteins and related bodies, 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 primarily to take' up different kinds of food material, 
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, proteins 
and their allies, the particular product is these cell receptors 
with which the chemical 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 ctually 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 actually needed. 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. THEORIES OF IMMUNITY 
237 
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, 
the substance introduced. Any substance which when 
introduced into the body causes the formation of an anti- 
body of any kind whatsoever is called an antigen, i. e., anti 
(body) former. 
If the three fundamental principles just discussed are 
thoroughly understood, the theory of the formation of 
different kinds of antibodies should not be difficult to 
comprehend. 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 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 intro- 
duced into the body its haptophore 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. Antitoxins 
are specific-that is, an antitoxin which will combine with 
the toxin of Bacillus tetani will not combine with that of 
Bacterium diphtherice, or of Bacillus botulinus, or of any 
other toxin, vegetable or animal. A NT1 TOXINS-A NTIENZ YMES 
239 
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 240 
RECEPTORS OF THE FIRST ORDER 
the toxins and antitoxins for diphtheria and tetanus. Anti- 
toxins have been prepared experimentally for a large number 
of both animal and vegetable poisons, including a number 
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. 252), antipollenin (sup- 
posed to be for the toxin of hay fever) and the antitoxins 
for the true bacterial toxins of Bacterium diphtherioe and 
Bacillus tetani. 
The method of preparing antitoxins is essentially the same 
in all cases, though differing in minor details. For commer- 
cial 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 c.c.) then the maximum amount 
of blood is collected from the jugular with sterile trocar and 
canula. 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 
sera which are put on the market must conform to definite 
standards of strength expressed in "units" as determined ANTITOXINS-ANTIENZ YMES 
241 
by the U. S. Hygienic Laboratory. In reality a "unit" of 
diphtheria antitoxin in the United States is an amount 
equivalent to 1 c.c. 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." Specifi- 
cally stated, a "unit" of antitoxin contains approximately 
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. I)., in reality would protect against about 200 M. L. I), 
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 gm. guinea-pig for 90 hours against the standard 
test dose" of the standard toxin. The standard test dose is 
100 M. L. I), of toxin for a 350 gm. guinea-pig. To express 
it another way, one could say that a "unit" of tetanus anti- 
toxin would protect one thousand 350 gm. guinea-pigs from 
1 M. L. I). each of standard tetanus toxin. 
Various methods have been devised for increasing the 
amount of antitoxin in 1 c.c. 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 content of " units" per c.c. 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 
pyocyaned) when mixed with the blood serum of an animal 
immunized against them. Gruber and Durham (189(5) 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 Bacterium mallei, 
"abortion agglutinin" for Bacterium abortus, "typhoid 
agglutinin" for Bacillus typhosus, etc. 
The phenomenon may be observed either under the micro- 
scope or in small test-tubes, that is, either microscopically or 
m acroscopical Iy. 
In this case the cells introduced, or more properly, some 
substances within the cells, probably protein in nature, act 
as stimuli to the body cells of the animal injected to cause 
them to produce more of the specific cell receptors which AGGLUTININS 
243 
respond to the stimulus. The substance within the intro- 
duced 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 exper- 
imentally with extracts of cells (bacterial and other cells) 
and the blood serum of the animal injected 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 agglutination. Hence 
it can be stated technically that an agglutinin possesses a 
haptophore group, and an agglutinating group. 
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 
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 244 
RECEPTORS OF THE SECOND ORDER 
cells, as well as for the cell introduced. The agglu- 
tinins for the cell introduced will be formed in larger quan- 
tity, 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 "coagglutinins") 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 testing against several known bacteria to 
determine for which it is specific, i. e., which is the cause 
of the disease in the animal. 
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 
against the blood sera of different animals, each of which has 
been properly inoculated with a different kind of known bac- 
teria. 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 had been inoculated, or immunized, as is usually 
stated. This method of identifying cultures of bacteria 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 PRECIPITINS 
245 
diagtiosis (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 protein in nature within the bacterial 
cell, it is reasonable 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 precipi- 
tate when mixed with the serum of an animal immunized 
against the particular bacterium and that the reaction is 
specific under proper conditions 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 sub- 
stance which causes a precipitate with solutions of the pro- 
tein used for immunization. The number of such precipi- 
tating serums that have been made experimentally is very 
large and it appears that protein 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 protein molecule (hapto- 
phore 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 pre- 
cipitation and agglutination are strictly analogous phenom- 
eifa. Precipitins act on proteins in (colloidal) solution and 
cause them to settle out, agglutinins act on proteins within 
cells which cells are in suspension in a fluid and cause the 
cells to settle out. Ehrlich's theory of the formation of 246 
RECEPTORS OF THE SECOND ORDER 
precipitins is similar to that of agglutinins, and need not be 
repeated. Substitute the corresponding 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 a sausage, and 
different kinds of milk in a mixture. An extract of the 
sausage is made and tested against the serum of an animal 
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 PRECIPITINS 
247 
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. A on Fodor (1885) 
showed that saprophytic bacteria injected into the blood 
are rapidly destroyed. Fliigge and his pupils, especially 
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 ward 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 exam- 
ining them ("Pfeiffer's phenomenon"). Belfanti and Car- 
bone and especially Bordet (1898) showed the specific dis- 
solving action of the serum of one animal on the blood cor- OY TOLY SI NS 
249 
puscles of another animal with which it had been injected. 
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 "cytotoxic" 
means "cell poisoning" or "injuring," while "cytolytic" 
means "cell dissolving." The latter term is the one gen- 
erally 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 cell which is dissolved, as a 
bacteriolysin, a hemolysin (red-corpuscle-lysin), epith elioly sin, 
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) then 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. Experiment has shown that it is the substance 
present in all serum that is the true dissolving body, while 
the immune substance serves merely to unite this body to the 
cell to be destroyed, i. e., to the antigen. Since the immune 
body has therefore two uniting groups, one for the dissolv- 
ing substance and one for the cell to be dissolved, Ehrlich 250 
RECEPTORS OF THE THIRD ORDER 
calls it the "amboceptor.'' He also uses the word "comple- 
ment" to denote the dissolving substance, giving the idea 
that it completes the action of dissolving after it has been 
united to the cell by the amboceptor, thus replacing 
Buchner's older term "alexin" for the same dissolving body. 
AMBOCEPTORS. 
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, probably protein, 
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 ambocep- 
tors. It will be noticed that these free receptors differ from 
either of the two groups 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 func- 
tion 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 amboceptor, and an active group which is 
usually called the zymophore or toxophore group. Comple- 
ments thus resemble either toxins, where the specific cell 
(antigen) is injured or killed, or enzymes, in case the cell is 
likewise dissolved. This action again shows the close rela- 
tion between toxins and enzymes. Complement may lose 
its active group in the same way that toxin does and becomes 
then compUmentoid. 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 COMPLEMENTS 
251 
an animal, the animal's blood will contain amboceptors for 
the cell used and also for others closely related to it. Immun- 
ization 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 
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 the 
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. 252 
RECEPTORS OP THE THIRD 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. 
FAILURE OF CYTOLYTIC SERUMS. 
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) 
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 COMPLEMENT-FIXA TION TEST 
253 
by using blood of an homologous ("same kind") animal. 
Hence Ehrlich suggested the use of apes for preparing bac- 
tericidal sera for human beings. (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 objec- 
tion. (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. 
Although little practical use has been made of bactericidal 
sera, the discovery of receptors of this class and the peculiar 
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-fixa- 
tion" or the "complement-deviation test" ("Wassermann 
test" in syphilis) and is applicable in a great variety of mi- 
crobial 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. 
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, 
COMPLEMENT-FIXATION TEST. 254 
RECEPTORS OF THE THIRD ORDER 
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 infected 
with a microorganism and a part of its defensive action con- 
sists in destroying the organisms in its blood or lymph, then 
it follows from the above discussion of cytolysins 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 war- 
ranted that the organism for which it is specific is the cause 
of the disease. Consequently what is sought in the "com- 
plement-fixation test" is a specific amboceptor. In carrying 
out the test blood serum from the suspected animal is col- 
lected, heated at 56° for half an hour to destroy any comple- 
ment 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 higher in complement 
than that of most animals. The blood of the gray rat con- 
tains practically as much. If the specific amboceptor is 
present, that is, if the animal is infected with the suspected 
disease, the complement will unite with the antigen-ambo- 
ceptor 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 physicochemical 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 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 therefore contains a hemolytic ambo- 
ceptor specific for the corpuscles, which is capable also of COMPLEMENT-FIXATION TEST 
255 
uniting with complement. The indicator is put into the 
first mixture and the whole is again incubated for at least 
two hours 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 ambo- 
ceptor) complex-and destroy the corpuscles. Hence if the 
complement is bound in the first instance, the specific ambo- 
ceptor for the first antigen must have been present in the 
blood, that is, the animal was infected with the organism 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." 
