PRINCIPLES 
OF 
BACTERIOLOGY 
BY 
ARTHUR A. EISENBERG, A.B., M.D., 
Director of Laboratories, St. John's Hospital; Pathologist to Lake- 
wood Hospital; Serologist to St. Ann's Hospital, Cleveland, 
Ohio; Director of Laboratories, Mercy Hospital 
(Canton, Ohio), Member Society of 
American Bacteriologists. 
THIRD EDITION 
ST. LOUIS 
THE C. V. MOSBY COMPANY 
1924 Copyright, 1918, 1923, 1924, By The C. V. Mosby Company 
(Printed in U. 8. A.) 
Press of 
The C. V. Mosby Company 
St. Louis, Mo. TO MY PARENTS 
Whose endless sacrifices have made my education pos- 
sible, and whose boundless love has made this a glorious 
world to live in, this book is dedicated as a token of 
undying affection and everlasting gratitude, by their son, 
THE AUTHOR PREFACE TO THIRD EDITION 
Continued favorable reception of the book by teachers 
of various training schools for nurses, necessitated a 
new edition within ten months after the appearance of 
the second. This flattering approval, while highly grat- 
ifying, has compelled me to subject the book to a most 
thorough scrutiny, in order to make it worthy of the 
favor which has been accorded it, and numerous addi- 
tions, alterations and corrections have been made 
throughout the entire text. It is earnestly hoped that 
these changes will enhance the usefulness of the book 
and will secure for it new friends as well as retain the 
old ones. 
I take great pleasure in expressing my gratitude to 
the reviewers, teachers and personal friends who kindly 
offered criticisms and suggestions-they have been very 
useful and valuable; I particularly wish to acknowledge 
my great obligation and offer my sincerest thanks to 
Dr. Kenneth L. Burdon, Instructor of Bacteriology, of 
the Washington University School of Medicine, who has 
given most generously of his time to go over the entire 
book, and whose constructive and searching criticism 
has greatly added to the value of the new edition. 
Among the most important changes to be found in this 
new edition are: 
1. Numerous alterations and corrections-these, as I 
have mentioned before-are due to Dr. Burdon's great 
kindness. 
2. A short chapter on "The Filtrable Viruses" which 
will be found to precede the discussion of the individual 
5 6 
PREFACE 
diseases grouped under the heading of "The Diseases of 
Unknown Causation. ' ' 
3. A clearer discussion of whether syphilis is an hered- 
itary or a congenital disease; a brief statement of the 
present status of our knowledge of the cause of influ- 
enza pandemic; a brief mention of Noguchi's work on 
infantile paralysis and yellow fever. 
It is my earnest hope that the new edition will meet 
with the approval of both the teachers and the students 
for whom this book is intended. 
Cleveland, Ohio. 
Arthur A. Eisenberg. 
PREFACE TO SECOND EDITION 
In preparing the second edition I have availed myself 
of the opportunity to correct whatever errors were made 
previously, and to follow the various suggestions kindly 
made by the author's friends, reviewers and others. 
The fundamental principles and the general plan of 
the book have been found by the teachers and the pupils 
alike to be quite satisfactory, especially the general sum- 
mary at the end of each chapter, and for this reason, 
the new edition contains additions rather than altera- 
tions. 
The new subject matter now includes: 
1. Additional information about the constancy and 
the mutation of bacteria. 
2. Discussion of the D'Herelle's phenomenon-one 
of the most important contributions since the days of 
Ehrlich and Bordet. 
3. Description of some of the newer technical pro- 
cedures, such as Paltouf's Modification of the Gram's PREFACE 
7 
method of staining, Pappenheim's method, and some cul- 
ture media. 
4. Thorough description of the rationale and the 
underlying principles of the Wassermann test. 
5. Description of the newer precipitation and floc- 
culation tests for the diagnosis of syphilis-Meinicke, 
Sachs-Georgi, Kahn and Bold tests. 
6. Discussion of the new-colorimetric-method of ti- 
trating culture media. 
7. Detailed description of taking blood cultures with 
the Keidel blood culture medium tubes. 
8. Detailed description of the pre-transfusion blood 
tests, including the discussion of blood typing of Jansky 
and Moss. 
9. More information about anaphylaxis, Besredka's 
desensitization methods, and the application of anaphy- 
laxis to the diagnosis of hay fever and bronchial asthma. 
10. The chapter on Influenza has been rewritten, 
and a working classification of streptococci has been 
added. 
11. The relation of leucocytes to infections. 
Arthur A. Eisenberg. 
Cleveland, Ohio. 
PREFACE TO FIRST EDITION. 
While lecturing to the nurses at the St. Vincent's Char- 
ity and St. John's Hospitals, of Cleveland, I have been 
frequently compelled to either modify or add new sub- 
ject matter to that contained in various textbooks of 
Bacteriology for Nurses, or face a very unpleasant sit- 
uation of being told that "we can't find in our book 
what you have told us," This could be remedied, of 8 
PREFACE 
course, to some extent, by referring the pupils to stand- 
ard books written for medical students, but, in the 
first place, the multiplicity of textbooks is hardly desir- 
able in teaching an elementary subject, and, in the 
second place, the information sought would usually 
be found intimately bound with much of what is de- 
cidedly "above their heads" and written in too technical 
a language to be readily accessible. 
I have felt, therefore, that it may be worth while to 
prepare a textbook which represents, with additions, my 
syllabus of lectures delivered at the above-mentioned 
training schools, and which, while prepared for the 
nurses and written in a very simple language, would be 
fairly complete and would incorporate the latest facts 
of Bacteriology-such facts as are recognized to be 
safely out of the zone of polemics and controversies; 
where the work described does not, as yet, warrant being 
regarded as an established fact mention is made to that 
effect. 
There are several features to which I have paid special 
attention, and the book is, perhaps, original to some ex- 
tent from the standpoint of the nature of the subject 
matter included as well as its arrangement. 
We begin to realize that it is while studying bacteriol- 
ogy that the rationale and the principles of bacterial 
prophylaxis are first brought to the nurse's attention, 
yet but very little information of this nature is usually 
given and but very little stress is laid on the connection 
between bacteriology and prophylaxis; it seemed to me 
that this would be a very appropriate place and a very 
opportune time to "drive home" the lessons, and for 
this reason, I have introduced in each chapter dealing 
with individual microorganisms, a section-"Mode of 
Infection, Disinfection and Prophylaxis"-as regards 9 
PREFACE 
the disease caused by particular microorganisms, giv- 
ing explicit instructions as regards the patient and those 
who mingle with him, including the nurse, the room and 
its contents. 
With the present need for more and more nurses, 
both here and "over there," a demand for nurses capa- 
ble of serving as laboratory assistants and technicians 
will be felt, and for this reason I have gone into the 
minute description of the simpler technical procedures, 
giving complete and detailed directions, taking nothing 
for granted and leaving nothing to one's imagination. 
For this reason I have laid much stress on cultural 
diagnosis giving much prominence to the several culture 
media used for the differentiation of the various mem- 
bers of the typhoid-colon-dysentery group. In each chap- 
ter a section is provided on Bacteriologic Diagnosis, 
under which all tests called for in connection with the 
particular disease are enumerated and complete technic 
is given, usually describing but one method-the one 
which has proved most serviceable in my hands. 
Sections on Immunotherapy of individual diseases are 
provided in each chapter wherever called for, and at 
the end of each chapter a summary of the most important 
characteristics is given. 
Since the bacteriology is usually taught to the first 
year pupils, the lecturer on bacteriology usually has to 
refer to other allied subjects, especially physiology and 
histology; I have endeavored to make such references as 
clear as I knew how by giving the full description and 
derivation of the terms used. 
A complete questionnaire is provided at the end of the 
book to facilitate the review of the subject. 
Numerous textbooks, monographs and magazine articles 
have been consulted-too numerous to be mentioned in 10 
PREFACE 
their entirety, but whenever special references were made, 
I have, at all times, endeavored to give full credit where 
it is due. 
In the section on history, I have briefly reviewed the 
most prominent achievements of the American bacteriolo- 
gists. 
I have tried to incorporate as much of the newer con- 
tributions to the theory and practice of bacteriology and 
serology as possible, including the problems of carriers, 
Schick's test, Plotz's work on the etiology of typhus, the 
work of Cole and his coworkers on pneumonia, the prom- 
ising work of Bull on the serum therapy of the gas bacil- 
lus infection, the complement-fixation test for tuberculo- 
sis, etc. 
My sincere thanks are due to my many friends for 
various favors conferred upon me in connection with 
the preparation of this work, and it affords me great 
pleasure to express my gratitude to Mr. Richard Fluent 
for the excellent photographs and to Mr. Daniel E. 
Quilter for his valuable aid in the preparation of the chap- 
ters on the 11 Bacteria in the Soil, Air, Water, and Milk." 
Mr. S. Coopersmith was very kind in arranging the 
different apparatus for the preparation of the photo- 
graphs. 
My sincere thanks are due to my very good friend, 
Dr. James E. Hallisy, for his kind help in the prepara- 
tion of the index as well as the reading of the final proof. 
Cleveland, Ohio. 
Arthur A. Eisenberg. CONTENTS 
SECTION I 
GENERAL BACTERIOLOGY 
CHAPTER 1 
PAGE 
Historical 19 
CHAPTER II 
General Information About Bacteria 24 
Bacterial Forms, 24; The Structure of the Bacterial Cell, 
25; Bacterial Reproduction, 29; Constancy of Type, 29; 
The Chemical and Physical Properties of the Bacterial 
Cell, 30; Nutrition of Bacteria, 31; Aerobes and Anaer- 
obes, 32; Longevity of Bacteria, 32; Parasites and Sapro- 
phytes, 32; Mutual Relations of Bacteria, 33; Relations 
of Bacteria to Physical Environment. 33; The Biological 
Activities of Bacteria, 35; The Distribution of Bacteria 
in the Animal Body, 38; Bacilli Carriers, 39. 
CHAPTER III 
The Destruction of Bacteria 40 
Bacterial Destruction by Physical Agents, 41; Bacterial 
Destruction by Chemical Agents, 45; Practical Disinfec- 
tion, 48. 
CHAPTER IV 
The Struggle Between Bacteria and the Body Infection 
and Immunity 52 
Infection, 52; The Infection Proper-Bacterial Poisons, 
54; Immunity, 56; Protective Substance or Immune 
Bodies, 60; Application of Hemolysis and Agglutination 
to Blood Transfusion Tests, 64; Theories of Immunity, 
66; D'Herelle's Phenomenon, 75; Anaphylaxis, 76; The 
Relation of Leucocytes to Infection, 78. 
CHAPTER V 
The Study of Bacteria and General Bacteriologic Technic 82 
The Study of Bacteria in Living State, 82'; Study of Bac- 
11 12 
PAGE 
teria in Stained Preparation, 83; Plating and Anaerobic 
Cultures, 92; Culture Media, 92; Actual Preparation of 
Various Culture Media, 102. 
CONTENTS 
Applied Bacteriology 113 
Examination of Material from Patients, 113. 
CHAPTER VI 
SECTION II 
SPECIAL BACTERIOLOGY 
CHAPTER VII 
The Staphylococcus Group 119 
Historical, 119; Morphology and Staining, 119; Cultural 
Characteristics, 119; Destruction, 120; Disease-Producing 
Properties, Mode of Infection, Disinfection and Prophy- 
laxis, 120; Infection and Immunity, 121; The Varieties 
of Staphylococci, 121; Bacteriologic Diagnosis, 122; Im- 
munity Treatment, 122; The Summary of Staphylococcus 
group, 124. 
CHAPTER VIII 
The Streptococcus Group 125 
Historical, 125; Morphology, 125; Cultural Characteristics, 
125; Destruction, 126; Disease Production, Mode of In- 
fection, Disinfection and Prophylaxis, 127; Mechanism 
of Infection and Immunity, 127; Bacteriologic Diagno- 
sis, 127; Immune Treatment, 128; Classification of 
Streptococci, 128; Summary of Important Characteristics, 
128. 
CHAPTER IX 
The Pneumococcus Group (Diplococcus Pneumoniae, Dip- 
lococcus, Lanceo-Latus-"Lance-Su aped") . . 129 
Historical, 129; Distribution, 129; Morphology, 129; Cul- 
tural Characteristics, 130; Destruction, 130; Disease Pro- 
duction, Mode of Infection, Disinfection and Prophylaxis, 
131; Mechanism of Infection and Immunity, 131; Bacte- 
riologic Diagnosis, 131; The Different Types or Strains 
of Pneumococci, 132; Immune Treatment, 133; Sum- 
mary of Important Characteristics, 133. CONTENTS 
13 
CHAPTER X 
PAGE 
The Meningococcus and Parameningococcus Group (Mi- 
crococcus Intracellularis Meningitidis . . 134 
Historical, 134; Morphology, 134; Cultural Characteris- 
tics, 134; Destruction, 135; Disease Production, Mode of 
Infection, Disinfection and Prophylaxis, 135; Mechanism 
of Infection and Immunity, 136; Bacteriologic Diagnosis, 
136; Immune Treatment, 136; Summary of Important 
Characteristics, 136. 
CHAPTER XI 
Gonococcus 138 
Historical, 138; Morphology, 138; Cultural Characteris- 
tics, 138; Destruction, 138; Disease Production, Mode of 
Infection, Disinfection and Prophylaxis, 139; Mechanism 
of Infection and Immunity, 140; Bacteriologic Diagnosis, 
140; Immune Treatment, 140; Summary of Important 
Characteristics, 140. 
Micrococcus Catarrhalis and Gram-negative Cocci . . 141 
Micrococcus Catarrhalis, 141; Gram-negative Cocci, 141. 
CHAPTER XII 
CHAPTER XIII 
The Colon-Typhoid-Dysentery Group B. Coli Communis . 142 
Historical, 142; Morphology, 142; Cultural Characteris- 
tics, 142; Destruction, 143; Disease Production, Mode of 
Infection, Disinfection and Prophylaxis, 143; Mechanism 
of Infection and Immunity, 144; Bacteriologic Diagnosis, 
144; Immune Treatment, 145; Summary of Important 
Characteristics, 145. 
CHAPTER XIV 
Bacillus Typhosus 146 
Historical, 146; Morphology, 146; Cultural Characteris- 
tics, 147; Destruction, 147; Disease Production, Mode of 
Infection, Disinfection and Prophylaxis, 148; Mechanism 
of Infection and Immunity, 149; Bacteriologic Diagnosis, 
151; Immune Treatment, 154; Summary of Important 
Characteristics, 155. 14 
CONTENTS 
CHAPTER XV 
। PAGE 
The B. Paratyphosus A and B. The B. Fecalis Alcali- 
genes. The B. Proteus. Dysentery Bacilli . 156 
B. Paratyphosus A and B, 156; B. Fecalis Alcaligenes, 
156; Bacillus Proteus, 156; The Dysentery Bacilli, 157. 
CHAPTER XVI 
Bacillus Mucosus Capsulatus (Friedlander's or Pneumo- 
Bacillus) 158 
CHAPTER XVII 
The Tetanus Bacillus (Lockjaw) 159 
Historical, 159; Morphology, 159; Cultural Characteris- 
tics, 159; Destruction, 159; Disease Production, Mode of 
Infection, Disinfection and Prophylaxis, 160; Mechanism 
of Infection and Immunity, 160; Bacteriologic Diagno- 
sis, 161; Immune Treatment, 161; Summary of Impor- 
tant Characteristics, 161. 
CHAPTER XVIII 
The Anthrax Bacillus. Bacillus Aerogenes Capsulatus. 
Bacillus of Malignant Edema 162 
The Anthrax Bacillus, 162; Bacillus Aerogenes Capsula- 
tus, 163; Bacillus of Malignant Edema, 164. 
CHAPTER XIX 
Bacillus Botulinus. Bacillus Mallei. Bacillus Pyocy- 
aneus 166 
Bacillus Botulinus, 166; Bacillus Mallei, 166; Bacillus 
Pyocyaneus, 166. CONTENTS 
15 
PAGE 
The Diphtheria Group 168 
Historical, 168; Morphology, 168; Cultural Characteris- 
tics, 168; Resistance, 169; Disease Production, Mode of 
Infection and Prophylaxis, 169; Mechanism of Infection 
and Immunity, 171; Bacteriologic Diagnosis, 171; Immu- 
nity Treatment, 172; Summary of Important Character- 
istics, 172. 
CHAPTER XX 
CHAPTER XXI 
Bacillus Tuberculosis 173 
Historical, 173; Morphology, 173; Cultural Characteris- 
tics, 173; Destruction, 174; Disease Production, Mode of 
Infection, Disinfection and Prophylaxis, 174; Mechanism 
of Infection and Immunity, 175; Bacteriologic Diagno- 
sis, 175; Immune Treatment, 175; Summary of Impor- 
tant Characteristics, 176. 
The Bacillus of Leprosy. The Smegma Bacillus . . . 177 
The Bacillus of Leprosy, 177; Smegma Bacillus, 178. 
CHAPTER XXII 
CHAPTER XXIII 
The Influenza Group. The Bacillus Pertussis. Bacillus 
Pestis. The Cholera Group. The Bacillus of 
Ducrey 179 
The Influenza Group, 179; The Bacillus Pertussis, 180; 
Bacillus Pestis, 180; The Cholera Group, 182; The Bacil- 
lus Ducrey, 183. 
CHAPTER XXIV 
The Spirochetal Diseases 184 
Syphilis, 184; Relapsing Fever, 189; Vincent's Angina, 
189; Yaws, or Frambesia, 189. 16 
CONTENTS 
PAGE 
Malaria. The Typhus Fever 190 
Malaria, 190; Typhus Fever, 191. 
CHAPTER XXV 
CHAPTER XXVI 
The Higher Bacteria. The Yeasts. The Molds . . . 192 
The Higher Bacteria, 192; Streptothrix, 192; Actinomy- 
cosis, 192; The Yeasts, 192; The Molds, 193. 
SECTION III 
CHAPTER XXVII 
Filterable Viruses. Diseases of Unknown Causation . 194 
Smallpox, 195; Measles, 195; Mumps, 195; Scarlet Fever, 
195; Trachoma, 196; Infantile Paralysis, 196; Yellow 
Fever, 196; Dengue, 197; Rocky Mountain Spotted Fever, 
197; Foot and Mouth Diseases, 197; Hydrophobia, or 
Rabies, 197. 
CHAPTER XXVIII 
Bacteria in Soil, Air, Water, and Milk 199 
Microorganisms of the Soil, 199; Microorganisms of the 
Water, 200; Bacteria of the Air, 203; Bacteria of Milk, 
204. 
General Care of the Laboratory 205 
CHAPTER XXIX 
List of Questions 206 
CHAPTER XXX ILLUSTRATIONS 
FIG. PAGE 
1. Bacilli, cocci, and spirilla 25 
2. Pneumococci, showing capsules 26 
3. Typhoid bacilli, showing flagella 27 
4. Tetanus bacilli, showing spores 28 
5. Hot-air sterilizer 42 
6. The Arnold sterilizer 43 
7. The autoclave 45 
8. Instrument employed in applying Dakin's solution to 
wounds 50 
9. Ehrlich's theory of immunity 68 
10. Side-chains, with bacterial poison attached, cast off free 
into circulation 69 
11. Illustration of Ehrlich's theory in case of diphtheria . 70 
12. Ehrlich's conception of a toxin molecule 72 
13. The receptor of second order (agglutinins and precipitins) 73 
14. The receptor of third order (bacteriolysis, hemolysin) . 74 
15. Apparatus for staining 83 
16. Microscope and artificial illumination 85 
17. Miscellaneous glassware 92 
18. Making a transfer of a culture 94 
19. Tubing the media 101 
20. Two types of water baths Ill 
21 A. The incubator 114 
21B. Special tube for obtaining blood for Wassermann . . 116 
22. Staphylococcus 120 
23. Staphylococcus pyogenes aureus 121 
24. Streptococcus pyogenes 126 
25. The meningococcus 135 
26. The gonococcus 139 
27. Bacillus coli 143 
28. Bacillus typhosus 146 
29. Dysentery bacilli 157 
30. Bacillus welchii 163 
17 18 
ILLUSTRATIONS 
31. The anthrax bacilli showing spores 164 
32. Diphtheria bacilli 169 
33. Pseudodiphtheria bacilli 171 
34. Tubercle bacilli, human 174 
35. Leprosy. Section of skin 177 
36. The smegma bacillus 178 
37. Influenza bacilli 181 
38. Bacillus pestis 181 
39. Spirillum cholerae 182 
40. Various types of spirochetae 188 PRINCIPLES OF BACTERIOLOGY 
SECTION I 
GENERAL BACTERIOLOGY 
CHAPTER I 
HISTORICAL 
While the existence of minute organisms and their 
causal relation to some diseases may have been suspected 
long before the actual proof came-thus the conception 
of contagion, i. e., transmission of a disease from man 
to man, is mentioned by Aristotle, Pliny, and also by some 
mediaeval scientists, it was but natural that their demon- 
stration should follow, above all things, the construction 
and improvement of magnifying instruments. The honor 
of having been first to actually see and describe living 
organisms, too small to be visible to the naked eye, be- 
longs to a Jesuit priest, Kircher (1659), who was fol- 
lowed, a few years later, by the Dutch linen-draper, Leeu- 
wenhoek (1675). There is very little doubt, however, 
that by far the greater number of the 1 ' animalcules ' ' seen 
by these observers were not bacteria but various protozoa 
(from Greek protos = first, and zoon = animal, the 
lowest form of animal life), as most of their studies were 
made on water. 
The period intervening between the end of the seven- 
19 20 
PRINCIPLES OF BACTERIOLOGY 
teenth and the beginning of the nineteenth century did 
not bring forth any startling discoveries, although some 
interesting observations were made by Plenciz, who ad- 
vanced the opinion that each infectious disease was due 
to a specific microorganism, by Otto Muller and Ehren- 
berg, the latter having contributed the classification which 
in its main points, is tenable even today. 
The next real advance came in 1838 when Cagniard- 
Latour, a physicist, showed the living nature of yeasts, 
found in fermenting substances, a fact corroborated by 
Schwann; this observation gave rise to much discussion 
and argument as the general opinion of the nature of 
fermentation at that time was that it was due to the 
decomposition of the protein matter, a process held to be 
essentially a chemical one. This new conception of fer- 
mentation was not, however, definitely accepted until 
Pasteur, the great French scientist, made known his class- 
ical studies on the fermentation in wine and beer. 
At this time the mooted and extremely important 
question was that of "spontaneous generation"-were 
the "animalcules" seen by Kircher and Leeuwenhoek and 
other microorganisms produced by others of their own 
kind or could they be produced from different "things," 
as, for example, mice were produced, according to Pliny, 
from dirty linen ? 
Supported by Buffon, Needham, in 1850, claimed that 
the theory of spontaneous generation was correct, as he 
could demonstrate that pieces of putrefying material, 
sealed in flasks, were found, a few days later, to contain 
enormous numbers of microorganisms; his experiments 
were repeated by an Italian investigator, Spallanzani, 
who having subjected the flasks to considerable heat, could 
not verify Needham's results. Then came the experi- 
ments of Schwann, Schultze, Schroeder and Dusch, who GENERAL BACTERIOLOGY 
21 
did not find any bacteria in infusions which had been 
boiled, but still many investigators clung to the "spon- 
taneous generation" until Pasteur did away with it for 
once and all. 
Pasteur showed that filtering air through cotton wool 
resulted in depositing enormous numbers of microorgan- 
isms, and when a single shred of such filter was placed 
in a sterile fluid, the latter would soon be teeming with 
bacteria, whereas, if entrance of air into these sterile 
fluids was prevented such fluids would remain sterile. The 
theory of spontaneous generation then received its death 
blow but for one detail-it could not be explained why 
the application of the same degree of heat did not always 
result in complete sterility; this last fact remained un- 
explained until 1870, when Cohn brought to light the ex- 
istence of bacterial spores and their very high powers of 
resistance to heat. 
Pasteur now turned his attention to the problem of 
fermentation and not only confirmed the earlier opinion 
of Cagniard-Latour and others, but pointed out a number 
of other fermentations, such as those of lactic acid, the 
decomposition of organic matter by putrefaction, etc. 
The dependence of the latter upon living agents suggested 
to the great English surgeon, Lister, the idea that the 
suppuration of infected wounds was a process identical 
with that of putrefaction, and working along the lines in- 
dicated by Pasteur's work, Lister introduced his anti- 
septic and aseptic methods and thus achieved immortal 
fame of having rendered possible modern surgery. 
And now we enter upon the era of discoveries of indi- 
vidual causative agents of various infectious diseases. 
In 1863, Davaine not only confirmed the earlier obser- 
vations of Brauell and Pollender (1855) on the anthrax 
bacillus, but proved that this disease could always be 22 
PRINCIPLES OF BACTERIOLOGY 
transmitted by injecting the blood containing these rod- 
shaped bacilli into other animals. In 1868, Obermeier 
discovered the spirillum (corkscrew-like bacterium) 
causing the relapsing fever. 
The discovery of the pus-producing bacteria was dem- 
onstrated by Koch, Pasteur, Ogston, Rosenbach, and 
others, about 1882. 
In the same year Koch made his epoch-marking dis- 
covery of the bacillus of tuberculosis; Koch has also in- 
troduced numerous technical procedures which have 
made possible the modern bacteriology. Very many 
pathogenic (from Greek pathos = disease, geunao = to 
give birth to, disease producing) bacteria were discovered 
in the succeeding years, until another epoch-marking 
discovery was made, that of Schaudinn and Hoffmann, 
in 1905, of the Spirocheta pallida-the organism caus- 
ing syphilis, one of the most ravaging diseases. 
Within the last thirty years a new branch of bacter- 
iology-the science of immunity (resistance to disease) 
or immunology has been created, chiefly through the 
work of Pasteur, Metchnikoff, Roux, Ehrlich, Behring, 
Bordet, and others. 
Our own country has contributed much to the science 
of bacteriology and immunology through the brilliant 
work of such men as Anderson and Rosenau (the work 
on anaphylaxis-increased susceptibility to disease, 
standardization of the diphtheria and the tetanus anti- 
toxins, etc.) ; Noguchi (the first successful cultivation 
of Spirocheta pallida, Wassermann test modification, 
etc.) ; Cole and his associates (the work on the differen- 
tiation of the various types of the pneumococcus and the 
successful serum treatment of pneumonia caused by 
Type I) ; Flexner (the antimeningitic serum) ; Russell 
(typhoid vaccination in the United States) ; Rosenow, 23 
GENERAL BACTERIOLOGY 
Hektoen, Vaughan (the work on toxins); Park, Plotz 
(the discovery of the organisms causing the typhus 
fever) ; Sewell (the work on the snake venom) ; Novy, 
Welch (the discovery of the gas bacillus) ; Gen. Stern- 
berg (the discovery of the pneumococcus); Bull (who is 
now doing a most promising work on the serum treat- 
ment of gas bacillus infection), and scores of others. CHAPTER II 
GENERAL INFORMATION ABOUT BACTERIA 
Bacteria belong to the vegetable kingdom, being minute 
unicelhdar organisms. 
Normally bacteria are of three different kinds so far 
as their form is concerned: 
1. Cocci (plural from Greek coccos, meaning kernel), 
which are round or oval; these usually occur either in 
clusters or pairs, being called diplococci (meaning double 
cocci) in the latter case. 
2. Bacilli (plural from Latin 'bacillus, meaning "a little 
rod"), which are straight, rod-shaped organisms, whose 
ends may be square or convex (more frequently) or con- 
cave (less frequently). 
3. Spirilla (plural from Latin spirillum, meaning a 
coil) which are curved or comma-shaped organisms (see 
Fig. 1). 
Of these three varieties the bacilli are by far the most 
frequent. 
The Size of Bacteria.-The unit of bacterial measure- 
ment is a micron (a Greek word meaning small) ; it is a 
thousandth part of a millimeter or 1/25000 of an inch, 
and is usually represented by the Greek letter y. Cocci 
usually vary from .1 /i to 2 in diameter, the average 
size being about .1 p in diameter. Bacilli vary from 1 
p to 3 or 4 p, some being very large, as, e. g., the an- 
I. Bacterial Forms 
24 GENERAL BACTERIOLOGY 
25 
thrax bacillus, which is from 5 to 10 /z long. The spiril- 
la vary greatly in size. 
The weight of bacteria is, of course, extremely light, 
the average being about 0.000000001 milligram, that is to 
say that sixteen hundred million bacilli weigh approxi- 
mately one milligram; a normal red blood cell is fifty 
thousand times as heavy as a single colon bacillus. 
II. The Structure of the Bacterial Cell 
The bacterial cell has an external membrane that is 
rigid and maintains the shape of the cell and is called 
ectoplasm (from the Greek ecton, meaning without, 
and plasma, meaning a formed thing). 
A B c 
Fig. 1.-A, bacilli; B, cocci; C, spirilla. 
The thickness of this cell wall varies, being thicker in 
the old than in the young organisms; special stains are 
required for the demonstration of the ectoplasm. 
The substance which comprises the interior of the bac- 
terial cell is called the endoplasm (from Greek endon, 
meaning within), and plasma, or cytoplasm (meaning cell 
substance) ; this is a clear, colorless, highly refractile sub- 
stance. 'Whether or not there is a nucleus (kernel) in the 
bacterial cell is not yet definitely settled, although the 
majority of bacteriologists are inclined to believe there 
is at least a nuclear substance if not a definite nucleus. 
Certain bacteria, notably the diphtheria group, exhibit 
one or more granules which are scattered irregularly in 26 
PRINCIPLES OF BACTERIOLOGY 
the endoplasm; they are called Ernst-Babes' or metachro- 
matic (from Greek meaning changing color) granules; 
nothing definite is known as to their significance. 
Capsules.-Quite a few bacteria are surrounded by an 
envelope or a halo which is called "capsule" (from Latin 
capsula, meaning a little box) ; the capsules are most fre- 
quently present in the organisms (e. g., pneumococcus) 
when they are first isolated from animal tissues or if 
Fig. 2.--Pneumococci, showing capsules. (Mallory and Wright-Pathological 
T echnic.) 
grown on substances rich in albuminous ingredients, and 
are frequently lost when bacteria are kept in the labo- 
ratory or if grown on ordinary culture media. As 
to the significance of the capsule, the most accepted opin- 
ion is that the capsule renders the bacteria more resistant 
against destruction and thereby more dangerous (virulent, 
from Latin vis, meaning power) ; the statement that pneu- 
mococcus has an especially well defined capsule when 
freshly isolated from animal tissues and is apt to lose it GENERAL BACTERIOLOGY 
27 
when artificially grown, certainly corroborates this view, 
as pneumococcus meets more resistance, when in the an- 
imal body, than when in a culture tube. With the ex- 
ception of pneumococcus mucosus capsulatus (pneumoeoc- 
Fig. 3.-Typhoid bacilli, showing flagella. (Mallory and Wright-Patho- 
logical Technic.') 
cus, Type III) bacteria have to be stained by special 
methods in order to demonstrate their capsules; this is 
a very important procedure in making bacteriologic diag- 
nosis in the case of pneumococcus, gas bacilli, etc. 28 
PRINCIPLES OF BACTERIOLOGY 
Flagella (from Latin flagellum, meaning whip).-All 
minute objects suspended in fluids are found to be in con- 
stant motion; this, however, is not true locomotion, but is 
irregular and jerky, and is called Brownian movement 
(after the Scotch investigator, Brown). Some bacteria, 
however, possess the property of true, independent mobil- 
ity which is due to the presence of one or more long, 
delicate, thread-like filaments called flagella (Fig. 3). 
They have to be stained by special methods. 
Spores.-Most of the bacteria when placed in unfavor- 
able environment, that is, when deprived of things es- 
Fig. 4.-Tetanus bacilli, showing spores. (Mallory and Wright--Patho- 
logical T echnic.) 
sential to their nutrition or in the presence of harmful 
substances, die and undergo slow disintegration. But a 
few bacteria are able to pass into a latent stage of exist- 
ence similar to hibernation of animals, during which the 
chemical interchange is at the lowest ebb. 
This is accomplished by the bacteria developing within 
their endoplasm highly refractile, oval bodies called spores 
(from Greek sporos, meaning seed). The spores are 
highly resistant to heat and disinfectants and thus endow 
the bacterium with a special resistance against destruc- 
tion. 
Spore formation is not a reproductive process. When GENERAL BACTERIOLOGY 
29 
placed in suitable surroundings the spores germinate and 
the original bacteria reappear. The spore formation is 
most frequent among those bacteria which live without 
air (anaerobes). Special staining methods are required 
for the demonstration of spores. 