In practice all the different ingredients must be accurately 
tested, standardized and used in exact quantities, and a test 
must also be run as a control with a known normal blood 
of an animal of the same species as the one examined. 
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 animals 
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 necessity and 
hence is rarely employed. It has been used, however, to 
detect traces of unknown proteins, particularly blood-serum 
proteins, in medicolegal 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 ascribed 
their formation to the phagocytes or to the same organs 
which form the leukocytes-lymphoid tissue generally, 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 exclusive. 
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 
proposed for these bodies the name opsonin, derived from a ELIE METCHNIKOFF PHAGOCYTOSIS-OPSONINS 
257 
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 present 
in normal serum and that in the serum of an animal which 
has been immunized against such bacteria the opsonins 
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 index 
of a given serum divided by the phagocytic index of a nor- 
mal serum gives the opsonic index of the serum tested. 
Assuming the normal opsonic index to be 1, Wright asserts 
that in healthy individuals the range should be not more 258 
PHAGOCYTOSIS-OPSONINS 
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 improper 
dosage in case attempts have been made to immunize 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 generally 
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 
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. 231. BACTERIAL VACCINES 
259 
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 undoubted. 
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 sub- 
stances. The second is that, on the other hand, it must con- 
tain 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 
per cent. NaCl). The number of bacteria per c.c. 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- 260 
PHAGOCYTOSIS-OPSONINS 
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 c.c., "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 c.c. of solution, when 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. 264 et seq.). Normal saline solution is isotonic with 
the body cells and hence is employed as the vehicle. 
Vaccines are either "autogenous" or "stock." An "autog- 
enous" 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 
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 sub- 
ject, but also from his own experience in animal, and espe- 
cially 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 streptococci, 
micrococci, and colon bacilli are the causative agents, but to BACTERIAL VACCINES 
261 
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. Indi- 
viduals 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 long gradu- 
ally has some of its antigens destroyed by the uninjured 
enzymes present with them, and so loses in potency. There- 
fore 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. 
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- 262 
PHAGOCYTOSIS-OPSONINS 
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 c.c. as before 
mentioned, and his method or some modification of it is still 
in general use. From what was said with regard to varia- 
tion, 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 (loses of micrococcus 
(staphylococcus) vaccines recommended by different writers, 
which vary from 50,000,000 to 2,000,000,000 per c.c. The 
author is decidedly of the opinion that there is no way of 
determining the dosage of a vaccine in the 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 gen- 
eral 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 reac- 
tion 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 disturbance, 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 dimin- 
ished. This is called the "negative phase," and Wright AGGRESSIN 
263 
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 opsonic 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 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 clinical 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 
prepares 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 
experience to date justifies them. It is worth the time and 
money 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 indi- 
vidual 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 was shown that it was the horse 
serum (foreign protein) that was the cause, and not the anti- 
toxin itself. In 1898 Richet 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 name "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 injec- 
tions of foreign proteins. It is now a well-established fact that ANAPHYLAXIS 
265 
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. Richet'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 ques- 
tion. The animal is said to be "sensitized" for that protein. 
The sensitization is specific since an animal injected with white 
of chicken's egg reacts to a second injection of chicken's 
egg only and not pigeon's egg or blood serum or any 
other protein. The specific poisonous substance causing the 
symptoms has been called " anaphylotoxin" 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 antigen. 
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 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 266 
ANAPHYLAXIS 
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 produced, 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 proteins from many different 
sources-animal, vegetable, pathogenic and saprophytic bac- 
teria-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 precipitating 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 common 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 connec- 
tion would be to split off the side chains, and since these are 
the specific parts of the protein, the enzyme must be spe- 
cific for each protein. The pepsin of the gastric juice and 
the trypsin of the pancreas split the native proteins only to 
peptones. As is well known these when injected in suffi- 
cient quantity give rise to poisonous symptoms, and will 
also give rise to anaphylaxis under properly spaced injec- 
tions. 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 essen- 
tial to the theory of anaphylaxis above outlined, i. e., a 
phenomenon due to the action of specific antibodies which are 
enzymes. On physiological grounds this appears the most 
rational of the few 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 ANAPHYLAXIS 
267 
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. 
As has been indicated "serum sickness" and sudden death 
following serum injections are probably due to a sensitization 
of the individual to the proteins of the horse in some unknown 
way. Possibly urticarial rashes and idiosyncrasies follow- 
ing the ingestion of certain foods-strawberries, eggs, oysters, 
etc., may be 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 
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 268 
ANAPHYLAXIS 
enzymes are not used up rapidly enough since the products 
of the bacillus are secreted slowly in such cases. lienee 
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 gm. of protein by this test. It is evi- 
dent that the test is applicable in medicolegal cases and in 
food examination and has been so used. 