III. Bacterial Reproduction 
The bacteria being minute unicellular asexual cells, the 
reproduction is a simple mechanical process, the cell hav- 
ing reached its maximum size (which is fairly constant 
for the species) a slight constriction appears, which deep- 
ens until a distinct partition or septum is formed, and 
the original cell is split into two cells, each being a com- 
plete organism, identical with the parent cell. Some of 
the higher bacteria (e. g., Spirocheta pallida, which 
causes syphilis) split not transversely but longitudinally 
(although many authors, including Noguchi, do not re- 
gard these organisms as bacteria). The rate of growth, 
that is the time elapsing between two successive cleav- 
ages, is about 15 to 20 minutes (Fischer), so that a 
single colon bacillus would yield about 1,500 trillion in 
a single day. This rate is never maintained for any 
length of time and by no means all bacteria multiply so 
rapidly. 
IV. Constancy and Mutation of Type 
While most of the bacterial properties remain constant, 
especially the disease-producing (pathogenic, from Greek 
pathos, meaning disease, and geneo, meaning to produce) 
properties, the beginner must remember that under certain 
conditions one or more characteristics of a given species 
of the bacterium may be lost, such variation has already 
been referred to in the connection with the capsule forma- 
tion. Pasteur succeeded in producing sporeless anthrax 30 
PRINCIPLES OF BACTERIOLOGY 
bacilli by growing them at 43° C. (instead of 37° C. 
which is the temperature at which they grow best nor- 
mally). 
The experience accumulated so far seems to point to 
the fact that the morphological changes alone should 
not be regarded as definite evidence of mutation, but 
one should take into consideration the reaction of the 
given bacterium to its culture media, staining, patho- 
genic properties and the various serological reactions 
(agglutination, complement fixation, precipitation, etc.). 
We know that the various members of the typhoid-colon- 
dysentery group cannot be differentiated morphologically, 
yet their disease-producing properties and the fer- 
mentation of sugars remain distinct. Even when changes 
in bacteria have been brought about by artificial means, 
these secondary changes will disappear, as shown by the 
work of Vaughan, Eisenberg, and others, when normal 
conditions have been restored. The most startling work 
along these lines is that of Rosenow, who claims to have 
among other things, mutated hemolytic streptococci into 
pneumococci; so far these observations seem to be ac- 
cepted as interesting bacteriological phenomena, but 
how true and permanent these mutations are, time alone 
will show. 
So far we may accept the statement that bacteria are 
quite constant as to type and that all secondary changes 
can be banished when normal conditions of bacterial 
development have again prevailed. 
V. The Chemical and Physical Properties of the Bac- 
terial Cell 
The various chemical constituents of bacteria may be 
tabulated approximately as follows: GENERAL BACTERIOLOGY 
31 
Water 85% 
Proteins 10-12% 
Fats 1% 
Ash 1.5%-2% 
Residue 1-1.5% 
(Kappes, Nencki and Scheffer.) 
This varies, as regards the individual constituents, to 
some extent; for example, the tubercle bacillus contains 
less proteins (8 per cent) but much more fats (3.5 to 4 
per cent). 
The proteins contained in its bacteria are the nucleopro- 
teins, globulins and special proteins. The bacterial ashes 
are mostly chlorides and phosphates of sodium, potassium, 
magnesium and calcium. 
Like other vegetable and animal cells, the bacterial 
cell reacts to the pressure which exists between, its own 
protoplasm and the surrounding medium-the. so-called 
osmotic pressure, which governs the exchange of the sub- 
stances within and without the bacterial cell, this depend- 
ing upon the permeability of the cell membrane (the 
ectoplasm), which permits certain substances to enter or 
leave the bacterial cell. 
VI. Nutrition of Bacteria 
In order that the bacteria may live and multiply they 
must have the following substances: 
Carbon.-This they may obtain from proteins, fats or 
carbohydrates. 
Oxygen.-This is obtained by the majority of bacteria 
directly from the atmosphere in the form of free oxygen. 
Not all bacteria, however, need free oxygen, and some, 
as will be mentioned later, can not live in its presence. 
Nitrogen.-This is taken in most cases, from proteins. 
There is quite an individual predilection for the special 
proteins required by different bacteria: the gonococcus, 
for example, grows best on uncoagulated human blood 32 
PRINCIPLES OF BACTERIOLOGY 
serum; the influenza bacillus requires hemoglobin (the 
coloring matter of the red blood cells) ; while the diph- 
theria bacillus grows best on the coagulated beef serum. 
A large number of bacteria may thrive without any pro- 
tein at all. 
Hydrogen.-Hydrogen is obtained largely in combina- 
tion with water. 
Salts.-Salts are absolutely necessary and are chiefly 
phosphates and chlorides of magnesium, calcium, potas- 
sium, and sodium, and iron (in some of the higher bac- 
teria). 
Those bacteria that must have oxygen and can not live 
without it are called obligatory aerobes. 
Those that can not live in the presence of oxygen (e. g., 
tetanus bacillus) are called obligatory anaerobes. Inter- 
mediate between these two extreme groups are those that 
prefer oxygen but can grow without it-faculative anae- 
robes-and those that prefer to grow without oxygen but 
can live in its presence-facultative aerobes. 
VII. Aerobes and Anaerobes 
VIII. Longevity of Bacteria 
The duration of bacterial life is unknown, but is, com- 
paratively speaking, brief. Dried spores may not only 
live, but retain their virulence (invasiveness), for years. 
IX. Parasites and Saprophytes 
Parasites are those bacteria which live and multiply in 
the human or animal body, while saprophytes are those 
which can not hold their own in animal tissues, but are 
found everywhere in the air, soil, water, and manure. Par- 
asitic bacteria are fastidious as regards their food, temper- 
ature, etc., while the saprophytes are easily satisfied and 
live on simplest media. The main distinction, however, lies 
in our conception that the parasitic bacteria arc those GENERAL BACTERIOLOGY 
33 
which produce disease, that is, are pathogenic, while the 
saprophytes live on dead matter and are of greatest value 
in the world's economy in breaking up the organic matter 
through the processes of fermentation and putrefaction. 
It is, however, well to qualify this statement, by point- 
ing out that there are "pure saprophytes" which live 
only on dead organic matter, and "facultative sapro- 
phytes" which usually lead saprophytic existence but, 
as a rule, live within living tissue; one should also bear 
in mind that there are bacteria, such, e.g., as B. coli 
communis, which, for most part, lead a saprophytic 
existence in the animal body and yet, not infrequently, 
become markedly pathogenic, causing appendicitis, per- 
itonitis, pyelitis, etc. 
X. Mutual Relations of Bacteria 
It is evident that in many cases several different bac- 
teria must live in the same place, or, in other words, the 
same surrounding must be favorable to several species. 
This possibility of several bacteria living and multiplying 
in the presence of each other is spoken of as symbiosis 
(from Greek syn, meaning with, and bios, meaning life), 
as exemplified by diphtheria and streptococci. Symbiosis 
is not so frequent, however, as the opposite fact-namely, 
the impossibility of one organism living in the presence 
of another-a condition which is known as antagonism 
(from Greek antagonisma, meaning struggle) ; examples 
of bacterial antagonism are gonococcus and bacillus pyocy- 
aneus, plague bacillus and streptococci, etc. 
XI. Relations of Bacteria to Physical Environment 
Temperature.-Bacteria, like other living beings, have 
their minimum (lowest), maximum (highest), and opti- 
mum (the best) temperature at which they will live, grow 
and multiply. 34 
PRINCIPLES OF BACTERIOLOGY 
For the large majority of the bacteria the optimum 
temperature is 37.5° C.; on the other hand, there are 
numerous bacteria in the water which grow at 20° C. 
Individual organisms have their limits, these varying 
with the environment adopted by them through many gen- 
erations; for example, the bacillus of the avian (bird) 
tuberculosis grows at 41° to 42° C. and can not grow 
at 37.5° C., while the bacillus of human tuberculosis grows 
best at the latter and will not grow at the former tempera- 
ture ; this difference in the optimum temperature of the 
two strains of the same organism is due and proportionate 
to the difference in the normal temperature of human 
beings and the birds. 
By gradual and persistent variation of the temperature 
it is possible to adapt certain bacteria to grow abundantly 
at the temperature several degrees higher or lower than 
their normal optimum temperature, but this is usually 
accompanied by the loss of certain of their characteristics, 
as in the case mentioned above-that of anthrax bacillus 
grown by Pasteur at the temperature of 42° C.-the 
bacilli finally grew well but lost their property of spore 
formation. 
Some common pathogenic organisms, such as colon bacil- 
lus, exhibit a very wide variation of temperature at which 
they may develop-from 10° C. to 40° C. The sapro- 
phytes exhibit an even higher variation. 
Ten to fifteen minutes' exposure to a temperature of 55° 
to 60° C. usually destroys the common pathogenic bacteria 
which are nonspore-bearing; the spores confer upon bac- 
teria enormous resistance to heat and the spore-bearing 
bacteria, therefore, are a much more formidable enemy 
than the other kind. 
Low temperatures are much less destructive than the 
high ones, and in the case of certain bacteria are useful GENERAL BACTERIOLOGY 
35 
in keeping cultures alive for a long period of time, e. g., 
pneumococcus and streptococcus. 
Moisture.-The presence of water is absolutely neces- 
sary for bacterial life. The effects of complete drying 
vary greatly with the different bacteria; thus gonococcus 
and cholera organisms die within a few hours while the ty- 
phoid and tubercle bacilli may withstand complete drying 
for two to three months. 
Light.-Most of the pathogenic bacteria are inhibited 
or destroyed by sunlight, although marked variation ex- 
ists among different bacteria as regards this. 
Electricity.-Electric light exerts a distinct bacterici- 
dal (bacteria killing) effect when applied in the strength 
of 800 to 900 candle power for six to nine hours. Roentgen 
or x-rays do not seem to have any bactericidal effect, 
while radium has both an inhibitory and bactericidal effect 
when applied at a distance of a few centimeters for a few 
hours. 
XII. The Biological Activities of Bacteria 
While the pathogenic, or disease-producing, properties 
are of immediate interest to us, we must not lose sight 
of the fact that these properties are merely a side action, 
so to speak, compared with other numerous bacterial 
activities, and that the pathogenic bacteria are the least 
numerous group of all the bacterial clans. The pro- 
duction of disease is merely incidental to the success- 
ful attempt on the part of the bacteria to establish a 
new domicile in the human or animal body. 
The much more important functions of the bacteria 
are those with which they serve the many important 
and useful purposes in the world's economy. 
It is absolutely no exaggeration to say that without 
bacteria all reduction of complex organic matter to 
simple compounds and the chemical interchange be- 
tween the animal and the vegetable kingdoms could 36 
PRINCIPLES OF BACTERIOLOGY 
not be carried out and all life on earth would cease, 
as Hiss and Zinsser so well say (Textbook of Bacteriology 
1914, p. 41) : "Far from being scourges, these minute 
microorganisms are paramount factors in the great 
cycle of living matter." 
The Katabolic (Breaking Down) Activities of Bac- 
teria.-The katabolic activities of bacteria consist in 
the fermentation of carbohydrates (sugars and starches) 
and in the splitting of fats and proteins. 
For our entire knowledge of fermentation we are 
indebted to the genius of Pasteur who was first to 
explain to us that the fermentation (as well as the 
splitting of proteins) is due to ferments or enzymes 
(from Latin and Greek respectively, meaning "lea- 
ven"). These are substances produced by a living 
cell, which bring about a chemical change without 
entering into the reaction themselves. The ferment is 
not attached to the end products of the reaction, and 
is not appreciably diminished during the reaction; in 
this way the bacterial ferments are like the chemical 
agents known as katalyzers--e. g., dilute acids, which 
bring about various chemical reactions and yet do not 
enter them themselves, as, for example, when a solu- 
tion of cane sugar brought into contact with dilute 
solution of sulphuric acid results in the formation of 
two single sugars in place of a double sugar r 
C12H22OU + H2O + h2so4 = c6h12o0 + c6h12o6 + h2so4. 
Cane sugar water sulphuric acid = grape sugar fruit sugar sulphuric acid 
The proper definition for ferments or enzymes is, 
therefore, "a substance which hastens a chemical re- 
action without itself taking part in it." The best 
conditions for the ferment action are the presence of 
moisture, a weakly acid or alkaline reaction, and a 
temperature ranging from 35° to 45° C. 
The presence of protein-splitting, or proteolytic, as they 
are called, ferments is shown by the power of bacteria to GENERAL bacteriology 
37 
liquefy gelatin, fibrin or coagulated blood serum. Among 
the important products of bacterial proteolysis are the 
ptomaines (from Greek ptoma meaning a dead body) 
which are highly poisonous and responsible for ptomaine 
poisoning. 
Certain bacteria produce the so-called coagulating, or 
"lab" ferments (from Latin lab, meaning rennet-a sub- 
stance found in the stomach, which coagulates milk) 
which causes coagulation of certain fluids, such as 
milk, blood, etc. Some bacteria produce fat-splitting 
ferments (cholera spirillum). 
As regards the fermentative properties of bacteria, 
the most important ferments are those which split the 
various sugars, cause the production of lactic acid, and 
the alcoholic fermentation (brought about by both 
bacteria and yeasts). 
A very important class of bacteria are the so-called 
dentrifying bacteria-those which reduce the nitrates to 
nitrites. 
The Anabolic (Upbuilding) Activities of Bacteria.- 
These consist in the property of some soil bacteria to 
accumulate large amounts of nitrogen from the air and 
thus to make up for the loss brought about by the ab- 
sorption of nitrogen by plants (Winogradsky). Even 
more important than this is the class of bacteria found 
in the root tubercles of certain plants (leguminosae), 
such as beans, peas, etc.; these bacteria not only do not 
withdraw nitrogen from the soil but enrich it, and 
upon this knowledge depends the well-known method 
of crop alternation used by farmers the world over. 
Certain bacteria bring about oxidation of ammonia 
to nitrites and nitrates; these are called nitrifying bac- 
teria. Some bacteria living in the salt water produce 
light, and a large number of bacteria (including the 
pathogenic bacteria) produce various pigments, thus 38 
PRINCIPLES OF BACTERIOLOGY 
staphylococcus aureus produces a yellow pigment, 
bacillus pyocyaneus produces a green pigment, while 
that produced by bacillus prodigiosus is red. 
XIII. The Distribution of Common Pathogenic Bacteria 
in the Animal Body 
While no organ in the animal body, except such 
structures as nails, is free from invasion from one or an- 
other kind of bacterium, still certain organs, in the 
course of evolution, have come to be preferred by cer- 
tain bacteria as their domicile, to the exclusion of other 
organs, and it may be useful to append the following 
table of bacterial distribution in the body. 
Shin.-Staphylococci and Streptococci. Tubercle, leprosy and smeg- 
ma bacilli. Tetanus, gas, and anthrax bacilli. 
Nose and Throat.-Staphylococci, streptococci and pneumococci, 
diphtheria, influenza and pertussis (whooping cough) 
organisms. Meningococci and catarrhalis groups. 
Tubercle bacillus and virus of poliomyelitis (infantile 
paralysis). 
Ear and Eye.-Streptococcus, staphylococcus and pneumococcus 
groups. Diphtheria and influenza groups. Koch- 
Weeks and Morax-Axenfeld bacilli. Gonococcus. 
Lungs.-Streptococcus and pneumococcus. Tubercle bacillus, staphy- 
lococcus, Friedlander's bacillus, influenza and pertussis 
groups, anthrax and plague bacilli, actinomyces and 
colon-typhoid group. 
Pelvic Organs.-Streptococcus and Staphylococcus groups. Gono- 
coccus and Spirocheta pallida. Tubercle and Smegma 
bacilli. 
Serous Fluids: 
1. Cerebrospinal fluid: (a) clear fluid, tubercle bacil- 
lus, Spirocheta pallida (syphilis), virus of poliomyeli- 
tis. (b) turbid fluid: pneumococcus, streptococcus, 
meningococcus, B. influenzae, typhoid-colon group. 
2. Pleural and pericardial fluids: (a) clear fluids; 
tubercle bacillus, (b) turbid fluid: pneumococcus, 
streptococcus, B'. influenza, typhoid, staphylococcus, 
3. Peritoneal fluid: Streptococcus group. Colon-typhoid 
group. Tubercle bacillus. GENERAL BACTERIOLOGY 
39 
Blood.-Streptococcus and pneumococcus groups. Typhoid group. 
Staphylococcus group. Recurrent fever and spirochetae. 
Plague bacilli. Friedlander's bacillus (rare). 
Intestinal Contents and Feces.-Colon-typhoid group, including the 
paratyphoids, the dysentery and B. fecalis alcaligenes. 
Mucosus capsulatus group. Tubercle bacilli, anthrax, 
tetanus and gas bacilli. 
(After Kendall, Bacteriology, 1917, p. 106.) 
Occasionally individuals are met with who, while ap- 
parently healthy, harbor pathogenic bacteria in their 
bodies. While they seem to suffer no ill effects from 
this, they are a source of danger to those who come in 
contact with them. Such individuals are called car- 
riers. In a majority of cases these carriers have had 
an attack of an infectious disease and have apparently 
recovered from it, but continue to eliminate the patho- 
genic bacteria which had caused the disease; some are 
but temporary carriers, while others may become 
habitual carriers, never ceasing to eliminate the bac- 
teria. This will be again referred to in the section deal- 
ing with infection and immunity, but it is well to call 
attention, at this time, to the very grave menace of 
such carriers. 
Many an epidemic, notably of typhoid fever and 
diphtheria, has been doubtless brought about by such 
carriers; in the case of typhoid fever, the bacilli 
appear to lodge in the gall bladder or the bile ducts, 
whence they pass into the intestinal canal and escape 
in feces. Women are more commonly typhoid carriers 
than men. Diphtheria carriers are most frequently 
found among children who often incite a diphtheria 
epidemic in schools. For this reason all cases of diph- 
theria and typhoid fever should have repeated examina- 
tion of throat cultures and feces cultures, respectively, 
after the patients have apparently recovered. 
XIV. Carriers CHAPTER HI 
THE DESTRUCTION OF BACTERIA 
The destruction of bacteria constitutes what prob- 
ably is the most glorious chapter of bacteriology-on 
that alone depended the birth and the development of 
modern surgery. 
Several terms, in this connection, are used, somewhat 
loosely, and the pupil should, at the outset, clearly under- 
stand the different ideas conveyed by them: by steriliza- 
tion is usually meant destruction of bacteria by heat, and 
is referred to in connection with the boiling of linen, gauze? 
bandages, infected dressings, instruments, cultures, etc.; 
disinfection usually means destruction of bacteria by the 
use of chemicals-bichloride of mercury, alcohol, car- 
bolic acid, etc.-and is used in connection with the ex- 
creta, urine, feces, or in speaking of destroying bacteria 
on surgeon's hands or patient's body; fumigation means 
bacterial destruction by means of certain gases, e. g., 
formaldehyde, and is used primarily in connection with 
the buildings or individual rooms; thus we sterilize 
bandages or the operating room gowns, or dressings; 
we speak of disinfecting hands with alcohol or the field 
of the operation-e. g., patient's skin with iodine or 
of disinfecting the patient's excreta with carbolic acid; 
and, finally, we fumigate the room with formaldehyde. 
These different terms refer, however, to the same pur- 
pose, accomplished by different means, namely, com- 
plete bacterial destruction, or the production of "asep- 
sis"; when a substance does not actually kill the 
microorganisms, but merely inhibits their growth 
40 41 
GENERAL BACTERIOLOGY 
and multiplication-such substances are spoken of as 
"antiseptics." 
I. Bacterial Destruction by Physical Agents 
1. Drying.-When complete, drying' destroys most of 
the pathogenic bacteria, but the individual bacteria dif- 
fer greatly from each other in this respect; thus, 
gonococcus and the bacillus of influenza are destroyed 
within a few hours, while the tubercle bacillus may live 
without a trace of moisture for months. 
2. Light.-Sunlight is a powerful germicide (from 
Latin meaning life killing), the different bacteria vary- 
ing from each other in this respect also, the tubercle 
bacillus dying within two hours; for this reason ' ' where 
there is sunlight there is no tuberculosis." (This, of 
course, is only a general statement, and should not be 
taken literally.) 
3. Electricity, Radium and Roentgen Rays.-See the 
preceding chapter. Briefly stated, x-rays have no in- 
fluence, while electricity and radium are both germicidal. 
The effects of electricity are not due to the direct action 
of the current, but to heat and the electrolytic changes 
in the culture liquid. 
4. Heat.-This is the standard and universal method 
of bacterial destruction. 
Dry Heat.-Burning, when we are dealing with ob- 
jects without value, such as, e. g., the sputum cups, is 
a very rapid and a sure means of sterilization. Hot air 
sterilization is carried out in the so-called "hot-air 
chambers" or sterilizers. (Fig. 5.) This is a double- 
walled, sheet-iron chamber, with the joints riveted in- 
stead of being soldered. The inner case is completely 
closed, while the outer one has a large opening in the 42 
PRINCIPLES OF BACTERIOLOGY 
bottom and two small ones at the top; the gas burners 
-one on each side-are so placed between the two 
walls that the flame plays directly on the inner case. 
This method of sterilization is used primarily for the 
glassware, and the temperature should be between 150° 
and 160° C. maintained for at least one hour. Remem- 
ber that if combustile articles are sterilized in this 
sterilizer, the temperature should not exceed 200° C. 
as cotton is browned at this temperature. 
Fig. 5.-Hot-air sterilizer. 
Moist Heat.-Boiling may be used for the steriliza- 
tion of instruments, syringes and other similar objects; 
vegetative forms (not spores) of bacteria may be de- 
stroyed by five minutes' boiling, while in order to de- 
stroy the spores one to two hours' boiling is sufficient. 
Live steam is the most practical of the methods of heat 
sterilization; while it may be improvised by use of 
almost any simple household makeshifts, such as a GENERAL BACTERIOLOGY 
43 
double steamer, wash boiler or potato steamer, in the 
laboratories it is carried out by means of so-called "Ar- 
nold" sterilizer (Fig. 6). This consists of a round or 
rectangular chamber with an outer cylinder well fitting 
over it, both set in a reservoir about 4 inches high, 
which has a shallow double bottom. The reservoir is 
filled with water which is heated by a gas flame; this 
rapidly vaporizes the thin layer of water contained in 
the double bottom of the reservoir, the steam rises rapid- 
ly and passes into the inner chamber containing the 
Fig. 6.-The Arnold sterilizer. 
articles to be sterilized, through its perforated bottom; 
the steam is condensed under the outer chamber and 
drops back into the reservoir. 
An exposure from fifteen to thirty minutes destroys 
all vegetative forms of bacteria. In order to get rid 
of spores a "fractional" method of sterilization is 
used; this consists in exposing the substances to be 44 
PRINCIPLES OF BACTERIOLOGY 
sterilized (usually culture media) from fifteen to thirty 
minutes on three successive days; the first exposure de- 
stroys the vegetative forms; by next day any spores 
which may have been present will have developed with 
the vegetative stage, and are killed by the second ex- 
posure ; the third exposure makes the sterilization com- 
plete. Remember that in noting the time of exposure 
it is necessary to calculate, not from the time of light- 
ing the flame, but from the time "the steam is up"; 
that is, from the time the temperature reaches 100° 
C. When substances to be sterilized can not be sub- 
jected to the temperature of 100° C., e. g., culture media 
containing albuminous materials (because they will 
coagulate above 60° C.) the principle of fractional 
sterilization may be adhered to by immersing the ob- 
jects in a water bath at a temperature of 55° to 60° C. 
for an hour on five or six consecutive days. 
Steam under pressure is the most powerful and cer- 
tain method of sterilization we possess. 
It is used when the objects can not be injured by mois- 
ture-operating room gowns, dressings, etc.; in the 
laboratory this method is used for infected glassware. 
The apparatus for steam sterilization under pressure 
is called an "autoclave" (Fig. 7). Exposure to steam 
at the pressure of fifteen pounds for fifteen to twenty 
minutes will destroy all bacteria and spores. 
Two precautions to be borne in mind in connection 
with the use of autoclave, are: 1. Allow all air to es- 
cape from the autoclave before closing the vent. 2. Do 
not open the door for at least fifteen to twenty minutes 
after the flame has been turned off, as the sudden re- 
lief of pressure thus produced may burn the operator 
and the stoppers (in tubes or flasks) will "pop out." GENERAL BACTERIOLOGY 
45 
II. Bacterial Destruction by Chemical Agents 
Many chemicals can either completely destroy the 
bacteria (bactericides or germicides) or merely inhibit 
their growth (antiseptics). Just how such injury is 
done to bacteria is, to a large extent, unknown; some 
substances such as strong acids, probably kill bacteria 
Fig. 7.-The autoclave. 
by rapid oxidation; some destroy them by coagulation 
of the bacterial protoplasm, some enter into chemical 
combination with the protoplasm and exert a poisonous 
action, some act by withdrawing water from the bac- 
terial cell, etc. Of the many chemicals used for the 
purposes of disinfection, the following are the most im- 
portant and the most widely used: 46 
PRINCIPLES OF BACTERIOLOGY 
Chloride of Lime or Bleaching Powder.-Chloride of 
lime is readily soluble in twenty parts of water. In 
dilution of 1:500 it will destroy the vegetative forms 
of bacteria in five to ten minutes. See the section on 
Practical Disinfection for a brief description of Dakin's 
solution. 
Iodine.-Tincture of iodine (7.5 per cent) has become 
in the last few years the standard disinfectant; in many 
hospitals it is the only agent used for disinfecting the 
field of the operation. It is most effective when freshly 
prepared; the parts on which it is to be applied should 
first be swabbed with alcohol and allowed to dry. 
Peroxide of Hydrogen.-As the presence of organic 
matter, such as pus, blood, etc., distinctly diminishes its 
effects, one should always remove these from wounds 
before applying the peroxide; it acts upon bacteria by 
liberating oxygen. 
Permanganate of Potassium.-Permanganate of 
potassium acts, as does the peroxide of hydrogen, and 
is a powerful germicide. 
Bichloride of Mercury (Mercuric Chloride.)-Bichlor- 
ide of mercury is a powerful germicide; a solution of 
1:1000 is commonly used; this will kill the vegetative 
forms in a few minutes, and in solution of 1:500 will 
kill spores in a few hours. The addition of 25 per cent 
alcohol greatly increases the germicidal action of bi- 
chloride of mercury. There are certain disadvantages: 
it is apt to cause local necrosis (destruction) of tissues 
because of its great affinity for proteins, or injure the 
kidneys by absorption; it is unreliable for disinfection 
of feces, sputum, etc., and can not be used for steriliza- 
tion of instruments. It is used almost exclusively for 
irrigating wounds and disinfecting the skin. 
Silver.-Silver preparations such as silver nitrate (0.1 GENERAL BACTERIOLOGY 
47 
to 4 per cent), argyrol, protargol, etc., are used prin- 
cipally upon mucous membranes and in the eye (e. g., 
in newborn babies to prevent ophthalmia. 
Alcohol.-Alcohol is an efficient germicide but only 
in dilute solutions, the strongest germicidal action be- 
ing exerted by 50 to 70 per cent solutions; those stronger 
than this are much less efficient, absolute alcohol being 
practically without any effects. 
Iodoform.-Iodoform is a weak germicide in itself, 
but when introduced into wounds, iodine is liberated 
and it is then a very efficient agent. 
Carbolic Acid.-Carbolic acid (phenol) and its de- 
rivatives-lysol, cresol, tricresol, creoline, etc., are very 
efficient germicides, especially the latter; they are not 
only germicidal but are also poisonous for human tis- 
sues ; for this reason they should not be applied to 
wounds in solutions stronger than ^-l per cent nor 
should they be left for any length of time, as gangrene 
is a common result of their prolonged application. They 
are the best agents for disinfecting feces, urine, sputum, 
soiled linen, etc., for which purpose a 5 per cent solu- 
tion should be used. 
Boric Acid.-Boric acid is not a germicide but an 
antiseptic, that is, it does not destroy the bacteria but 
prevents their development; it is used upon mucous 
membranes and in the eye. 
Formaldehyde.-Formaldehyde is used either as a 
gas for fumigation or a 40 per cent solution in water 
("formalin") ; in the latter form it is an excellent sub- 
stance to be used for sputum, urine, feces, etc., in 
solution 2 to 5 per cent. For the use of formaldehyde 
in fumigation see the next section. 
Sulphur.-Sulphur is used chiefly for destroying the 
insects. 48 
PRINCIPLES OF BACTERIOLOGY 
III. Practical Disinfection 
Sputum is a very difficult thing to disinfect because 
the bacteria contained in it are protected by a thick 
envelope of mucus; a 5 per cent solution of carbolic 
acid is very efficient; sputum cups had best be burned; 
sputum napkins can either be soaked in a 5 per cent 
solution of carbolic acid or immersed in boiling water 
for a half hour. Do not use bichloride of mercury as 
it forms a thick layer of albuminated mercury around 
the bacteria. 
2. Feces should be received in a porcelain or metal 
container and immediately mixed with large amounts 
of 5 per cent carbolic acid or formalin or 10 per cent 
chloride of lime, and allowed to remain in contact with 
these substances for at least one hour before disposing. 
The soiled parts of the patient should be wiped with 
a cloth dipped in 2 per cent carbolic acid or cresol, 
then with water to remove the disinfectant. 
3. Urine may be disinfected in a manner similar to 
that in which feces should be disinfected. 
4. Cloth material, linen, napkins, etc., which have 
been handled by the patient should be soaked for at 
least two hours, either in 2 per cent formalin or in 5 
per cent carbolic acid, before it is taken out to be boiled 
-be careful not to remove any infected linen from the 
patient's room in a dry state. 
5. Bath water should not be allowed to drain before 
it has been mixed with an ounce or two of chlorinated 
lime for at least an hour. 
6. Skin and hands should be scrubbed with a brush 
and green soap and then soaked for a few minutes in 
1:1000 solution of bichloride of mercury. 
A patient recovering from a disease such as smallpox, 
scarlet fever, measles, and other "eruptive" fevers GENERAL BACTERIOLOGY 
49 
should receive a bath in 1:1000 solution of bichloride 
of mercury (taking care not to have the solution enter 
the patient's mouth, nose, ears, or eyes). 
As to the disinfection of the operator's hands, there 
still exists but little uniformity, some surgeons using 
the permanganate-oxalic acid, bichloride method 
(Welch's method)-others using the alcohol-carbolic 
acid method (Fiirbringer) while still others use the 
bichloride solution only. 
As a matter of fact, the belief is gaining ground that 
the particular antiseptic used plays but an unimportant 
part, the two main considerations being the use of hot 
running water, soap and brush for at least 5 minutes, 
and wearing sterile rubber gloves; during the opera- 
tion the surgeon usually dips his gloved hands into a 
basin containing 1:1000 solution of bichloride of mer- 
cury. 
7. Surgical instruments, catgut, etc., are sterilized in 
different ways in different hospitals. The usual way is 
to boil the instruments in soda solution. 
8. Thermometers should be kept in a 5 per cent solu- 
tion of formalin; when needed, they should be thor- 
oughly rinsed in water. 
9. Rooms, closets, etc., had best be fumigated by 
formaldehyde. The best and the simplest method is 
that of Russell and Evans (Report of State Board of 
Health of Maine, 1904). For each thousand cubic feet 
of space ten ounces of formalin and five ounces of 
potassium permanganate crystals are placed in a two- 
or three-gallon galvanized iron pail which must have 
flaring sides because there is much spattering when the 
reaction between permanganate and formalin takes 
place; for this reason it is also advisable to place 
some heavy paper under the pail. Have all closets, 
drawers, etc., wide open, while all doors, windows, 50 
PRINCIPLES OF BACTERIOLOGY 
sashes, key-holes, etc., should be tightly plugged--the 
fumes of formaldehyde being extremely irritating and 
dangerous. As soon as potassium permanganate and 
"V/ocheh 
CINCINNATI 
Fig. 8.-Instrument employed in applying Dakin's solution to wounds. 
It consists of five glass distributing tubes for using single or large number 
of rubber conducting tubes, the flask is used for mixing the solution and 
the syringe with rubber bulb is used in testing the permeability of the 
conducting tubes. 
formalin are mixed, give the pail a good kick where- 
upon evolution of the formaldehyde gas will take place. 
Leave the room instantly and lock the door, leaving GENERAL BACTERIOLOGY 
51 
the room undisturbed for at least six hours, at the end 
of which period all windows should be opened. 