Summary. 
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 specific, 
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. ANAPHYLAXIS 
269 
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 
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 flagella. 
Exercise 25. Study of individual species. 
Exercise 26. Determination of thermal death-point. 
Exercise 27. Action of disinfectants on bacteria. 
Exercise 28. Action of sunlight on bacteria. INDEX. 
A 
Abbe, 28 
condenser, 184-185 
Abilgaard, 24 
Abortion, contagious, 224, 226, 253 
Abrin, 239 
Absorption of free nitrogen, 105 
Accidental carriers, 220 
structures, 40 
Acetic acid, 88 
fermentation, 29 
Achorion schoenleinii, 25 
Acid, acetic, 88 
fermentation, 29 
butyric, 88 
fermentation, 29 
carbolic, first used, 26 
fast bacilli, staining of, 193 
bacteria, 73 
fermentation, 82 
hydrochloric, 225 
lactic, fermentation, 85-88 
production of, 98 
soils, 70 
Acquired immunity, 230 
Actinomyces bovis, 27 
Actinomycosis, 223 
Actions, reducing, 101 
Activating enzymes, 113 
Active immunity, 230 
production of, 230-232 
Activities, physiological, definition 
of, 76 
in identification, 199 
Activity, overproduction, of cells, 
235, 236 
Acute coryza, 223 
disease, 214 
Aerobe, facultative, 66 
Aerobes, 66 
Aerobic, 66 
Agar medium, 163-164 
Agent, choice of, for disinfection, 
151-156 
Agents, physical, for disinfection, 
119-143 
Agglutinating group, 243 
Agglutination, 242 
macroscopic, 242 
microscopic, 242 
Agglutinin, 242 
bacterial, 242 
hema-, 242 
Agglutinins, 242-245 
anti-, 246 
co-, 244 
compared with precipitins, 245 
functions of, 243 
normal, 243 
specificity of, 244 
theory of formation of, 242 
use of, 243, 244 
Agglutinogen, 243 
Aggressin, 263 
Aids in isolation of bacteria, 182, 
183 
Air, bacteria in, 61 
filtration, 141 
Albumin in bacteria, 73 
Alcohol as disinfectant, 148 
Alcoholase, 113 
Alcoholic fermentation, 29, 89 
Alexin, 248 
Algae, relation to bacteria, 34 
Alimentary tract as channel of 
infection, 225 
Amboceptor, 250 
hemolytic, 254 
partial, 251 
specific, 254 
Amboceptorogen, 251 272 
INDEX 
Amboceptors, 250 
anti-, 251, 252 
co-, 251 
specificity of, 250 
Ameba coli, 27 
Ameboid colony, 207 
Ammoniacal fermentation, 29 
Amphitrichic, 43 
Amylase, 112 
Anaerobe, facultative, 66 
Anaerobes, 66 
Anaerobic, 66 
bacteria, first discovered, 29 
organisms, cultivation of, 172- 
176 
Analysis, chemical, of tubercle 
bacilli, 74 
of ash, 71 
Anaphylactic, anti-, 265, 267 
phenomena, 267 
reaction, uses of, 268 
Anaphylatoxin, 265 
Anaphylaxis, 264-268 
anti-, 267 
antibodies in, 266 
theory of, 266 
Anderson, 264 
Andry, Nicholas, 24 
Anilin dyes, 27, 188 
fuchsin, 189 
gentian violet, 189 
Animal inoculation, 210 
Animalcules, bacteria considered to 
be, 19 
Animals, disinfection of, 155 
experimental, 210 
Ankylostomum duodenale, 26 
Anthrax, 26, 218, 222, 225, 226 
spores of, 27 
vaccine, 231 
Antiagglutinins, 246 
Antiaggressin, 263 
Antiamboceptors, 251, 252 
Antianaphylactic, 265 
Antianaphylaxis, 267 
Antibacterial immunity, 232 
Antibodies, 237 
in anaphylaxis, 266 
place of production of, 269 
Antibody formation, 116 
Anticomplement, 251 
Antidiphtheritic serum, 240 
Antienzyme, 239 
Antienzymes, 238-241 
Antigen, 237 
syphilitic, 254 
Antipollenin, 240 
Antiprecipitins, 246 
Antisepsis, 119 
Antiseptic, 119 
methods introduced, 26 
Antisnake venoms, 252 
Antitetanic serum, 240 
Antitoxic immunity, 232 
Antitoxin, 238 
diphtheria, 28 
preparation of, 240 
standard, 241 
unit of, 241 
Antitoxins, 238-241, 268 
Antivenin, 240 
Apex of lung, 227 
Apparatus of Barber, 180 
Appearance of growth in identifi- 
cation, 200 
Appert, 20 
Aqueous gentian violet, 189 
Arborescent growth, 204 
Aromatic compounds, 93 
production of, 99 
Arthus, 264 
Arthus's phenomenon, 264 
Articles, unwashable, disinfection 
of, 155 
washable, disinfection of, 155 
Artificial immunity, 230 
Ase, termination of name of 
enzyme, 112 
Asepsis, 119 
Aseptic, 119 
Ash, analysis of, 71 
Asiatic cholera, 227 
Attenuated, 231 
Autoclave, 125 
Autogenous vaccine, 260 
Autoinfection, 216 
Autolysis, 114 
Autotrophic, 75 
Azotobacter, 106 
B 
Babes-Ernst corpuscles, 42 
Bacilli, butter, 193 
colon, 226 
grass, 193 
tubercle, chemical analysis of, 74 INDEX 
273 
Bacillus, 49, 59 
botulinus, 93, 116, 218, 238 
coli, 67, 84, 148, 161, 216, 224 
Ducrey's, 224 
enteritidis, 93 
fluorescens, 67 
of blue milk, 29 
paslorianus, 106 
prodigiosus, 67 
subtilis, 67, 72, 193 
tetani, 116, 193, 215, 238, 240 
typhosus, 63, 224, 242 
vulgaris, 67 
Bacteria, acid-fast, 73 
aids in isolation of, 182, 183 
anaerobic, first? discovered, 29 
as scavengers, 95 
chemical elements in, 71 
classed as fungi, 34 
considered to be animalcules, 19 
definition of, 37 
energy relationships of, 36, 37 
first classed as plants, 30 
seen, 18 
shown to cause disease, 27 
in feces, 62 
in the air, 61 
in the sea, 61 
in the soil, 61 
in urine, 62 
in water, 61 
iron, 75, 78 
isolation of, 178-183 
measurement of, 37 
metabolism of, 75-81 
morphology of, 39 
motile, 42 
nitric, 103 
nitrous, 102 
occurrence, 61 
pathogenic, outside the body, 
218 
position of, 34 
rate of movement of, 42 
related to protozoa, 37 
relation to algae, 34 
to molds, 34 
to yeasts, 34 
relationships of, 34 
size of, 37 
staining of, 188-195 
study of, 157 
of physiology of, 196-209 
sulphur, 75, 94 
Bacteriacea, 59 
Bacterial agglutinin, 242 
vaccines, 258 
preparation of, 259, 261 
Bacterin, 231 
Bacteriocidin, 249 
Bacteriological examination, ma- 
terial for, 211 
microscope, 184 
Bacteriology, pathogenic, defini- 
tion of, 213 
reasons for study, 217 
Bacteriolysin, 249 
Bacteriotropin, 257 
Bacterium, 59 
abortus, 242 
acidi oxalici, 72 
anthracis, 148, 193 
diphtheria, 116, 215, 216, 238, 240 
lepra, 193 
mallei, 242 
of Johne's disease, 193 
smegmatis, 193 
study of, necessary steps for, 158 
tuberculosis, 72, 160, 193 
xylinum, 72 
Barber and Webb, 231 
apparatus, 180 
Barnyards, disinfection of, 153-155 
Baskets, wire, 168 
Bassi, 25 
Bastian, Dr. Charlton, 23 
Baumgartner, 234 
Beaded growth, 204 
Bed-bugs as carriers, 220 
Beds, contact, 104 
Beer, pasteurization of, 129, 133 
Beggiatoa, 60 
Beggiatoacea, 60 
Behring, 28 
Belfanti, 248 
Berg, 25 
Berkefeld, 142 
Bichloride of mercury as disinfec- 
tant, 146 
Bilharz, 26 
Bilharzia disease, 26 
Biochemical reactions, definition of, 
76 
Bipolar germination of spore, 46' 
Black-leg, 218, 222, 226 
vaccine, 231 
Bleaching powder as disinfectant, 
146 274 
INDEX 
Blood, detection of, 246 
serum, Loeffler's, 166 
medium, 166, 167 
Blue milk bacillus, 29 
fermentation, 29 
Boehm, 25 
Boils, 216, 222 
Bollinger, 27 
Bonnet, 19 
Bordet, 248 
Botrytis bassiana, 25 
Bottles, staining, 190 
Bougie, 141 
Bouillon, 159 
Bread, salt rising, 84 
Bronchopneumonia, 215, 224 
Broth, glycerin, 160 
meat, 159 
nitrate, 161 
sugar, 160 
Brownian motion, 44 
movement, 44 
Buchner, 248 
Budding of yeasts, 34 
Burning, 120 
Burying, 142 
Butter bacilli, 193 
rancidity of, 90 
Butyric acid, 88 
fermentation, 29 
Buzzards as carriers, 220 
C 
Caignard-Latour, 29 
Calcium hypochlorite as disinfec- 
tant, 146 
oxide as disinfectant, 146 
Candles, filter, 141 
Canned goods, spoiling of, 48 
Canning, 68 
Capsule, 41 
of the spore, 45 
Capsules, staining of,5194 
Carbohydrates, fermentation of, 82 
in bacteria, 73 
Carbol-fuchsin, 189 
Carbolic acid as disinfectant, 147 
first used, 26 
Carbon, circulation of, 96 