10. Dakin's Solution.-Within the last six years a 
most successful method of treating infected wounds 
has been devised by Dakin and Carrel (both of the 
Rockefeller Institute for Medical Research, of New 
York) and has been extensively applied on battle fields, 
in industrial plants, hospitals, etc. The preparation of 
Dakin's solution is somewhat complicated and the 
original articles by Dakin and Carrel should be con- 
sulted ; the solution consists of calcium hypochlorated 
(bleaching powder), sodium carbonate and sodium bi- 
carbonate ; these when properly mixed (this means a 
careful titration of the first mentioned ingredient) 
cause slow generation of chlorine; the effects of this 
nascent chlorine differ materially from those ordinary 
effects of chlorine, since not only the destruction of 
bacteria in the tissues is ever so much more thorough 
and rapid but the effect on the dead tissue in the 
wound is extraordinary-the most infected wounds 
with dead ragged pieces of tissue become clean and 
active cicatrization (healing) rapidly takes place. The 
application of Dakin's solution should be carried out 
most faithfully by the Carrel method if these unusual 
results are to be obtained; a special apparatus devised 
by Carrel must be used, and all the directions given by 
Dakin and Carrel should be carried out to the letter, 
without the least deviation. CHAPTER IV 
THE STRUGGLE BETWEEN BACTERIA AND THE 
BODY INFECTION AND IMMUNITY 
I. Infection 
By infection we understand more than mere entrance 
of bacteria to the animal body; an infection means the 
entrance and successful multiplication of bacteria in 
the animal body, with the subsequent development of 
a disease (which is then called infectious disease). 
That the mere presence of bacteria in the animal body 
does not mean infection or infectious disease is obvious 
from the fact that several species of bacteria are con- 
stantly present in certain parts of the body; the colon 
bacilli are always present in the intestines, the bacillus 
xerosis is very frequently found in the lining of the 
eyelids (conjunctiva), staphylococci, streptococci, and 
pneumococci are very often found in normal mouths. 
In order that the infection should take place the 
following conditions should be fulfilled: 
1. Bacteria should gain entrance to the body by a 
path specially adapted to their requirements; thus, cer- 
tain bacteria invariably attack through the gastroin- 
testinal tract, e. g., the typhoid bacillus and cholera 
spirillum; some bacteria enter the body only through 
the skin, e. g., staphylococci, streptococci, tetanus bacil- 
lus, etc. These different paths are so necessary for 
their successful invasion that should the typhoid bacil- 
lus (which is a gastrointestinal invader) be rubbed into 
the skin, or, conversely, should the staphylococcus 
52 53 
GENERAL BACTERIOLOGY 
(which is a skin invader) be swallowed, no harm 
would, in all probability, result. 
2. Bacteria should invade the body in sufficiently 
large numbers. 
3. They should find an environment which is favor- 
able to their nutritional requirements. 
4. The resistance of the individual attacked should 
be sufficiently weakened to permit the bacteria to de- 
velop in his body. (This is discussed fully in a subse- 
quent section on Immunity.) 
5. We have mentioned in a preceding chapter that 
all bacteria are divided into two classes, according to 
their activities, the saprophytes, those which feed on 
dead organic matter and are highly useful in maintain- 
ing the chemical balance between the animal and the 
vegetable kingdoms, and the parasites or pathogenic 
(disease-producing) bacteria which feed on the liv- 
ing body and produce disease; although this division 
is not absolute, since many saprophytes may become 
under special conditions pathogenic (for example, they 
may gain entrance to a gangrenous limb and while 
feeding on dead organic matter will, nevertheless, 
cause disease by producing poisonous substances), yet 
it is convenient to separate the bacteria into these two 
classes; it is evident that in order that the infection 
may take place, the bacteria must be pathogenic. 
6. Even the pathogenic bacteria differ very much 
within the same species as to the degree of their power 
to incite disease; such power is spoken of as virulence; 
for example, pneumococci kept artificially (that is, in 
culture media) are much less virulent than those freshly 
isolated from the animal tissues. 
For a successful infection it is necessary, therefore, 
that the pathogenic bacteria shall possess sufficient 
virulence. 54 
PRINCIPLES OF BACTERIOLOGY 
II. The Infection Proper-Bacterial Poisons 
. When all of the above conditions have been complied 
with, we will have an infection, which may be either 
local or general. If only inflammation of a part or an 
abscess (pus accumulation) in a certain part results, 
we speak of this as a local infection; on the other hand 
from such a local infection-or without it-the bacteria 
may gain entrance to the lymph and blood vessels and 
be carried into the circulation and thus be distributed 
through the entire body-this is called "septicemia" 
(from Greek septikos, meaning putrid and aima, mean- 
ing blood) or (by the laity) blood poisoning. If the bac- 
teria thus carried to other organs form abscesses there, 
we speak of this condition as "pyemia" (from Greek 
pion, meaning pus, and aima, meaning blood). 
Septicemia and pyemia are caused in a large majority 
of cases by streptococci and staphylococci. 
It is evident, however, that mere local injurious effects 
or blocking of the small blood vessels (capillaries, from 
Latin meaning hair-like) are not sufficient to account 
either for severe general effects following infection or 
for the fact that the same bacteria produce the same in- 
jury no matter where they lodge (within their special 
path of entrance, of course)-in other words it is evident 
that bacteria produce their effects not mechanically, but 
chemically, by means of the so-called bacterial poisons, 
which are called toxins (from Greek toxicon, meaning 
poison). 
Those toxins are of three kinds: 1. Exotoxins ("ex- 
ternal" poison) which are separable* poisons, secreted 
by bacteria just as sweat is secreted by the sweat 
*1 believe the usual terms "soluble" and "insoluble'' had better be 
dismissed, as (in my experience, at least) they usually are a source 
of confusion to the pupil. "How do the insoluble poisons do any 
harm if they are insoluble?" is a favorite question. GENERAL BACTERIOLOGY 
55 
glands; such separable poison is given off by the bac- 
terium into the surrounding culture medium if it is 
grown artificially or into the circulation if it lodged 
in the body. For example, if bacteria are grown in 
liquid culture, such as broth, and this broth is filtered, 
it will be found that, although the bacteria will be left 
on the filter, the liquid substance, which had filtered 
through, although free from bacteria, will be found 
to be just as poisonous as the original broth in which 
the bacteria were grown. 
Very few bacteria produce such separable poisons, 
or exotoxins, the most important bacteria which belong 
to this class are the diphtheria, the tetanus, and the 
gas bacilli. 
2. Endotoxins ("internal" poisons) are inseparable 
poisons which are firmly attached to the bacterial cell 
and are not secreted by it but are liberated only upon 
the death and disintegration of the bacterium. 
The greater number of the pathogenic bacteria pro- 
duce such poisons, the endotoxins; as, for example, the 
typhoid and the colon bacilli, the staphylococci and 
the streptococci, and the cholera spirilla, etc. 
3. In addition to these two classes of poisons, there 
is a third kind, present in all bacterial bodies, after 
the removal of exotoxins and endotoxins, a certain pro- 
tein residue, which differs from both of the above in 
that it is not specific (that is, whether it is derived 
from the diphtheria or the typhoid bacilli, it will pro- 
duce the same effects when injected into an animal) 
and in that its action is very mild, since its injection 
causes only mild local inflammations or abscesses. 
Besides the difference in the mode of production the 
exotoxins differ from the endotoxins in that the former 
are destroyed by lower temperature than the latter. 
Now that we know that the bacteria act not by their 56 
PRINCIPLES OF BACTERIOLOGY 
mere presence, but through the production of different 
poisons, the next question which suggests itself is: 
How do the bacterial poisons act? 
It is one of the most widely accepted facts in the 
whole domain of bacteriology that the bacterial poisons, 
whether exotoxins or endotoxins, have a more or less 
definite selective chemical action on special tissues and 
organs, thus the exotoxins of the tetanus bacillus acts 
specifically on the nervous tissue, the endotoxin of the 
streptococcus, and staphylococcus act on the red blood 
cells, and so forth. 
The special selective action, or, to borrow the term 
from chemistry, affinity, which seems to exist between 
certain bacteria and certain tissues, depends upon the 
physical and chemical ability of the poisons to enter 
into union with the tissue cells. The famous experi- 
ment brought forth as a proof of this contention is 
that of Wassermann and Takaki; viz., if brain tissue is 
allowed to remain in the solution of the tetanus exo- 
toxin, and is then removed, the remaining fluid is free 
from any poisonous effects, while the injection of the 
brain tissue into an animal will result in the produc- 
tion of tetanus, thus showing that the brain tissue had 
actually absorbed the tetanus exotoxin from the solu- 
tion. 
III. Immunity (from Latin "immunis," meaning safe) 
When we stop to consider that so many people har- 
bor enormous numbers of bacteria while only a small 
number of individuals actually develops infection and 
a still much smaller number dies of infections, we are 
brought face to face with the fact that there is some 
peculiarity about some people that, under similar cir- 
cumstances, permits them to escape the infection while 57 
GENERAL BACTERIOLOGY 
others succumb to it, or permits them to recover from 
infections while others die from the same infection. 
This "something" or "vitality," as we frequently 
speak of it, is that peculiarity in the animal body which 
is spoken of as immunity, and we may define it as a 
lack of susceptibility to bacterial disease (which pre- 
vents us from contracting it) and a power of resistance 
which enables us to recover from bacterial disease (if 
the latter develops). 
It is evident, therefore, that the term "immunity" 
expresses the idea that the resistance is not absolute 
since in some people it is so strong as to prevent them 
from infections, while in others it is only strong enough 
to secure for them a recovery from infections, while in 
still others, it is not even strong enough to do that, and 
such individuals die. 
Classification and Varieties of Immunity 
Since the term "immunity" itself is only relative it 
is evident that the classification of its various types 
will also be, at best, only relative, yet it is convenient 
to consider the immunity as being of several distinct 
types. 
1. Natural, inherited or congenital immunity is that 
which exists in entire races, species or individuals at 
their very birth and is just as much one of their char- 
acteristics as their anatomic make-up. For example, in- 
fluenza and leprosy never occur-either spontaneously 
or through artificial inoculation-among animals. 
Among the different animal species there are great dif- 
ferences in resistance; for example, rats and dogs are 
remarkably immune to anthrax and the common fowl 
can not be infected with tetanus. 
Furthermore, within one and the same species the 58 
PRINCIPLES OF BACTERIOLOGY 
different races show different degrees of resistance to 
the same infection; our own domestic sheep are much 
less resistant to anthrax than the Algerian sheep; 
among the human beings we know that while the Amer- 
ican Indians and negroes are much more susceptible 
to tuberculosis than the white men, the latter are much 
more susceptible to yellow fever than are the negroes. In 
connection with this racial immunity the fact must not 
be lost sight of that we are also dealing with the dif- 
ferent hygiene conditions and customs which doubtless 
play a part in this question. 
Finally within the same race there exists great varia- 
tion in susceptibility and resistance among the individ- 
ual members, as seen, for example, in schools, when 
during an outbreak of an epidemic only a few pupils 
contract the infection. . 
2. Acquired immunity is one which does not exist in 
individuals at birth, but which develops in them either 
as a result of having had an attack of an infectious 
disease or is brought about by artificial means. 
It is well known that many infectious diseases occur 
but once in the same individual, one attack usually con- 
ferring a lasting immunity against subsequent attacks; 
the following table from Zinsser's hi feet ion and Resist- 
ance (published by the Macmillan Co., 1914, p. 60) may 
be referred to in this connection. 
Infectious diseases in which one at- 
tack usually confers lasting im- 
munity: 
Plague 
Typhoid fever 
(Second attacks rare-in 2.4 
per cent cases) 
Cholera 
Infectious diseases in which one at- 
tack does mot confer lasting im- 
munity : 
Pyogenic infections. 
Gonorrhea 
Pneumonia 
Influenza 
Dengue fever 
Diphtheria 
Recurrent fever 
Tetanus 
Erysipelas 
Beriberi 
Malaria 
Tuberculosis 
Smallpox 
Chickenpox 
Scarlet fever 
Second attack 
very rare 
Measles-Secondary attacks 
quite rare 
Yellow fever 
Typhus fever 
Syphilis-Reinfection rare 
Mumps-Secondary attack rare GENERAL BACTERIOLOGY 
59 
Such immunity-not congenital but brought about 
by having had one attack of an infectious disease-is 
called "naturally acquired immunity." 
If it is acquired by artificial means, it is spoken of 
as "artificial acquired immunity," and may be either 
"active or passive." 
Active immunity is that variety of the artificially ac- 
quired immunity in which the patient receives an in- 
jection of live, attenuated or dead bacteria (as in ty- 
phoid vaccination) or of the matter from a sore (as in 
smallpox vaccination) ; in such cases the patient is really 
made to undergo a very mild attack of the disease 
against which he is vaccinated, with the purpose of pro- 
ducing protective substances which will render him in- 
susceptible to the disease proper; this immunity is called 
"active" because the patient takes an active part in 
tbe production of immunity-he himself elaborates the 
protective substances. 
Passive immunity is that variety of the artificially ac- 
quired immunity in which the patient receives, in the 
injection, not the dead bacteria or their poisons, but 
the protective substances themselves; for example, in 
the case of diphtheria, a horse is injected with the 
diphtheria exotoxin (in gradually increasing doses) 
until he has a vast number of protective substances, 
then his blood (in reality the blood serum) containing 
these protective substances is injected into a patient 
suffering from diphtheria; since the patient did not 
take an active part in the elaboration of the protective 
substances, this variety of immunity is called "pas- 
sive" (on the other hand, the immunity produced in 
the horse by the injection of the diphtheria exotoxin is, 
of course, active, since the animal had elaborated its 
own protective substances). 60 
PRINCIPLES OF BACTERIOLOGY 
These varieties of immunity are graphically repre- 
sented in the following diagram. 
Immunity 
Natural (C o n- 
genital or 
inherited). 
Species -- (fowl 
to anthrax). 
Racial - (White 
men more re- 
sistant to tu- 
fa e r c u 1 osis 
than Indians 
or negroes). 
Individual. 
Naturally Ac- 
quired (b y 
having had 
one attack 
of the in- 
fectious dis- 
ease). 
Acquired 
Artificially Acquired 
(through protective substances) 
Active 
(when the pa- 
tient produces 
his own pro- 
tective s u b- 
stances, as, e. 
g., following 
typhoid vacci- 
nation). 
Passive 
(when the pa- 
tient receives 
protective sub- 
stances p r o- 
duced in some 
animals; as, e. 
g., when he re- 
ceives d i p h- 
theria a n t i- 
toxin). 
To make this still clearer let us take as an example 
the typhoid fever and various types of immunity which 
may exist against it: if a person is born immune to it, 
this will be natural immunity; if he develops immun- 
ity after birth, this is acquired immunity; if immunity 
was the result of this person's having had an attack of 
typhoid fever, we will call this naturally acquired im- 
munity ; if the person developed it after he had received 
an injection of dead typhoid bacilli, he has the active 
artificial immunity; but if he had been injected with 
the blood serum of an animal which had received dead 
typhoid bacilli, then his would be the passive artificial 
immunity. 
IV. Protective Substance or Immune Bodies 
Now that we have considered the various types of 
immunity we have to study just what the protective 
substances, which constitute the immunity, are: GENERAL BACTERIOLOGY 
61 
After all the conditions necessary for a successful in- 
fection have been satisfied (see the section on Infec- 
tion) and the patient has become a victim of an in- 
fectious disease, his organism makes an attempt to 
overcome it by producing the substances which will 
destroy the infective agent, i. e., the bacteria and their 
poisons. 
These protective substances are called immune bodies 
or antibodies; just where in the body they are pro- 
duced (that is, in what particular organ or organs) is 
unknown, but they are always found in the blood 
serum. 
A few words as to the blood: the blood of the living 
person is always liquid and consists of a fluid part called 
plasma; in this plasma are three kinds of blood cells: 
the red cells ( erythrocytes, from Greek erytheos, meaning 
red, and kytos, meaning cell), the white cells (leucocytes, 
from Greek leukos, meaning white), and the blood plate- 
lets. 
When blood is shed it ceases, after a few minutes, 
being liquid and becomes solid, or, as we usually say, 
it clots (coagulates) ; after a few hours a straw-colored 
liquid begins to separate from the part which remains 
clotted, this liquid part of the clotted part is called 
the blood serum; so that the liquid part of living (i. 
e., unshed) blood is plasma, while the liquid part of the 
clotted blood is blood serum. 
Now after this short digression, let us return to the 
consideration of the immune bodies or antibodies. 
The first statement to make in this connection is that 
the normal blood has the power to destroy bacteria; 
this power varies in different individuals; if the blood 
serum is kept for some time it will be noticed that this 
power rapidly diminishes. The substance responsible 
for this effect of blood is called complement. 62 
PRINCIPLES OF BACTERIOLOGY 
We now know that there are two chief kinds of bac- 
terial poisons-the exotoxins and the endotoxins, the 
former being the separable poisons, secreted by bac- 
teria and circulating in the blood throughout the en- 
tire body, while the bacteria themselves do not enter 
the blood circulation and usually remain where they 
have become lodged; the latter are inseparable poisons 
and only are liberated from the bacterial body after 
the bacteria have died, the bacteria themselves having 
circulated in the blood (the third kind of bacterial 
poisons, namely, the bacterial proteins are unimportant 
since they are not specific). It is evident that since the 
poisons liberated by the bacteria are different, the anti- 
bodies must be different, and such is the fact. 
Let us first consider the case of the exotoxin-producing 
bacteria, such as the diphtheria or tetanus bacilli; when 
these have lodged in the body, within a short time there 
will be produced an enormous amount of exotoxin; if 
the body should produce such substances that would 
kill the bacteria, this would be of very little use, be- 
cause w'hile it would prevent further elaboration of 
exotoxin, nevertheless, there has already accumulated 
enough poison to kill the body; for this reason a sub- 
stance is produced that does not attack the bacteria 
but neutralizes the exotoxin; this substance is called 
"antitoxin. " 
It is very important to know that the early opinion 
that the antitoxin actually destroys the exotoxin is er- 
roneous ; it prevents the action of exotoxin not by de- 
stroying it, but by neutralizing it, the mixture of exo- 
toxin and antitoxin being neutral much in the same 
manner as the mixture of an acid or alkali is. 
When bacteria act by producing not the exotoxin but 
the endotoxin, it is apparent that it would be useless 
for the body to produce antitoxins, and the body in GENERAL BACTERIOLOGY 
63 
such cases produces several antibodies (not one, as in 
the case of exotoxins). 
1. Bacteriolysins (bacteria, and Greek lysis, mean- 
ing solution) ; these are the substances which cause 
the bacteria to swell up, become granular, and finally 
undergo a complete solution. 
2. Agglutinins (from Latin, meaning glue) ; these are 
substances which cause the bacteria to lose their motil- 
ity if they are motile, and then clump in small or large 
groups. 
Agglutinins are not really immune bodies, because 
clumped bacteria are just as dangerous and virulent as 
those that are not agglutinated, and are mentioned here 
because of their importance in diagnosis of certain in- 
fectious diseases. 
3. Precipitins are substances which cause bacterial 
culture filtrates when mixed with the blood serum to 
form precipitates. Like agglutinins they are not true 
immune bodies, but are formed by bacterial invasion, 
and for this reason are mentioned here. 
4. Opsonins (from Greek opsonein, meaning prepare 
food for) are substances which so act on bacteria as to 
make them more easily destroyed by the white blood 
cells (leucocytes), as will be mentioned later. 
It is very important to remember that antitoxins are 
formed by injection of not only bacteria and their prod- 
ucts, but of many other poisons of plant and animal 
origin. 
Likewise lysins, agglutinins and precipitins may be 
produced by injection of numerous different sub- 
stances; one of the most common examples is the injec- 
tion of one animal with the red blood cells of another, 
whereupon the former will produce antibodies (lysins) 
against the red blood cells of the latter; such antibodies 64 
PRINCIPLES OF BACTERIOLOGY 
instead of being called bacteriolysins are called 
hemolysins ("blood dissolvers"). 
V. Application of Hemolysis and Agglutination to 
Blood Transfusion Tests 
Before transfusion of blood is resorted to in the treat- 
ment of such conditions as severe hemorrhage, pernicious 
anemia, shock, etc., the blood of the patient (who now 
is called "the recipient") and that of the one who gives 
the blood ("the donor") must be "matched," that is, 
we must ascertain that neither one of the two bloods 
dissolves (hemolysis) or clumps (agglutinates) the other. 
The blood is secured from both the donor and the 
recipient either from a vein by means of a syringe or 
from a finger by means of a spring lancet; a part of each 
blood is received into a small test tube and allowed to 
clot; the remainder of each blood is received into a 
test tube containing about 10 c.c. of normal salt solution 
(0.85% N'aCl) or 2% sodium citrate solution. 
Two c.c. of blood from each-donor and recipient- 
are sufficient for the test. 
The clotted portions of the blood are stirred up with 
a wire and put in an ice box for the serum to separate 
from the clot. The portions received in the citrate or 
normal salt solution are "washed" three times ("wash- 
ing" red blood cells consists of centrifugalizing the 
tubes for two or three minutes when the cells will be 
found packed on the bottom of the tube, pouring off 
the supernatant fluid, then pouring on some more of 
the normal salt solution and centrifugalizing again) ; 
after the third washing, enough salt solution is added 
to make the cells a 10% solution. 
The actual test is carried out as follows: Six 4x% 
centimeter test tubes (the so-called Wassermann tubes) GENERAL BACTERIOLOGY 
65 
are placed in a test tube rack, and to each one 1 c.c. of 
normal salt solution is added. 
The first tube receives 0.1 c.c. of the recipient's 
(patient's) cell emulsion and 0.2 of the donor's serum. 
The second tube receives 0.1 c.c. of the donor's cell 
emulsion and 0.2 of the recipient's serum. 
The above two tubes are for the actual test; the 
following four tubes are the controls: 
The third tube receives 0.1 c.c. of the recipient's cell 
emulsion and 0.2 c.c. of the recipient's serum. 
The fourth tube receives 0.1 c.c. of the donor's cell 
emulsion and 0.2 c.c. of the donor's serum. 
The fifth tube receives only 0.1 c.c. of the recipient's 
cell emulsion. 
The sixth tube receives only 0.1 c.c. of the donor's cell 
emulsion. 
The tubes are now incubated for thirty minutes at 
37.5° C. At the end of this time they are taken out, 
shaken throughly, and re-incubated for thirty minutes 
longer, and examined again. The last four tubes-the 
control tubes-will show neither hemolysis nor agglutina- 
tion on shaking. If both tubes one and two do not 
show hemolysis or agglutination, the blood of the donor 
is suitable for transfusion; if either tube one or two 
shows hemolysis (disappearance of red blood cells and 
the contents of the tube assuming clear golden or Bur- 
gundy color) or agglutination (as shown by cell clumps 
not disappearing on vigorous shaking) the donor's blood 
is not suitable for transfusion. 
Moss's Classification.-When transfusion must be done 
at once, and every hour counts, then a very rapid method 
of blood matching must be resorted to. 
The work of Landsteiner, Jansky and Moss has estab- 
lished that all human bloods belong, so far as agglutina- 
tion is concerned, to one of four groups. 66 
PRINCIPLES OF BACTERIOLOGY 
Blood serums of each group are kept in stock; the 
donor's red blood cells are added to a drop of each of 
the four serums on a glass slide, and, under the micro- 
scope, one easily determines which type the blood be- 
longs to. The donor and the recipient must belong to 
the same group; remember that in spite of the state- 
ments that group 4 is a universal donor (that is, the 
blood of one who belongs to group 4 may be used for 
transfusing the person who belongs to any group), se- 
vere reactions have followed its use. Always have the 
recipient and the donor belong to the same group. 
VI. Theories of Immunity 
There are two distinct explanations offered to account 
for the various phenomena of immunity, one of Metch- 
nikoff, called "cellular theory" (because he ascribes the 
most important part to the action of various cells of the 
body) or "theory of phagocytosis" (from Greek phagein, 
meaning to eat and kytos meaning a cell) ; and the other 
of Ehrlich, called "humoral theory" because the greatest 
importance is ascribed to body fluids (humoral is from 
Latin hiimor, meaning fluid). 
1. Metchnikoff's Cellular Theory, or Phagocytosis.- 
This theory is based on Metchnikoff's earlier observa- 
tions that ameba (a unicellular organism) when about 
to receive its nourishment would flow about the food 
particle, surround and engulf it, and then ingest it, and 
under the microscope the gradual disappearance of the 
food particle could be observed. 
Metchnikoff next observed that, when anthrax bacilli 
were injected into frogs, leucocytes (which possess 
ameboid movement) would engulf and destroy the 
bacilli. This line of observations, continued in the 
animals and human beings, soon left no room for doubt 67 
GENERAL BACTERIOLOGY 
that a similar process frequently took place; in fact, 
the actual engulfment and disintegration of gonococci, 
meningococci, staphylococci, and pneumococci is easily 
demonstrable. 
Metchnikoff divides the phagocytic cells into two 
groups: 1. Macrophages ("big eaters") which are 
large mononuclear leucocytes and certain fixed tissue 
cells, such as those lining serous cavities (pericardium,* 
peritoneum,** and pleura,!) cells of the spleen, lymph 
glands, etc. This takes place especially in chronic in- 
fectious diseases, such as tuberculosis, syphilis, etc. 
2. Microphages ("small eaters") are chiefly polymor- 
phonuclear leucocytes,! they are especially concerned in 
acute infectious diseases, such as meningitis, etc. 
Among the substances mentioned under the anti- 
bodies formed against the endotoxin-producing bac- 
teria were the so-called opsonins; it is now well to em- 
phasize their importance in connection with phagocyto- 
sis. The reason that not all bacteria are destroyed by 
phagocytosis is twofold: in some diseases, such as 
pneumonia, meningitis, etc., the number of leucocytes 
is increased from the normal 7,500 to 15,000, 20,000, 
and upwards per cubic millimeter; this is called 
leucocytosis; in some diseases there is a diminution of 
leucocytes, and this is called leucopenia; thus, first of 
♦Pericardium is the sac in which the heart is found. 
♦♦Peritoneum is the lining of the abdominal cavity. 
tPleura is the covering of the lungs. , 
^Leucocytes (the white blood cells) are of seven different kinds: 
1. Polymorphonuclear leucocytes or neutrophiles-these contain a 
multishaped (not multiple) nucleus. 
2. Small lymphocytes--these are the size of a red blood cell, the 
nucleus almost completely filling the cell. 
3. Large lymphocytes-differing from (2) in being larger (as large 
as the neutrophiles). 
4. Large mononuclears-as large as (3) but the nucleus is pushed 
to one side. 
5. Transitionals-as large as (4), but usually have crescent or 
horseshoe shaped nuclei. 
6. Eosinophiles-as large as (5) and contain red granules. 
7. Basophile or mast cells-as large as (6) and contain blue 
granules. 68 
PRINCIPLES OF BACTERIOLOGY 
all, for a successful phagocytosis there must be a 
leucocytosis; but even that alone is insufficient, as bac- 
teria may repel the leucocytes; this is called negative 
chemotaxis (chemical attraction) ; this is the reason 
opsonins are so important because they help attract the 
leucocytes (i. e., exert positive chemotaxis) and render 
bacteria more easily digestible by leucocytes. 
2. Ehrlich's Humoral Theory.-According to Ehr- 
lich every body cell consists of a central part which is 
concerned in its function (that is, if it is a muscle cell 
its function is contractile, if it is a gland cell its func- 
Fig. 9.-Ehrlich's theory of immunity, a, body cell; b, the receptor, or 
immune body; c and d bacterial poison; c, haptophore (anchoring) group; 
d, toxophore (poisonous) group. 
tion is to secrete, etc.), and another part which is con- 
cerned in receiving food particles; for the latter pur- 
pose it is conceived that each cell possesses chemical 
affinities or as Ehrlich calls them "side chains," which 
are the means of attracting to the cell the various nutri- 
tive substances. 
The poisonous material, such as products of bacteria, 
may also become attached to the'body cell through its 
side chains, and, in this manner, injure the cell (Fig. 9). 
If the poison is severe enough the body cell so injured 
will die; if not, it will only be damaged; since the body GENERAL BACTERIOLOGY 
69 
cell must have food, and since its side chains are not 
useful any more (having combined with the bacterial 
poisons), these side chains with the bacterial poisons 
attached to them, will be cast off into the blood stream, 
and new side chains will be formed (Fig. 10). But 
when new side chains are produced, not only enough 
are formed to replace the old ones, but many more 
than are really necessary, according to Weigert's law 
of overproduction or overcompensation-if, for ex- 
ample, only six side chains have combined with bac- 
terial poison, and, having become useless to the body 
Fig. 10.-Side-chains, with bacterial poison attached, cast off free into 
circulation, a, body cell; b, receptor or immune body (side-chain); c and d, 
bacterial poison. 
cell, were cast off, then, when regeneration of side 
chains takes place, several hundred will be formed in- 
stead of just six. The body cell does not need them; 
what becomes of them? Not being needed, they are 
cast off into the circulation and there combine with the 
poisons which originally were the cause of their forma- 
tion (Fig; 10). In order to understand the importance 
of this, let us take a concrete example: let us suppose 
that a body cell was injured by diphtheria exotoxin; 
let us further suppose that four side chains combined 
with this exotoxin; the body cell is injured but re- 70 
PRINCIPLES OF BACTERIOLOGY 
covers; these four side chains combined with the exo- 
toxin are cast off; new side chains are formed-not 
four but many more, according to Weigert's law; four 
of these new side chains are retained by the body cell 
to replace the old ones, the rest are thrown off into 
the circulation; having been formed as a result of in- 
jury to the cell by diphtheria exotoxin, these free side 
ABC 
0 E 
Fig. 11.-Illustration of Ehrlich's theory in case of diphtheria. 
A. A body cell (a) is injured by diphtheria toxin; (cd) through the union 
of the latter with the receptor (immune body on side chain, b). 
B. As a result of this the toxin and the receptor (immune body or side 
chain) are thrown off. 
C. New receptors are formed to replace the one which had been thrown 
off, but, according to Weigert's law of overproduction or overcompensation, 
instead of one receptor many more (three in the illustration) are produced. 
D. Only one receptor is needed to replace the lost ones, and the other 
two receptors are cast off. 
B. These two free receptors (immune bodies or side chains) having been 
produced as a result of stimulation by diphtheria toxin, have now a special 
affinity for it and will, whenever they meet it unite with it, thus protecting 
the body cell by intercepting the toxin (cd). 71 
GENERAL BACTERIOLOGY 
chains, circulating in the blood, have the same affinity 
for the diphtheria exotoxin as if they were attached 
to the body cell, and because of this affinity, wherever 
they will meet the diphtheria exotoxin, they will com- 
bine with it, and, in this way, protect the body cell; 
because if it were not for their combining with the 
exotoxin, the latter would reach the body cell and 
would there combine with it by means of the side chains 
which remained there (to replace the four original 
side chains), and would do the same damage to the 
cell as it had done before (Fig. 11). 
It is quite clear, therefore, that these free side chains 
are the antitoxins-they neutralize or combine with the 
exotoxins. This explains how a patient recovers from 
diphtheria; it explains why he remains immune (for 
some time or forever) after one attack of the disease; 
and, finally, it explains why such blood if injected into 
another animal would protect the latter from such in- 
fection. After the horse has been several times in- 
jected with the diphtheria exotoxin, his blood is full of 
these antitoxins (the side chains) and when injected 
into the patient, these side chains combine with the 
exotoxins in the patient's blood and thus protect him, 
in not allowing the exotoxins to reach the body cells 
and injure them. If a certain individual has no cor- 
responding side chains for a certain exotoxin, in the 
first place, he will never be affected by this kind of 
exotoxin-natural or inherited immunity. 
From his observations Ehrlich concluded that the 
toxin molecule consists of two parts: the haptophore 
group (the "carrying" part) which unites with the 
side chain of the body cell, and the toxophore group 
(the "poisoning" part), which does the actual dam- 
age (Fig. 12). 
Some of the poisonous substances are attached di- 72 
PRINCIPLES OF BACTERIOLOGY 
rectly to the side chain, as we have seen in the example 
of the diptheria exotoxin, such side chains, as for ex- 
ample, antitoxins, are called receptors of the first order. 
Such side chains as agglutinins and precipitins arc 
called receptors of the second order and differ from 
the antitoxins in that they have not only the combin- 
ing group but also a digesting group (because the ag- 
glutinins and the precipitins not only unite with bac- 
terial substances, as the antitoxins do with exotoxins, 
Fig. 12.-Ehrlich's conception of a toxin molecule. The toxin molecule 
(cd) consists of two parts: c, which combines with the receptor or side- 
chain b and is called the haptophore (the carrying or anchoring) group, and 
d, which contains the poisonous part and is called the toxophore group. 
but also cause clumping and precipitating; hence the 
need of the digesting group). (Fig. 13). 