uses of, 77 
Carbone, 248 
Carriers, 219 
Carriers, accidental, 220 
Cars, stock, disinfection of, 155 
Catalase, 113 
Catalyzer, 111 
Causation of disease, 23, 116 
Cell contents, 39 
forms, 49, 55 
staining for, 195 
groupings, 52-55 
staining for, 195 
structures, 39 
wall, 39 
composition of, 72 
Cells, chemical stimuli of, 235 
overproduction activity of, 235, 
236 
specific chemical stimuli of, 
235 
Cellular theory of immunity, 234 
Cellulose, 72 
Chain, 52 
Channels of infection, 222 
alimentary tract, 225 
conjunctiva, 223 
external auditory meatus, 223 
genitalia, 224 
intestines, 225 
lungs, 224 
milk glands, 223 
mouth cavity, 223 
mucosae, 223 
nasal cavity, 223 
pharynx, 223 
skin, 222 
stomach, 225 
tonsils, 223 
Chaos, 24 
Characteristics of toxins, 114, 115 
Charrin, 242 
Chart, descriptive, 200 
Chauveau, 234 
Cheese, eyes in, 85 
poisoning, 93 
ripening, 29 
Chemical analysis of tubercle 
bacilli, 74 
composition, 70-74 
of bacteria, 36 
elements in bacteria, 71 
stimuli, effect of, 236 
of cells, 235 
specific, 236 
theory of immunity, 234 
Chemotherapy, 227, 232 INDEX 
275 
Chevreuil and Pasteur, 21, 25, 
29 
Chicken-pox, 224 
Chitin, 72 
Chlamydobacteriacece, 60 
Chlamydothrix, 60 
Chloride of lime as disinfectant, 146 
Chlorine as disinfectant, 145 
Chloroform as disinfectant, 150 
Chlorophyl, absent in bacteria, 34 
Chlorosis, Egyptian, 26 
Cholera, Asiatic, 227 
hog, 220, 226, 230 
Choleras, 225, 226, 227 
Chromogenesis, 100 
Chromoparic, 100 
Chromophoric, 100 
Chronic disease, 214 
Chronological index, 31 
Circulation of carbon, 96 
of nitrogen, 96 
of phosphorus, 96 
of sulphur, 97 
Cladothrix, 60 
Classification, first attempt at, 30 
Migula's, 59, 60 
Cleaning of slide, 191 
Clostridium, 47, 106 
Clothing for disinfection, 156 
Coagglutinins, 244 
Coagulases, 112 
Coagulating enzymes, 112 
Coamboceptors, 251 
Cobra, 252 
CoccacecE, 59 
Coccus, 49 
Coenzymes, 110 
Cohn, 26, 30 
Cold, 136 
incubator, 198 
Colds, 223 
Colon bacilli, 226 
Colonies, plate, study of, 209 
Colony, 159 
ameboid, 207 
effuse, 207 
punctiform, 206 
rhizoid, 206, 208 
Combustion, spontaneous, 104 
Commensal, 76 
Complement, 250 
anti-, 251 
deviation test, 253 
fixation test, 253-255 
Complement, lecithin as, 251 
source of, 254 
Complementoid, 250 
Complementophil haptophore, 250 
Complements, 251 
Composition, chemical, 70-74 
of bacteria, 36 
Compounds, aromatic, 93 
Compressed oxygen, 67 
Condenser, Abbe, 184, 185 
Conditions for formation of spores, 
48 
for growth, general, 62 
maximum, 62 
minimum, 62 
optimum, 62 
Conjunctiva as channel of infec- 
tion, 223 
Contact beds, 104 
Contagion, direct and indirect, 23 
Contagious abortion, 224, 226, 253 
diseases, 25, 214 
Contagium vivum, 23 
Contents, cell, 39 
Continuous pasteurization, 129 
Corpuscles, Babes-Ernst, 42 
red, 227 
Corrosive sublimate as disinfectant, 
146 
Coryza, acute, 223 
Cotton plugs, 20, 168 
Coughing, 226 
Crateriform liquefaction, 205 
Cream, ripening of, 86 
Creite, 248 
Crenothrix, 60 
Creolin as disinfectant, 148 
Creosols as disinfectants, 147 
Cultivation of anaerobic organisms, 
170-176 
Cultural characteristics as physio- 
logical activity, 117 
Culture medium, 157 
essentials of, 158 
gelatin, 161-163 
inoculation of, 170, 176 
pure, 157 
tubes, 168 
Cultures, mass, 172 
plate, 164, 172 
puncture, 169 
pure, methods of obtaining, 178- 
183 
slant, 170 276 
INDEX 
Cultures, slope, 170 
stab, 169 
Curled edge, 208 
Cutaneous inoculation, 211 
Cycle, nitrogen, etc., 96, 97 
Cytolysin, 249 
Cytolysins, 248-255 
Cytolytic, 249 
serums, failure of, 252 
substances, 268 
Cytophil haptophore, 250 
Cytoplasm, 39 
Cytotoxic, 249 
D 
Dallera, 264 
Davaine and Payer, 26 
Death-point, thermal, 65 
determination of, 198 
Decomposition of urea, 95 
Deep culture tubes, 174 
Definition of bacteria, 37 
of immunity, 217 
of pathogenic bacteriology, 213 
of spore, 48 
of unit, 241 
Degeneration forms, 51 
Denitrification, 102 
Deodorant, 119 
Deposits of sulphur, 104 
Descriptive chart, 200 
Detection of blood, 246 
Determination of oxygen relations, 
198 
of thermal death-point, 198 
Deviation test, complement, 253 
Diastase, 112 
Diffusion, food of bacteria by, 36 
Digestion of milk, 91 
Diluting blood serum, necessity 
for, 244 
Dilution plates, 178 
Diphtheria, 223, 227 
antitoxin, 28 
Diplobacillus, 52 
Diplococcus, 53 
Diplospirillum, 52 
Discharges, intestinal, 226 
nasal, 226 
urethral, 226 
vaginal, 226 
Discontinuous sterilization, 121 
Disease, acute, 214 
Bilharzia, 26 
causation of, 23, 116 
chronic, 214 
contagious, 25 
foot-and-mouth, 223, 226 
germs, 23 
hookworm, 26 
Johne's, 225, 226 
non-specific, 215 
of flies, 26 
of silkworms, 26 
silkworm, 25 
Diseases, contagious, 214 
infectious, 213 
skin, 222 
specific, 215 
Dishes, Petri, 164 
Disinfectant, 119 
Disinfectants, factors affecting, 
152-156 
Disinfection, 118 
and sterilization, practical, 152- 
156 
chemical agents, 144-150 
choice of agent, 151-156 
clothing for, 156 
of animals, 155 
of barnyards, 153-155 
of harness, 155 
of manure, liquid, 155 
of rooms, 153 
of stables, 153-155 
of stock cars, 155 
of surgical instruments, 153 
of unwashable articles, 155 
of vehicles, 155 
of washable articles, 155 
physical agents for, 119-143 
steam and formaldehyde, 156 
Dissemination of organisms in the 
body, 225 
Distilling, sour mash, 87 
Division, planes of, 53 
rate of, 80 
Dosage of vaccines, 262 
Dose, minimum lethal, 241 
standard test, 241 
Douglas, 256 
Dourine, 224, 226 
Drumstick spore, 47 
Dry heat, 121 
Drying, 119 
Ducrey's bacillus, 224 INDEX 
277 
Dunham's peptone solution, 161 
Durham, 242 
Dyes, anilin, 28, 188 
Dysenteries, 225, 226, 227 
Dysentery, tropical, 27 
E 
Ectoplasm, 39 
Edge, coarsely granular, 208 
curled, 208 
entire, 208 
lacerate, 207 
rhizoid, 208 
Edema, malignant, 218, 222 
Effuse colony, 205 
Ehrenberg, 30 
Ehrlich, 234 
Ehrlich's theory of formation of 
precipitins, 245 
of immunity, fundamental 
principles of, 235 
summary of, 237 
Eichsted, 26 
Electric milk purifier, 139, 140 
Electricity, 68, 138 
Elements, chemical, in bacteria, 71 
Elimination of pathogenic organ- 
isms, 226 
paths of, gall-bladder, 226 
kidneys, 226 
saliva, 226 
urine, 226 
Empusa mucoe, 26 
Endo-enzymes, 114 
Endogenous infection, 216 
Endoplasm, 39 
Endotoxins, 116 
Energy relationships of bacteria, 
36, 37 
Ensilage, 87 
Enteritis, 215 
Entire edge, 208 
Entrance, paths of, of pathogenic 
organisms, 222 
Enzyme, final test for, 111 
Enzymes, 109-114, 268 
activating, 113 
and toxins compared, 114, 115 
as catalyzers, 111 
chief characteristics of, 111 
classification of, 112, 113 
co-, 110 
Enzymes, coagulating, 112 
name of, 112 
oxidizing, 113 
produced by all cells, 112 
production of, 109 
reducing, 113 
similar to living organisms, 109, 
110 
splitting, 112 
Enzymoid, 239 
Epitheliolysin, 249 
Equatorial germination of spore, 45 
Erysipelas, hog, 226 
Essential structures, 39 
Essentials of a culture medium, 158 
Esters, production of, 98 
Ether as disinfectant, 150 
Eubacteria, 59 
Examination, bacteriological, mate- 
rial for, 211 
Exanthemata, 226 
Exhaustion theory of immunity, 
234 
Exo-enzymes, 114 
Exogenous infection, 216 
Exotoxins, 116 
Experimental animals, 210 
Explanation of natural immunity, 
216 
External auditory meatus as chan- 
nel of infection, 223 
genitalia, 227 
as channels of infection, 224 
Eyes in cheese, 85 
F 
Factors affecting disinfectants, 
151, .152. 
modifying immunity, 228 
Facultative, 66 
aerobe, 66 
anaerobe, 66 
parasite, 76 
Failure of cytolytic serums, 252 
of vaccines, 261 
Fats in bacteria, 73 
splitting of, 90 
Favus, 25 
Feces, bacteria in, 62 
Fermentation, 82 
acetic acid, 29, 88 
acid, 82 278 
INDEX 
Fermentation, alcoholic, 29, 89 
ammoniacal, 29 
and putrefaction, 28 
blue milk, 29 
butyric acid, 29, 88 
gaseous, 82 
lactic acid, 85-88 
of carbohydrates, 82 
tubes, 68, 174 
Ferments, organized, 114 
unorganized, 114 
Fever, Malta, 245 
recurrent, 27 
scarlet, 224, 226 
Texas, 214, 215, 220 
typhoid, 214, 226 
yellow, 221 
Filament, 52 
Filiform growth, 204 
Film, fixing of, 191 
preparation of, 190 
Filter, Berkefeld, 142 
candles, 141 
Pasteur-Chamberland, 142 
sprinkling, 104 
Filterable organisms, 216 
virus, 216 
Filtration, 140, 141 
air, 141 
water, 141 
First order, receptors of, 239 
Fixation test, complement, 253-255 
Fixed, 254 
Fixing of film, 191 
Flagella, position of, 42, 43 
staining of, 194 
Flagellum, 42 
Flash process, 133 
Fleas as carriers, 220 
Flexner, 253 
Flies as carriers, 220 
disease of, 26 
Flugge, 248 
Fodor, von, 248 
Food taken in by diffusion, 36 
uses of, 75 
Foot-and-mouth disease, 223, 226 
Foreign-body pneumonia, 224 
Formaldehyde and steam disinfec- 
tion, 156 
as disinfectant, 148-150 
Formation of agglutinins, theory 
of, 242 
of antibodies, 116 
Formation of precipitins, Ehrlich's 
theory, 245 
of spores, 45 
conditions for, 48 
Forms, cell, 49, 55 
degeneration, 51 
growth, 51 
involution, 51 
study of, 30 
Foxes as carriers, 220 
Fracastorius, 23 
Free receptors, 237 
spore, 45 
Fuchs, C. J., 29 
Fuchsin, anilin, 189 
carbol, 189 
Functions of agglutinin, 243 
Fungi, bacteria classed as, 34 
Funnel-shaped liquefaction, 205 
G 
Gabbet's blue, 190 
method of staining, 193 
Gall-bladder as a path of elimina- 
tion, 226 
Gas, natural, 84 
production of, 98 
Gaseous fermentation, 82 
Gaspard, 25 
Gelatin culture medium, 161-163 
liquefaction of, 92 
plates first used, 27 
General conditions for growth, 62 
infections, vaccines in, 261 
Generation, spontaneous, 17 
Generic names introduced, 30 
Genitalia, external, 227 
as channels of infection, 224 
Gentian violet, anilin, 18 
aqueous, 189 
German measles, 214 
Germination of spore, 45 
bipolar, 46 
equatorial, 45 
oblique, 45 
polar, 45 
Germs, disease, 23 
Gescheidel, 248 
Glanders, 215, 223, 224, 226, 227, 
253 
infectious, 24 
mallease, reaction in, 246 INDEX 
279 
Glands, mammary, 226 
salivary, 226 
Gleichen, 30 
Globulin in bacteria, 73 
Glycerin broth, 159 
Glycerinized potato, 166 
Gonococcus, 224 
Gonorrhea, 226, 227 
Good health, 269 
Grain rust, 25 
Gram-negative, 192 
positive, 192 
Gram's method of staining, 191 
solution, 192 
Granular, coarsely, edge, 208 
Granules, metachromatic, 42 
Neisser, 42 
polar, 42 
Grape juice, pasteurization of, 129, 
132 
Grass bacilli, 193 
Green plants, nitrogen nutrition of, 
107 
Griesinger, 26 
Group, agglutinating, 243 
haptophore, 238, 239, 243, 247, 
250 
precipitating, 246 
toxophore, 250 
zymophore, 239, 247, 250 
Groupings, cell, 52, 55 
Growth, appearance of, in identifi- 
cation, 200 
arborescent, 204 
beaded, 204 
filiform, 204 
forms, 51 
papillate, 204 
villous, 204 
Gruber, 242 
Gruber-Widal test, 244 
Gruby, 25 
H 
Haeckel, 256 
Hanging drop slide, 185 
Hansen, Emil Chr., 27 
Haptophore, complementophil, 250 
cytophil, 250 
group, 238, 239, 243, 247, 250 
Harness, etc., disinfection of, 155 
Health, good, 269 
Heat, 119-135 
dry, 121 
moist, 121 
production of, 104 
Heated serum, 248 
Hemagglutinin, 242 
Hemicellulose, 72 
Hemolysin, 249 
Hemolytic amboceptor, 254 
Hemorrhagic septicemias, 225 
Henle, 25, 215 
Hericourt, 264 
Herpes tonsurans, 25 
Hesseling, von, 29 
Heterologous, 252 
Heterotrophic, 75 
Hill, 30 
Hoffman, 23 
Hog cholera, 220, 226, 230 
erysipelas, 226 
Homologous, 253 
Hookworm disease, 26 
Host, 76 
Hot beds, 105 
Hydrochloric acid, 225 
Hydrogen peroxide as disinfectant, 
145, 150 
uses of, 78 
Hydrophobia, 227 
Hydrostatic pressure, 69 
Hypochlorites as disinfectant, 145 
I 
Ice-cream poisoning, 93 
Identificati on, appearance of growt h 
in, 200 
of blood, 246 
of meat, 246 
of milk, 246 
physiological activities in, 199 
Immunity, 228 
acquired, 230 
active, 230 
production of, 230-232 
antibacterial, 232 
antitoxic, 232 
artificial, 230 
cellular theory of, 234 
chemical theory of, 234 
definition of, 213 
Ehrlich's theory of, fundamental 
principles of, 235 280 
INDEX 
Immunity, Ehrlich's theory of, 
summary of, 237 
exhaustion theory of, 234 
inherited, 230 
natural, 230 
explanation of, 269 
noxious retention theory of, 234 
outlines of, 229 
passive, 230 
phagocytosis theory of, 234 
relative, 228 
serum-simultaneous method in, 
231 
side-chain theory of, 234 
summary of, 268 
theories of, 234 
to protein, 265, 267 
unfavorable environment theory 
of, 234 
Inactivated, 249 
Incubation, period of, 214 
Incubator, cold, 198 
Incubators, 196 
Index, chronological, 31 
opsonic, 257, 258 
phagocytic, 257 
Indicator, 254 
Indol, 93 
Infection, 214 
auto-, 216 
channels of, 222 
endogenous, 216 
exogenous, 216 
mixed, 216 
primary, 216 
secondary, 216 
wound, 25, 27, 215 
caused by bacteria, 27 
Infections, general, vaccines in, 261 
localized, vaccines in, 261 
Infectious diseases, 213 
Infective organisms, specificity of 
location of, 227 
Infestation, 214 
Infested, 214 
Influenza, 224, 226, 227 
Infusoria, 30 
Inherited immunity, 230 
Inoculation, 210 
by feeding, 211 
by inhalation, 211 
cutaneous, 211 
intracardiac, 211 
intramuscular, 211 
Inoculation, intra-ocular, 211 
intraperitoneal, 211 
intraspinal, 211 
intrathoracic, 211 
intravenous, 211 
needles, 176 
of culture medium, 170, 176 
subcutaneous, 210 
subdural, 211 
Inoculations, protective, first, 26 
Instruments, surgical, disinfection 
of, 153 
Intestinal discharges, 226 
Intestine, large, 227 
small, 227 
Intestines as channel of infection, 
225 
Intracardiac inoculation, 211 
Intramuscular inoculation, 211 
Intra-ocular inoculation, 211 
Intraperitoneal inoculation, 211 
Intraspinal inoculation, 211 
Intrathoracic inoculation, 211 
Intravenous inoculation, 211 
Invasion, 214 
Invertase, 112 
Involution forms, 51 
Iodine as disinfectant, 145 
Iron bacteria, 75, 78 
uses of, 78 
Isolation of bacteria, 178-183 
aids in, 182, 183 
J 
Jablot, 30 
Jack o'lanterns, 94 