Of the greatest interest and importance are the side 
chains, such as lysins (baeteriolysins, hemolysins, etc.), 
which are called receptors of the third order; we know 
experimentally that when an animal is injected with 
endotoxin-producing bacteria, e. g., cholera spirilla, its 
blood serum contains baeteriolysins; that means that 
if this animal's blood serum is mixed with cholera 
spirilla the latter will be destroyed-just what happens 
in the animal's blood; now, if this animal's blood serum 73 
GENERAL BACTERIOLOGY 
should be heated to 56° C. for thirty minutes, and 
then be mixed with cholera spirilla no such bacterial 
destruction will take place; this would seem to indi- 
cate that the bacteriolysins must have been destroyed 
by heat; yet, if to this heated blood serum a little nor- 
mal blood serum from another animal is added, prompt 
destruction of bacteria will take place; this means that 
bacteriolysis is produced by the joint action of two sub- 
stances : the specific immune body, produced by the animal 
as a result of injection (the antibody or the bacteriolysin) 
Fig. 13.-The receptor of second order (agglutinins and precipitins). 
a, body cell; b, receptor (immune body or side-chain), consisting of two 
parts; c,. anchoring group, d, digesting group; e, bacterial poison. 
which is not destroyed by heating the blood serum up 
to 56° C. for thirty minutes, and a nonspecific sub- 
stance, present in all normal as well as immune blood 
serum, and which is destroyed by heating up to 56° 
C.; this normal substance is the same substance which 
has been mentioned before in the section on immunity 
and to which a slight normal bactericidal (bacteria- 
killing) property of the blood is due; it is called com- 
plement. 
We thus see that the receptors of the third order, 
such as bacteriolysins or hemolysins (which destroy the 74 
PRINCIPLES OF BACTERIOLOGY 
red blood cells), do not act upon bacteria or red blood 
cells, as the case may be, alone, but in conjunction with 
the complement; because of this the side chain receptor 
of the third order has two "chains," one by which it 
combines with the bacterium or red blood cell or what- 
ever had been injected into the animal, and the other 
for combining with the complement. (Fig. 14.) 
While Ehrlich's humoral theory has very satisfactor- 
ily explained the production of all varieties of anti- 
bodies, for both the exotoxin and the endotoxin pro- 
Fig. 14.--The receptor of third order (bacteriolysis, hemolysin). The 
receptor (side-chain or immune body), b consists of two parts; c, which 
unites with bacterium e and d which unites with complement (/). 
ducing bacteria, it fails to take into consideration the 
phagocytosis of Metchnikoff-a phenomenon which 
doubtless plays a tremendously important part in many 
infections. 
On the other hand, Metchnikoff's theory of 
phagocytosis does not explain as many of the phe- 
nomena of immunity, as does Ehrlich's, and it is neces- 
sary to use both theories in order to have a clear pic- 
ture of immunity. 
3. D'Herelle's Phenomenon of Bacteriophage.-As a 
result of extensive experimental studies D'Herelle of GENERAL BACTERIOLOGY 
75 
Pasteur Institute claims that he discovered an ultra- 
microscopic organism (one which cannot be seen with 
an ordinary microscope), which he claims is the main 
source of our immunity. According to him, this ultra- 
microscopic organism or, as he calls it, bacteriophage, 
is parasitic upon (that is, lives at the expense of) ordi- 
nary bacteria (just as bacteria are parasites upon human 
body), and during the disease development destroys the 
bacteria which produces the given disease; he demon- 
strated the presence of bacteriophage by mixing a part 
of dysenteric stool with the dysentery culture where- 
upon in the latter bacteriolysis took place, and the bac- 
teriolytic substance could then be transmitted and trans- 
ferred from this tube to others. D'Herelle's idea, there- 
fore, is that in all infectious diseases the question of 
recovery depends upon the fact that in the human and 
animal intestines there are the minute (ultramicro- 
scopic) bacteria which get into the disease producing 
bacteria and secrete a dissolving (lytic) substance on 
these bacteria, and then recovery takes place; there are 
bacteriophages for all bacteria. 
In D'Herelle's monograph just published under the 
title "The Bacteriophage" evidences are given where 
the administration of the properly produced bacterio- 
phage (contained in the filtrates of such material as pus, 
stool, infected fluids) has produced definite improvement 
and recovery in numerous infectious diseases. 
No matter what the nature of the bacteriophage is- 
whether it is, as D'Herelle claims, "a bacterium within 
a bacterium," or a ferment produced by intestinal 
mucous membrane, as claimed by Kabeshima, or an 
autolytic ferment produced by bacteria themselves, as 
claimed by Bordet-D'Herelle's phenomenon is one of 76 
PRINCIPLES OF BACTERIOLOGY 
the greatest bacteriological discoveries of recent years, 
and one which promises to revolutionize our conception 
of immunity, infection and treatment of infectious 
diseases. 
VII. Anaphylaxis 
Anaphylaxis is the opposite of prophylaxis-the lat- 
ter is the prevention of infection, while anaphylaxis 
(from Greek ana, meaning against or no, and phylaxis, 
meaning protection) means an abnormal sensitiveness to 
infection. 
By anaphylaxis we mean sensitiveness due, not only 
to bacteria, but to any other proteins; it explains why 
some people are left more susceptible to infectious dis- 
ease after one attack than ever before; why some people 
can not eat certain kinds of food, as e. g., shell fish; why 
in some people injection of a serum (not only immune 
serum such as diphtheria, but even a normal horse serum 
which is given to control hemorrhage) produces danger- 
ous symptoms of shock, and, at times, even death. 
The main facts about anaphylaxis can be summed up 
as follows: 
1. Any protein-whether bacterial plant or animal- 
can produce anaphylaxis. 
2. The second dose of such protein may be infinitesi- 
mally small. 
3. If the animals do not develop anaphylaxis after the 
second injection, they remain forever immune. 
4. The greatest liability for the development of anaphy- 
laxis is between the tenth and fifteenth day after the 
first injection. 
5. Anaphylaxis can be prevented by giving the second 
injection before the tenth day. 
6. Anaphylaxis can be passively transferred from GENERAL BACTERIOLOGY 
77 
mother to her offspring, that is, if mother should be given 
one injection, and her offspring should be injected with 
her blood, and then with an injection of the sensitizing 
substance, the offspring will develop anaphylaxis. 
The experiments of Pearce and Eisenbrey, and par- 
ticularly those of Schultz, Dale, and Weil, seem to es- 
tablish definitely that the anaphylactic reaction takes 
place not in the blood stream but in the fixed tissue 
cells. 
Practically, the knowledge of anaphylaxis is very im- 
portant. 
1. Some diseases can be diagnosed by taking advantage 
of the fact that if a person is unusually sensitive to a 
certain organism because he is infected with it, an in- 
jection of the specific bacterial protein will cause a 
sharp reaction and will help diagnose his infection; on 
this principle are based such tests as tuberculin; luetin 
(for syphilis), and mallein (for glanders); a small 
amount of specific bacterial protein is injected into the 
skin, and if a person suffers from an infection caused 
by this particular bacterium, a marked reaction shows 
itself both at the site of injection and in the general 
condition of the patient. 
2. It explains the mysteries of the so-called "serum- 
disease"-disease which follows the administration of 
various serums. In many cases the serum should be given 
in very small doses before the full amount given in order 
to avoid the possibility of anaphylaxis. 
Besredka, of Paris Pasteur Institute, developed a 
more perfect technic of "desensitization," that is, avoid- 
ing the anaphylaxis, by testing the sensitiveness of a 
patient by first introducing minute quantities of the 
serum subcutaneously. 
Diagnosis of Hay Fever and Asthma by Anaphylactic 78 
PRINCIPLES OF BACTERIOLOGY 
lieaction.-Through the work of Dunbar, Cooke, Vander 
Veer, and particularly of Walker, it is now possible to 
establish the particular pollen causing in a given patient 
hay fever or the particular protein (either food, bac- 
teria, or animal appendages) in asthmatic patients. A 
little powdered protein (prepared from a pollen, a bac- 
terium, an article of food, or such material as chicken 
feathers, animal dandruff, etc.,) is put over a shallow 
abrasion on a patient's arm, and a drop of decinormal 
sodium hydroxide solution is placed over it (to dissolve 
the protein) quickly, if the patient is susceptible to the 
given protein; within one to ten minutes a fairly large 
urticarial wheal (depending on patient's susceptibility) 
develops at the site of the test. When the proteins re- 
sponsible for the patient's condition are established, a 
series of injections with the protein is given, usually 
with very good results. 
VIII. The Relation of Leucocytes to Infections 
The human blood is composed of the liquid part called 
plasma, and three types of cells, called, respectively: 
white blood cells or leucocytes, red blood cells or eryth- 
rocytes, and blood platelets; the number of erythro- 
cytes is 5,000,000 per cu. mm., that of the leucocytes is 
7,000-8,000 per cu. mm., and that of blood platelets is 
300,000 per cu. mm. 
While the erythrocytes are all alike-just as the blood 
platelets are all alike-the leucocytes are of seven dif- 
ferent kinds, as follows: 
1. Polymorphonuclear leucocytes or neutrophiles: 
these constitute about 70 per cent of all leucocytes. 
2. Small lymphocytes make up 20 per cent. 
3. Large lymphocytes-about 3 per cent. 
4. Large mononuclear leucocytes-about 3 per cent. GENERAL BACTERIOLOGY 
79 
5. Transitional leucocytes-about 2 per cent. 
6. Eosinophile leucocytes-about 1 per cent. 
7. Basophile leucocytes or mast cells-about 1 per 
cent. 
Generally speaking, in most infections there is an in- 
crease in the number of leucocytes-leueocytosis, al- 
though in a few acute infectious diseases the reverse is 
the rule, the leucocytes being diminished-a condition 
called leucopenia. 
Leucopenia occurs, quite characteristically, in ty- 
phoid fever, tuberculosis, malaria, dengue, measles, in- 
fluenza, and such tropical diseases as Kala-azar. 
Leueocytosis occurs with especial regularity in pneu- 
mococcic, septic (streptococcic and staphylococcic), 
meningococcic and colon bacillus infections. 
What is just as important as, or even more important 
than, the white cell count (to determine the presence 
of leueocytosis or leucopenia) is a differential count by 
which is understood the counting of the percentages of 
the various leucocytes in a stained blood film prepa- 
ration. 
Again, in a general way, one may say that in acute 
infections the neutrophiles are increased while in a 
chronic infection the lymphocytes are (the neutrophiles 
being the microphages and the lymphocytes the macro- 
phages of Metchnikoff) increased. 
This increase or decrease of neutrophiles or lympho- 
cytes is independent of the total leueocytosis or leuco- 
penia-that is, one may have an increase of total number 
of leucocytes and a decrease of any given kind of leu- 
cocytes, or one may have a decrease of total number 
of leucocytes with an increase of any one or more types 
of leucocytes; for example, one may have 15,000 leu- 
cocytes and only 50 per cent neutrophiles or 30 per cent 80 
PRINCIPLES OF BACTERIOLOGY 
lymphocytes, or one may have 5,000 leucocytes and 80 
per cent neutrophiles or 50 per cent lymphocytes-such 
increase or decrease in the percentages of the different 
types of leucocytes in the presence of the opposite con- 
ditions of the total leucocytes is called relative increase 
or decrease: if for example one has leucocytosis of 
15,000 and only 50 per cent neutrophiles (instead of 
70 per cent) this decrease is relative (because there 
really is an absolute increase of neutrophiles--50 per 
cent of 15,000 being 7,500, whereas normally one has 
only 70 per cent of 8,000, which is 5,600) ; again if one 
has a leucopenia of 5,000 with 40 per cent (instead of 
20 per cent) of lymphocytes, this increase in lympho- 
cytes is only relative (since there really is an absolute 
decrease of lymphocytes, 20 per cent of 8,000 is 2,500, 
whereas 40 per cent of 5,000 is 2,000). 
It is of the greatest importance to determine the dif- 
ferential count, especially the percentage of neutro- 
philes, because while increase in the total leucocyte count 
gives us an idea of the protection or defense of the body, 
the increase of neutrophiles corresponds to the severity 
of the infection-the student should always remember 
this: total leucocytosis refers to protection, increase of 
neutrophiles refers to severity of infection. 
The following combinations may, therefore, be 
present: 
1. Leucopenia (e.g., 5,000) or slight leucocytosis (e.g., 
10,000) with high percentage of neutrophiles (e.g., 90 
per cent)-this is the most unfavorable combination, as 
it means low protection against a very severe infection. 
2. High leucocytosis (e.g., 20,000) with high percent- 
age of neutrophiles (e.g., 90 per cent); this is not quite 
as bad, as it means a strong protection against a very 
severe infection. GENERAL BACTERIOLOGY 
81 
3. Leucopenia (e.g., 5,000) or low leucocytosis (e.g., 
10,000) with a low percentage of neutrophiles (e.g., 70 
per cent)-this shows a weak protection against a weak 
infection. 
4. High leucocytosis (e.g., 20,000) and a low percent- 
age of neutrophiles (e.g., 75 per cent)-the most favor- 
able combination, as it means a very strong protection 
against a mild infection. 
Walker has summarized the various combinations into 
what is known as the Walker's Index: he regards the 
normal leucocyte count as 10,000, and the normal per- 
centage of neutrophiles as 70 per cent; he further says 
that so long as the neutrophiles (the index of the severity 
of infection) increases 1 per cent for every 1,000 of the 
total leucocyte count (the index of protection), there is 
no cause for alarm;, in other words if total leucocyte 
count is 10,000, percentage of neutrophiles may be 70; 
if total leucocyte count is 15,000, percentage of neutro- 
philes may be 75; if total leucocyte count is 20,000, 
percentage of neutrophiles may be 80; if total leucocyte 
count is 25,000, percentage of neutrophiles may be 85; 
if total leucocyte count is 30,000, percentage of neutro- 
philes may be 90; and so forth, without causing any 
alarm 
If, for example, the total count is 20,000 and neutro- 
philes are 80 per cent, the index is 1. If the total 
count is 20,000 and neutrophiles are 70 per cent, the 
index is +10 (that is, neutrophiles are 10 per cent less 
than they could be with safety). If the total count is 
20,000 and neutrophiles are 90 per cent, the index is 
-10 (that is. neutrophiles are 10 per cent too much for 
safety). The more negative the index, the graver the 
outlook. CHAPTER V 
THE STUDY OF BACTERIA AND GENERAL 
BACTERIOLOGIC TECHNIC 
I. Study of Bacteria in Living State 
The study of bacteria in living state is carried out in 
what is called the "hanging drop" preparation: A 
"hollow" slide is used-an ordinary glass slide, usually 
3 inches by 1 inch, in the center of which a circular depres- 
sion is made, the edges of which are smeared with vase- 
line, then a drop of fluid medium in which the bacteria 
have been grown is placed in the center of an ordinary 
cover-glass (this is a piece of very thin glass, about 1 
inch square) ; the majority of books then suggest to lift 
up the cover-slip and then invert it over the concavity 
of the "hollow" slide, so that the drop hangs over the 
depression of the slide; [my custom is to do just the oppo- 
site ; namely, without disturbing the cover-glass, I lift up 
the slide and invert it over the cover-glass so that the 
vaseline around the depression of the slide just touches 
the cover-slip, and then put the slide down on the table 
with the cover-glass uppermost; in this way the danger 
of breaking the very thin cover-glass in handling it with 
fingers is eliminated (the danger of crushing it when 
inverting the glass slide over the cover-glass does not 
exist since the vaseline over the depression of the slide 
acts as a buffer between it and the cover-glass) ; besides, 
another source of danger is done away with; namely, 
that of spilling or smearing the bacterial drop while in- 
82 GENERAL BACTERIOLOGY 
83 
verting it over the glass slide-a very real danger, too, 
when handling virulent bacteria.] 
If the bacteria have been grown on a solid medium, 
some growth should be taken from the culture tube with 
a platinum needle and emulsified with physiologic salt 
solution (0.8 per cent solution of NaCl) and a drop of 
this emulsion should then be transferred to the cover 
Fig. 15.--A, staining support with a gas burner underneath; B, staining 
bottles; C, Petri dishes. 
slip. The preparation is then ready for the microscopic 
examination. 