Jar, Novy, 175 
Johne's disease, 225, 226 
K 
Kette, 29 
Kidneys as path of elimination, 226 
Kinase, 113 
Kircher, 18, 23 
Klebs, 27 
Klencke, 26 
Koch, Robert, 17, 27, 31, 215, 248 
Koch's postulates, 215 
Kra vs, 245 
Kruse, 231 
KtlCHENMEISTER, 26 INDEX 
281 
L 
Lacerate edge, 207 
Lactacidase, 113 
Lactic acid fermentation, 85-88 
Lancisi, 24 
Landois, 248 
Large intestine, 227 
Latour, 29 
Laveran, 24, 28 
Lecithin as complement, 251 
Leeuwenhoek, Anthony van, 18 
Legumes, 106 
Leidy, Joseph, 30 
Leprosy, 215, 223, 227 
Lesser, 30 
Lethal dose, minimum, 241 
Leukocytes, washing of, 257 
Lice as carriers, 220 
Liebert, 26 
Light, 65, 136 
production of, 99 
Linnaeus, 24 
Lipase, 112 
Liquefaction, crateriform, 205 
funnel-shaped, 205 
of gelatin, 92 
saccate, 205 
stratiform, 205 
Liquid manure, disinfection of, 155 
media, 158 
Lister, 26 
Litmus milk, 161 
Lobar pneumonia, 224 
Localized infections, vaccines in, 
261 
Location, specificity of, of infective 
organisms, 227 
Loeffler's blood serum, 166 
blue, 189 
Losch, 27 
Loop needles, 177 
Lopotrichic, 43 
Lung, apex of, 227 
Lungs, 227 
as channels of infection, 224 
Lysol as disinfectant, 148 
M 
McCoy, Vera, 148 
Macrococcus, 49 
Macroscopic agglutination, 242 
Malaria, 24, 214, 220, 227 
Malarial parasite, 28 
Mallease reaction in glanders, 240 
Mallein test, 267 
Malignant edema, 218, 222 
Malta fever, 245 
Mammary gland, 226 
Manure, liquid disinfection of, 155 
Margaropus annulatus, 220 
Martin, 29 
Mass cultures, 172 
Material for bacteriological exami- 
nation, 211 
Maximum conditions, 62 
Measles, 224, 226 
German, 214 
Measly pork, 26 
Measurement of bacteria, 37 
Meat broth, 159 
identification of, 246 
poisoning, 93 
Meatus, external auditory, as chan- 
nel of infection, 223 
Mechanical vibration, 69 
Media, liquid, 158 
selective, 182, 183 
solid, 158 
synthetic, 158 
Medium, agar, 163, 164 
blood serum, 166, 167 
culture, 157 
essentials of, 158 
inoculation of, 170, 176 
gelatin, 161-163 
large quantities of, 172 
potato, 164-166 
reaction of, 70 
synthetic, 167 
Meningitis, 223 
Meningococcus, 223 
Mercuric chloride as disinfectant, 
146 
Merismopedia, 54 
Metabiosis, 92 
Metabolism of bacteria, 75-81 
relative to man, 81 
Metachromatic granules, 42 
Metastases, 216 
Metatrophic, 75 
Metchnikoff, 234, 256 
Methods, antiseptic, 26 
of obtaining pure cultures, 178- 
183 
Microbiology, 213 282 
INDEX 
Micrococci, 226 
Micrococcus, 49, 59, 223, 224 
pyogenes aureus, 148 
Micromillimeter, 37 
Micron, 37 
Microscope, bacteriological, 184 
improvements in, 28 
invented, 18 
Microscopic agglutination, 242 
Microspira, 60 
comma, 63 
Microsporon furfur, 26 
Migula's classification, 59, 60 
Milk, 161 
blue, bacillus of, 27 
fermentation in, 29 
digestion of, 91 
glands as channel of infection, 
223 
identification of, 246 
litmus, 161 
pasteurization of, 129, 132, 133, 
134, 135 
purifier, electric, 139, 140 
souring of, 29 
tuberculous, 226 
Minimum conditions, 62 
lethal dose, 241 
Mixed infection, 216 
vaccine, 260 
Mixotrophic, 75 
M. L. D., 241 
Moist heat, 121 
Moisture, 63 
Molds, relation to bacteria, 34 
Monas, introduced, 30 
Monotrichic, 42 
Mordants, 188 
Morphology of bacteria, 39-55 
Mosquito theory of malaria, 24 
Motile bacteria, 42 
Motion, Brownian, 44 
Mouth cavity as channel of infec- 
tion, 223 
Movement, Brownian, 44 
rate of, in bacteria, 42 
M, 37 
Mu, 37 
Mucosa; as channels of infection, 
223 
Muller, 30 
Muntz, 30 
Mycoproteid, 72 
Myxomycetes, 36 
N 
Nageli, 30 
Names, generic, introduced, 30 
Nasal cavity as channel of infec- 
tion, 223 
discharges, 226 
Natural gas, 84 
immunity, 230 
explanation of, 269 
Needham, 19 
Needles, inoculation, 176 
loop, 17 
platinum, 176 
straight, 176 
Negative phase, 262, 263 
test, 255 
Neisser granules, 42 
Nephrolysin, 249 
Neufeld, 257 
Neurotoxin, 249 
Nichrome wire, 177 
Nitrate broth, 161 
Nitric bacteria, 103 
Nitrification, 103 
due to organisms, 30 
Nitrifying organisms, 30 
Nitrogen, absorption of, 105 
circulation of, 96 
nutrition of green plants, 107 
uses of, 78 
Nitrous bacteria, 102 
N on-pathogenic, 76 
Non-specific disease, 215 
Normal agglutinins, 243 
Nosema bombyds, 27 
Novy jar, 175 
Noxious retention theory of im- 
munity, 234 
Nuclein, 40 
in bacteria, 73 
Nucleus, 40 
Nutrition of green plants, nitrog- 
enous, 107 
Nuttal, 248 
O 
Obermeier, 27 
Objective, oil immersion, 184, 185 
Oblique germination of spore, 45 
Occurrence of bacteria, 51 
Oidium albicans, 25 INDEX 
283 
Oil immersion objective, 184, 185 
Opsonic index, 257, 258 
value of, 258 
power, 257 
Opsonin, 256 
Opsonins, 256-263, 268 
antibodies of second order, 257 
specificity of, 257 
Optimum conditions, 62 
Order, first, receptors of, 239 
second, receptors of, 242, 247 
third, receptors of, 248, 250 
Organisms, anaerobic, cultivation 
of, 172-176 
dissemination of, in the body, 225 
filterable, 216 
infective, specificity of location, 
.227 
nitrifying, 30 
pathogenic, elimination of, 226 
paths of entrance, 222 
ultramicroscopic, 215 
Organized ferments, 114 
Osmotic pressure, 68, 137 
Otitis media, 223 
Otto, 264 
Outlines of immunity, 229 
Overproduction activity of cells, 
235, 236 
Oxidation, 102 
Oxidizing enzymes, 113 
Oxygen, 66 
as disinfectant, 144 
compressed, 67 
pressure, 66, 67 
relations, determination of, 198 
uses of, 77 
Oznam, 24 
Ozone, 67, 138 
as disinfectant, 145 
P 
Pancreas, 226 
Papillate growth, 204 
Parasite, 76 
facultative, 76 
of malaria, 28 
strict, 76 
Partial amboceptor, 251 
Passive immunity, 230 
Pasteur, 21, 25, 26, 27, 28, 29, 231, 
234 
Pasteur flask, 22, 23 
treatment of rabies, 231 
Pasteur-Chamberland filter, 142 
Pasteurization, 127-135 
continuous, 129 
of beer, 129, 133 
of grape juice, 129, 132 
of milk, 129-132, 133, 134, 135 
of wine, 129 
Pathogenic, 76 
bacteria, definition of, 213 
outside body, 218 
bacteriology, reasons for study, 
217 
organisms, elimination of, 226 
paths of entrance, 222 
Paths of elimination, gall-bladder, 
226 
kidneys, 226 
saliva, 226 
urine, 226 
of entrance of pathogenic organ- 
isms, 222 
Pebrine, 26 
Peptone solution, Dunham's, 
161 
Period of incubation, 214 
Peritrichic, 43 
Peronospora infestans, 26 
Perty, 30 
Petri dishes, 164 
Petroleum, 84 
Pfeiffer, 248 
phenomenon, 248 
Phagocytes, 234 
Phagocytic index, 257 
Phagocytosis, 256-263, 268 
theory of immunity, 234 
Pharynx as channel of infection, 
223 
Phase, negative, 262, 263 
positive, 263 
Phenol as disinfectant, 147 
Phenomena, anaphylactic, 267 
Phenomenon, Arthus's, 264 
Pfeiffer's, 248 
Phosphate rock, 103 
Phosphine, 94 
Phosphorescence, 99 
Phosphorus, circulation of, 96 
uses of, 79 
Photogenesis, 99 
Physical agents for disinfection, 
119-143 284 
INDEX 
Physiological activities, definition 
of, 76 
use in identification, 199 
Physiology of bacteria, study of, 
196-209 
Pigment, production of, 100 
Pimples, 216, 222 
Piroplasma bigeminum, 215, 220 
Piroplasmoses, 220, 222 
Piroplasms, 227 
von Pirquet, 264 
Pityriasis versicolor, 26 
Plague, 224 
Planes of division, 53 
determination of, 54 
Pianococcus, 59 
Planosarcina, 59 
Plasmodiophora brassicoe, 27 
Plasmolysis, 39 
Plasmoptysis, 40 
Plate colonies, study of, 209 
cultures, 164, 172 
Plates, dilution, 178 
gelatin, first used, 27 
Platinum needles, 176 
Plectridium, 47 
Plenciz, 24, 28 
Plugs, cotton, 168 
Pneumococcus, 224 
Pneumonia, broncho-, 224 
foreign body, 224 
lobar, 224 
Pneumonias, 226 
Poisoning, cheese, 93 
ice-cream, 93 
meat, 93 
Polar germination of spore, 45 
granules, 42 
Poliomyelitis, 223 
POLLENDER, 26 
Polyvalent vaccine, 260 
Pork, measly, 26 
Position of bacteria, 34 
of flagella, 42, 43 
of spore, 46, 47 
Positive phase, 263 
test, 255 
Postulates, Koch's, 215 
Potato, glycerinized, 166 
medium, 164-166 
rot, 26 
Power, opsonic, 257 
Practical sterilization and disinfec- 
tion, 152-156 
Pragmidiothrix, 60 
Precipitatory group, 246 
Precipitin, 245 
Precipitinogen, 245 
Precipitins, 245-247 
anti-, 246 
compared with agglutinins, 245 
formation of, Ehrlich's theory, 
245 
uses of, 246 
Preparation of antitoxin, 240 
of bacterial vaccine, 259, 261 
of film, 190 
Preservative in vaccine, 260 
Pressure, hydrostatic, 69 
osmotic, 68, 137 
oxygen, 66, 67 
steam, 124 
Preventatives, stock vaccines as, 
260 
Prevost, 25 
Primary infection, 216 
Production of acid, 98 
of active immunity, 230-232 
of antibodies, place of, 269 
of aromatic compounds, 99 
of enzymes, 109 
of esters, 98 
of gas, 98 
of heat, 104 
of light, 99 
of pigment, 100 
of toxins, 114 
Prophylaxis, 264 
Protamin in bacteria, 73 
Protease, 112 
Protective inoculations, first, 28 
Protein immunity, 265, 267 
split products, 266 
Proteins, putrefaction of, 91-97 
Proteus, introduced, 30 
Protoplasm, 39 
Prototrophic, 75 
Protozoa related to bacteria, 37 
Pseudomonas, 59 
pyocyanea, 116, 242 
radidcola, 106 
Ptomaines, 92 
Puccinia graminis, 25 
Punctiform colony, 206 
Puncture cultures, 169 
Pure culture, 157 
methods of obtaining, 178-183 
Purin bases in bacteria, 73 INDEX 
285 
Putrefaction, 25, 91 
and fermentation, 28 
of proteins, 91-97 
Q 
Quick lime as disinfectant, 146 
Quinsy, 223 
R 
Rabies, 226 
Pasteur treatment of, 231 
Rabiger's method of staining, 194 
Radiations, 69 
Radium, 69 
Rashes, serum, 264 
Rate of division, 80 
of movement in bacteria, 42 
Rats as carriers, 220 
Rattlesnake, 252 
Rayer, 26 
Reaction, anaphylactic, use of, 268 
mallease, in glanders, 246 
of medium, 70 
surface, 81 
Reactions, biochemical, definition 
of, 76 
Reaumur, 30 
Receptor, 238 
Receptors, 235, 236 
free, 237 
of the first order, 239 
of the second order, 242, 247 
of the third order, 248, 250 
Recurrent fever, 27 
Red corpuscles, 227 
Redi, Francesco, 18 
Reducing actions, 101 
enzymes, 113 
Relapses, 216 
Relationships of bacteria, 30 
Renucci, 25 
Resistance of spores, 47 
Retarder, 131 
Rheumatism, 223 
Rhizoid colony, 206, 208 
edge, 208 
RhodobacteriaceoB, 60 
Richet, 264 
Ricin, 239 
Rimpau, 257 
Rindfleisch, 27 
Ringworm, 26 
Ripening of cheese, 29 
of cream, 86 
Robin, 239 
Rock, phosphate, 103 
Rods in blood, 26 
Rogers, 242 
Rooms, disinfection of, 153 
Root tubercles, 105 
Rosenau, 264 
Rot, potato, 26 
Roup, 223 
Roux, 28 
Rust, grain, 25 
S 
Saccate liquefaction, 205 
Saliva as path of elimination, 226 
Salivary glands, 226 
Salt rising bread, 84 
Saprogenic, 91 
Saprophilic, 92 
Saprophyte, 76 
Sarcina, 29, 54 
lutea, 67 
Sarcoptes scabiei, 25 
Sauer kraut, 87 
Scarlet fever, 224, 226 
Scavengers, bacteria as, 95 
Schick, 264 
Schistosomum hematobium, 26 
Schizomycetes, first used, 30 
Schonlein, 25 
Schosing and Muntz, 30 
Schroeder and Dusch, 20 
experiment, 20, 21 
Schultze, 20 
experiment, 20 
Schwann, 20, 29 
experiment, 20, 21 
Sea, bacteria in, 61 
Sealing air-tight, 19 
Second order, receptors of, 242, 247 
Secondary infection, 216 
Selective media, 182, 183 
Sensitization, 265 
Sensitized, 265 
vaccine, 232 
Septicemias, hemorrhagic, 225 
Serum, antitetanic, 240 
blood, necessity for diluting, 244 286 
INDEX 
Serum, diphtheritic, 240 
heated, 248 
rashes, 264 
sickness, 264, 267 
Serum-simultaneous method in im- 
munity, 231 
Serums, cytolytic, failure of, 252 
Shape of spore, 46 
Sickness, serum, 264, 267 
Side-chain theory of immunity, 234 
Side-chains, 236 
Silkworm disease, 25, 26 
Size of bacteria, 37 
Skatol, 93 
Skin as channel of infection, 222 
diseases, 222 
Slant cultures, 170 
Slide, cleaning of, 191 
hanging drop, 185 
Slope, cultures, 170 
Small intestine, 227 
Smallpox, 224, 226, 230, 231 
vaccine, 231 
Smith, Theobald, 264 
Snake venoms, anti-, 252 
Sneezing, 226 
Soap as disinfectant, 148 
Sodium hypochlorite as disinfec- 
tant, 146 
Soil, bacteria in, 61 
Soils, acid, 70 
Solid media, 158 
Solution, Gram's, 192 
stock, of stains, 189 
Sore throat, 223 
Sound, 69 
Sour mash distilling, 87 
Source of complement, 254 
Souring of milk, 29 
Spallanzani, 19 
Specific amboceptor, 254 
chemical stimuli of cells, 235 
disease, 215 
Specificity of agglutinins, 244 
of amboceptors, 250 
of location of infective organisms, 
227 
of opsonins, 257 
Spermotoxin, 249 
Spherotilus, 60 
Split products of protein, 266 
Splitting enzymes, 112 
of fats, 90 
Spirillaceoe, 60 
Spirilloses, 220, 222 
Spirillum, 50, 60 
Spirosoma, 60 
Spirochceta, 60 
Spirochaeta obermeieri, 27 
Spirochete, 50 
Spirochetes, 221 
Spoiling of canned goods, 48 
Spontaneous combustion, 104 
generation, 17 
Spore capsule, 45 
definition of, 48 
free, 45 
germination of, 45 
of anthrax, 27 
position of, 46, 47 
shape of, 46 
Spores, 44 
cause of spoiling of canned 
goods, 48 
conditions for formation of, 48 
first recognized, 30 
formation of, 45 
resistance of, 47 
staining of, 192 
two, in bacterium, 47 
Sprinkling filters, 104 
Stab cultures, 169 
Stables, disinfection of, 153-155 
Stain, anilin-fuchsin, 189 
gentian violet, 189 
aqueous gentian violet, 189 
carbol-fuchsin, 189 
Gabbet's blue, 190 
Loeffler's blue, 189 
Staining bottles, 190 
for cell forms, 195 
groupings, 194 
Gabbet's method, 193 
Gram's method, 191 
of acid-fast bacilli, 193 
of bacteria, 188-195 
of capsules, 194 
of flagella, 194 
of spores, 192 
Rabiger's method, 194 
reasons for, 188 
result of physiological activity, 
117 
Welch's method, 194 
Ziehl-Neelson method, 193 
Stains, stock solutions, 189 
Standard antitoxin, 241 
test dose, 241 INDEX 
287 
Standard toxin, 241 
Standardization of vaccines, 259, 
260 
Staphylococcus, 54 
Starin, W. A., 181 
Steam, 122 
and formaldehyde disinfection, 
156 
sterilizers, 122 
under pressure, 124 
Slegomyia, 221 
Sterile, 119 
Sterilization, 118 
and disinfection, practical, 152, 
156 
discontinuous, 121 
Sterilizers, steam, 122 
Stimuli, chemical, effect of, 236 
of cells, 235 
specific chemical, 235 
Stock cars, disinfection of, 155 
solutions of stains, 189 
vaccine, 260 
as preventatives, 260 
in treatment, 260 
Stomach as channel of infection, 
225 
Straight needles, 176 
Stratiform liquefaction, 205 
Streptobacillus, 52 
Streptococcus, 53, 59, 223, 224 ' 
Streptospirillum, 52 
Streplothrix bovis, 27 
Strict parasite, 76 
Structures, accidental, 40 
cell, 39 
essential, 39 
Study of bacteria, 157 
of bacterium, necessary steps for, 
158 
of forms, 30 
of pathogenic bacteriology, rea- 
sons for, 217 
of plate colonies, 209 
of physiology of bacteria, 196-209 
Subcutaneous inoculation, 210 
Subdural inoculation, 211 
Substances, cytolytic, 268 
Substrate, 111 
Sugar broth, 160 
Sulphur bacteria, 94 
circulation of, 97 
deposits of, 104 
uses of, 78 
Summary of Ehrlich's theory of 
immunity, 237 
of immunity, 268 
Surface reaction, 81 
Surgical instruments, disinfection 
of, 153 
Susceptibility, 216 
Symbionts, 76 
Symbiosis, 76 
Synthetic media, 158 
medium, 167 
Syphilitic antigen, 254 
Syphilis, 215, 226, 227, 253 
T 
Tapeworm, 26 
Temperature, 64 
Tenia solium, 26 
Test, complement-deviation, 253 
complement-fixation, 253-255 
dose, standard, 241 
for enzymes, 111 
for toxins, 115 
Gruber-Widal, 244 
mallein, 267 
negative, 255 
positive, 255 
tuberculin, 267 
Wassermann, 253 
Widal, 244 
T^pefiplp 997 
Tetanus', 214, 218, 222, 227 
Tetracoccus, 54 
T^pfporl ^4- 
Texas fever, 214, 215, 220 
Thaer, 29 
Theories of immunity, 234 
Theory, cellular, of immunity, 234 
chemical, of immunity, 234 
contagium vivum, 23, 24 
Ehrlich's, of formation of precipi- 
tins, 245 
of immunity, fundamental 
principles of, 235 
summary of, 237 
exhaustion, of immunity, 234 
for use of vaccines, 261 
mosquito, of malaria, 24 
noxious retention, of immunity, 
234 
of anaphylaxis, 266 
of formation of agglutinins, 242 288 
INDEX 
Theory, phagocytosis, of immu- 
nity, 234 
side-chain, of immunity, 234 
unfavorable environment, of im- 
munity, 234 
Thermal death-point, 65 
determination of, 198 
Thermophil, 65 
Thiobacteria, 60 
Thiothrix, 60 
Third order, receptors of, 248-250 
Thread, 52 
Thrush, 25, 223 
Ticks as carriers, 220 
Titration, 160 
Tonsillitis, 223 
Tonsils, 227 
as channels of infection, 223 
Touissant, 259 
Toxin, 227, 238 
final test for, 115 
standard, 241 
Toxins and enzymes compared, 114, 
115 
characteristics of, 114, 115 
other than bacterial, 115 
production of, 114 
true, 116 
Toxoid, 239 
Toxophore group, 250 
Tract, alimentary, as channel of 
infection, 225 
Traube, 248 
Treatment of rabies, Pasteur, 231 
stock vaccines in, 260 
Treponema pallidum, 224 
Trichinella spiralis, 26 
Trichinosis, 26 
Trichophyton tonsurans, 26 
Tropical dysentery, 27 
True toxins, 116 
Trypanosomes, 221 
Trypanosomiases, 220, 222 
Tubercle bacilli, chemical analysis 
of, 74 
Tubercles, root, 105 
Tuberculin test, 267 
Tuberculosis, 215, 223, 224, 225, 
226, 227 
due to bacteria, 28 
infectious nature of, 26 
Klencke's experiment, 26 
Villemin's experiment, 26 
Tuberculous milk, 226 
Tubes, culture, 168 
deep, 174 
fermentation, 168, 174 
Vignal, 173 
Two spores in bacterium, 47 
Tyndall, John, 23 
Tyndall's box, 22, 23 
Typhoid fever, 214, 226 
transmission by flies, 220 
U 
Ultramicroscopic organisms, 215 
Ultra-violet rays, 138 
Unfavorable environment theory 
of immunity, 234 
Unit, definition of, 241 
of antitoxin, 241 
Unorganized ferments, 114 
Unwashable articles, disinfection 
of, 155 
Urea, decomposition of, 95 
Urease, 113 
Urethral discharges, 226 
Urine as path of elimination, 226 
bacteria in, 62 
Use of agglutinins, 243, 244 
of anaphylactic reaction, 268 
Uses of carbon, 77 
of food, 75 
of hydrogen, 78 
of iron, 78 
of nitrogen, 78 
of oxygen, 77 
of phosphorus, 79 
of precipitins, 246 
of sulphur, 78 
of vaccines, theory of, 261 
V 
Vaccination, 231 
Vaccine, 231 
anthrax, 231 
autogenous, 260 
bacterial preparation of, 259, 261 
black-leg, 231 
mixed, 260 
polyvalent, 260 
preservative in, 260 
sensitized, 232 
smallpox, 231 INDEX 
289 
Vaccine, stock, 260 
Vaccines, bacterial, 258 
dosage of, 262 
failure of, 261 
in general infections, 261 
in localized infections, 261 
standardization of, 259, 260 
stock, as preventatives, 260 
in treatment, 260 
theory for use of, 261 
Vacuoles, 41 
Vaginal discharges, 226 
Value of opsonic index, 258 
Varo, 23 
Vaughan, 266 
Vehicles, disinfection of, 155 
Venoms, antisnake, 252 
Viborg, Eric, 24 
Vibration, mechanical, 69 
Vibrio, 50 
introduced, 30 
Vignal tubes, 173 
Villemin, 26 
Villous growth, 204 
Vinegar, 88 
Virulence, 216 
Virus, filterable, 216 
Vultures as carriers, 220 
W 
Wall, cell, 39 
composition of, 72 
Washable articles, disinfection of, 
155 
Washing of leukocytes, 257 
Wassermann test, 253 
Water, bacteria in, 61 
filtration, 141 
Webb and Barber, 231 
Weigert, 28, 235 
Welch's method of staining, 194 
Whooping-cough, 224 
Widal, 242 
test, 244 
Widal-Gruber test, 244 
Will o' the wisp, 94 
Wine, pasteurization of, 129 
Winogradsky, 30, 60 
Wire baskets, 168 
nicrome, 177 
Wollstein, 24 
Woronin, 27 
Wound infection, 25, 27, 215 
caused by bacteria, 27 
Wright, 256 
X 
I 
X-RAYS, 69 
Y 
Yeast, 29 
relation to bacteria, 34 
Yellow fever, 221 
Z 
Zansz, Hans and Zacharias, 18 
Zenker, 26 
Ziehl-Neelson method of staining, 
193 
Zooglaea, 41 
Zootoxins, 116 
Zymase, 113 
Zymases, 113 
Zymogen, 113 
Zymophore group, 239, 247, 250