The main object to be accomplished by studying the 
bacteria in this way is to determine whether or not they 
are motile. 
II. Study of Bacteria in Stained Preparation 
For this purpose a drop of fluid culture or a drop of 
bacterial growth emulsified in a little physiologic salt 84 
PRINCIPLES OF BACTERIOLOGY 
solution, is spread thinly on a glass slide or a cover-slip, 
is allowed to dry in the air, is then rapidly passed several 
times through the flame of a Bunsen burner (this process 
fixes the preparation), and is now ready for staining. 
(Fig. 15.) 
Ordinary Stains (Gruebler's or Merck's) 
The stains used for bacteriologic purposes are anilin 
dyes, which are prepared from saturated solutions cither 
alcoholic or aqueous (in water). 
The most commonly used stains are: 
Methylene blue: aqueous saturated solution is 6.7 per cent. 
Methylene blue: alcoholic saturated solution is 7 per cent. 
Gentian violet: aqueous saturated solution is 1.5 per cent. 
Gentian violet: alcoholic saturated solution is 4.8 per cent. 
Basic Fuchsin: aqueous saturated solution is 1.5 per cent. 
Basic Fuchsin: alcoholic saturated solution is 3 per cent. 
The alcohol used here is understood to be 95 or 96 per 
cent alcohol. 
After the bacterial preparation has been fixed, the 
stain is poured on the slide, and is allowed to remain 
there for two to five minutes; most frequently methylene 
blue is used for routine examinations. The stain is then 
poured off the slide, the preparation is washed in water, 
dried on filter paper, a small drop of cedar oil is placed 
on the cover-glass or slide, or, if the preparation is to 
be preserved, a drop of Canada balsam is to be put 
between the glass slide and the cover-glass, and a drop 
of cedar oil is then placed on the cover-glass; the 
preparation is then ready for microscopic examination. 
(Fig. 16.) 
No directions for the use of the microscope are given, 85 
GENERAL BACTERIOLOGY 
for it is my conviction that no directions will ever do any 
good; the pupil must be individually taught just how 
to handle the microscope and care for it. 
Special Staining Methods 
A. Gram's Method.-This is an extremely important 
method which permits differentiation of bacteria into two 
classes: the so-called Gram-positive and Gram-negative 
bacteria; this will be made clear immediately after the 
Fig. 16.-Microscope (A) and artificial illumination (B). 
description of the method of staining which is as fol- 
lows : 
1. Prepare the bacterial smear in the usual manner on 
a glass slide or a cover-glass, as described in the section 
on the Study of Bacteria in Stained Preparation. 
2. Cover the preparation for five minutes with the 
anilin gentian violet stain, which is prepared as follows: 
5 c.c. of anilin is shaken thoroughly with 125 c.c. of dis- 86 
PRINCIPLES OF BACTERIOLOGY 
tilled water and the mixture is filtered through moist 
filter paper. 
To 108 c. c. of the mixture add 12 c. c. of the saturated 
alcoholic solution of gentian violet. 
3. Pour off the stain and cover it for thirty seconds 
with Gram's iodine solution, which is prepared as fol- 
lows : 
Iodine 1 gram 
Potassium iodide 2 grams 
Distilled water 300 c.c. 
4. Pour off the iodine solution and decolorize with 
95%-98% alcohol until it ceases to discharge the blue 
color. 
5. Wash the preparation in water, and leave some water 
on the slide. 
6. Pour a few drops of carbol-fuchsin stain and im- 
mediately wash the preparation in water. 
7. Dry on a filter paper and put a drop of cedar oil. 
The carbol-fuchsin stain is prepared as follows: Dis- 
solve 1 gram of basic fuchsin in 10 c. c. of absolute alcohol, 
and mix the 10 c.c. of this alcoholic solution of fuchsin 
with 90 c. c. of 5 per cent aqueous solution of carbolic 
acid. 
When stained by this method, it will be found that 
some bacteria have retained the gentian violet stain, 
while others have lost it and have taken up the carbol- 
fuchsin stain. Those bacteria which, when stained by 
this method, have retained the gentian violet (i. e., are 
stained violet) are called Gram-positive, while those 
which have lost it and have taken up the carbol-fuchsin 
(i. e., are stained red) are called Gram-negative. The 
following table shows the most important bacteria as 
belonging to either one or the other class: GENERAL BACTERIOLOGY 
87 
Bacterial Classification According to Gram's Method 
GRAM-POSITIVE 
(Retain the gentian violet) 
Staphylococci 
Streptococci 
Pneumococci 
Bacillus of anthrax 
Bacillus diphtheria 
Bacillus of tetanus 
Bacillus of tuberculosis 
Bacillus aerogenes 
capsulatus 
("Gas" bacillus) 
GRAM-NEGATIVE 
(Lose gentian violet, take carbol 
fuchsin and appear red) 
Bacillus typhosus 
Bacillus coli 
Meningococcus 
Gonococcus 
Glanders bacillus 
Bacillus pyocyaneus 
Bacillus of influenza 
Plague bacillus 
Cholera spirillum 
Friedlander's bacillus 
Paltauf's Modification.-This staining fluid retains its 
power for a much longer period, and is prepared as 
follows: 
Four c.c. aniline oil is mixed with 90 c.c. of distilled 
water and 7 c.c. of absolute alcohol. Shake well and 
filter through a moist filter paper until clear, and add 
2 grams of Gruebler's gentian-violet; allow to stand 24 
hours, filter before use. This will keep at least 6 to 8 
weeks. 
In staining proceed as follows: 
1. Prepare the smear in the usual manner. 
2. Pour the stain and allow to stand three minutes. 
3. Gram's iodine solution, two minutes. 
4. Absolute alcohol thirty seconds. 
5. Counterstain with carbol fuchsin (ten seconds) 
without washing. 
Many workers have lately recommended the use of 
acetone instead of the absolute alcohol. It is well worth 
trying. 88 
PRINCIPLES OF BACTERIOLOGY 
B. Stain for Spores.- 
1. Make and fix the preparation in usual manner. 
2. Cover with Loeffler's alkaline methylene blue and 
heat the stain until it boils, remove it from flame, then 
heat again; repeat this for one minute (Loeffler's alkaline 
methylene blue is prepared by mixing 30 c.c. of saturated 
alcoholic solution of methylene blue with 100 c.c. of 
1:10,000 solution of potassium hydroxide in water). 
3. Rinse in water. 
4. Decolorize with a mixture of 98 c.c. of 80 per cent 
alcohol and 2 c.c. of nitric acid, until all blue has dis- 
appeared. 
5. Rinse in water. 
6. Dip three to five seconds in a mixture of 10 c. c. 
of saturated alcoholic solution of eosin and 90 c.c. of 
water. 
7. Rinse in water and blot. 
Spores are stained blue and the body of bacteria are 
stained pink. (See Fig. 4.) 
C. Stain for Capsule.- 
1. Smear the bacteria in a drop of beef blood serum, 
dry and fix by heat in the usual manner. 
2. Stain for a few seconds with a mixture of 5 c.c. of 
saturated solution of gentian violet and 95 c.c. of dis- 
tilled water. Hold the preparation flooded in this over 
a flame until it steams. 
4. Wash off the stain with 20 per cent aqueous solu- 
tion of copper sulphate. 
5. Blot (do not wash in water). 
The capsule appears as a blue halo around a dark pur- 
ple cell body. (See Fig. 2.) 
D. Stain for Flagella.- (See Fig. 3.) 
Use bacterial emulsion from young cultures grown on 
agar media (see section on Culture Media). 89 
GENERAL BACTERIOLOGY 
1. Dry the preparation in air and fix by heat. 
2. Pour the following mixture, freshly filtered: 10 c.c. 
of 20 per cent aqueous solution of tannic acid, 5 c. c. of 
saturated aqueous solution of ferrous sulphate, and 1 c. c. 
of saturated alcohol solution of fuchsin. 
Allow this mixture to remain thirty to forty-five sec- 
onds, heating it gently. 
3. Wash thoroughly in water. 
4. Stain for one to two minutes, warming gently, with 
the following freshly filtered mixture: 
50 c.c. of 5 per cent anilin gentian violet. 
0.05 gram of sodium hydroxide. 
5. Wash in water and blot. 
E. Stain for Bacilli Tuberculosis and Other "Acid- 
Fast" Bacilli 
The Ziehl-Neelsen Method. 
1. Make the preparation (from the culture, urine, 
sputum, etc.) in the usual manner, dry in air and fix with 
heat. 
2. Pour the carbol-fuchsin stain (its preparation has 
been given in the section on Gram's Method), and heat 
gently until it steams; continue this for three to five 
minutes, this had best be done on a special staining 
support under Which there is a gas pipe with numerous 
perforations, connected by a piece of rubber tubing with 
the source of gas supply; this method permits of regu- 
lating the amount of heat just sufficient for steaming, 
thus preventing the boiling. (See Fig. 15.) 
3. Decolorize with 1 per cent hydrochloric acid or 5 
per cent nitric acid in alcohol, then with 90 per cent 
alcohol until no pink color is discharged. 
4. Wash in water. 
5. Stain in aqueous saturated solution of methylene 
blue for one to two minutes. 
6. Rinse in water, and blot. 90 
PRINCIPLES OF BACTERIOLOGY 
By this method the tubercle bacilli are stained red, 
while everything else (including other bacteria) is stained 
blue. 
The principle of this stain is as follows: the tubercle 
bacilli do not stain with ordinary dyes-for this reason, 
hot carbol-fuchsin is used; but other bacteria which may 
be present (in the sputum, urine, etc.) are also stained 
(red). When strong decolorizing acid is applied, how- 
ever, all other bacteria lose the carbol-fuchsin stain, and 
take the methylene blue, while the tubercle bacillus does 
not give up the carbol fuchsin in spite of the action of 
the strong acid solutions (for this reason it is called an 
"acid-fast" organism), and remains red. Other acid- 
fast bacteria are the smegma and the leprosy bacilli. 
Pappenheim's Method 
This is used in order to differentiate between the 
tubercle and the smegma bacilli, which are stained 
alike; this is especially important in the examination of 
urine, as smegma bacilli, derived from the genitals, may 
be the source of confusion. 
Preparations are made in the usual way: Stain with 
hot carbol fuchsin for two minutes. Pour off the stain 
without washing, and pour on the following mixture: 
Rosolie Acid 1 gram. 
Absolute Alcohol 100 c.c. 
Methylene-blue to saturation. 
Glycerine 20 c.c. 
This is poured on and drained off slowly, four or five 
times, then washed in water. 
In this way the smegma bacilli are decolorized, while 
the tubercle bacilli are stained bright red. 
F. Stain for Malaria and Blood Films-The best one 
is Wright's stain; which is prepared as follows (avoid 
using the commercial stain, as the various specimens vary 91 
GENERAL BACTERIOLOGY 
greatly in strength, and each one has to be tried out 
before it can be safely used) : 200 c.c. of 1 per cent solu- 
tion of methylene blue in 0.5 per cent solution of sodium 
bicarbonate in distilled water are steamed in Arnold's 
sterilizer for one hour. 
Upon cooling, 1,000 c.c. of 0.1 per cent aqueous solu- 
tion of eosin (water soluble) is added, until a metallic 
scum appears on the surface of the mixture. The pre- 
cipitate which has been formed is collected by filtration, 
dried, and a satured solution is made in methyl (wood) 
alcohol. This is filtered and diluted with one-fourth its 
bulk of methyl alcohol. 
To look for malarial organisms, make a preparation 
in the same manner as for the study of blood, the so- 
called preparation for a differential count: clean the pa- 
tient's ear or the tip of a finger with alcohol, dry and 
prick with a needle or a blood lancet; wipe away the 
first drop of blood, collect the next drop on a scrupulously 
clean glass slide; apply another slide, held at angle of 
about 45° so that the edge of the second slide just touches 
the first slide where the drop of blood is; this will 
cause the latter to spread, by capillary attraction, along 
Ihe edge of the second slide; drag it along the first slide, 
holding it firmly against it, and you will get a thin, even 
"blood film." This is allowed to dry, and the slide 
is then covered with the stain for one to two minutes 
(this does not stain but merely fixes the preparation be- 
cause of the methyl alcohol present in the stain). Now 
add a few drops of distilled water to the film covered 
with stain until a distinct metallic luster appears on the 
surface. Leave this on for five to ten minutes. Wash 
in distilled water and dry. 
In such a preparation the red blood cells appear pink 
(if they contain the malarial parasites the latter are seen 92 
PRINCIPLES OF BACTERIOLOGY 
as being in-not on-the red blood cells and have the 
various appearances as described in the section on Ma- 
laria) ; the leucocytes appear as follows: the polymor- 
phonuclears are body pink and the nuclei (from one to 
five segments) purple, the small and the large mononu- 
clears (the lymphocytes) show a narrow rim of pink 
protoplasm and large purple nucleus, the mast cells 
show purple granules, and the eosinophiles show red 
granules. 
Fig. 17.-Miscellaneous glassware. A, Erlenmeyer flask; B, graduate 
glass cylinder; C, anaerobic apparatus; D, fermentation tube; E, boiling 
flask. 
III. Plating and Anaerobic Cultures 
See Section on Applied Bacteriology. 
IV. Culture Media 
This is one of the most important things in bacteri- 
ology, not only because we can not study bacteria un- GENERAL BACTERIOLOGY 
93 
less we grow them, but because the different ways in 
which the different bacteria grow on media and the dif- 
ferent changes which they produce in the culture media 
(fermentation of sugars, the production of acid, coagu- 
lation of milk, liquefaction of gelatin, etc.) constitute one 
of the most valuable means of bacterial differentiation 
and identification. (Fig. 18.) 
By a culture medium (plural: culture media) we mean 
any substance on which bacteria grow outside the animal 
body. 
Before the various culture media can be prepared, sev- 
eral other things have to be attended to. 
Preparation of Glassware.-If the glassware is new, 
immerse it in 1 per cent solution of hydrochloric acid, 
then wash it in 1 per cent sodium hydroxide, and, finally, 
wash it in running water. Old glassware containing in- 
fectious material should first be autoclaved for one hour, 
then emptied, and boiled for one hour in soap suds, then 
cleaned with a brush, and sterilized for one hour in the 
hot-air sterilizer at 150° C. If it is very dirty, it may, 
after it has been autoclaved, be immersed for twelve to 
twenty-four hours in a mixture of three parts of saturated 
aqueous solution of potassium bichromate and one part 
of sulphuric acid. 
The usual glassware for culture media consists of test 
tubes, Erlenmeyer and Florence flasks, and Petri dishes, 
which arc shown in accompanying illustrations; flasks 
and tubes should all be plugged with nonabsorbent cot- 
ton ; the best and easiest way to plug the test tubes, is 
to take a piece of cotton, two inches square, put over 
the mouth of the test tube and push it in for a distance 
of one inch with a pencil or a glass rod. 
The Composition of Culture Media.-Most culture 
media contain meat, agar, peptone, salt, and water. 94 
PRINCIPLES OF BACTERIOLOGY 
With these substances as a basis, many so-called "enrich- 
ing" substances may be added, in order to enable us to 
grow Ihe bacteria which require especially rich food- 
stuffs ; of such fastidious bacteria we may mention 
pneumococci, streptococci, gonococci, influenza bacilli, 
etc. Of the "enriching" substances those most frequently 
used are various sugars, blood (either whole or "whipped" 
-defibrinated in order to prevent the coagulation), as- 
Fig. 18.-Making a transfer of a culture. 
citic fluid (the fluid removed from the patients suffer- 
ing from dropsy), etc. 
Speaking generally, the following requirements con- 
cerning the general nature of the ingredients used in 
preparing the culture media should be borne in mind: 
1. Distilled water should be used unless tap water is 
specified. 
2. All chemicals used should be of the highest grade 
obtainable (the so-called "chemically pure," C. P.) GENERAL BACTERIOLOGY 
95 
3. The peptone should be Witte's or American pep- 
tone. 
4. The meat should be lean, fresh beef. 
Titration of Media.-Titration of media is a very im- 
portant step. By titration of media is understood the 
adjustment of the reaction of the media, that is, whether 
they are acid or alkaline; the bacteria are, as a rule, very 
sensitive to this, and for this reason titration should be 
most carefully carried out in all cases. 
The color indicator used is 0.5 per cent solution of 
phenolphthalein in 50 per cent alcohol.* 
To determine whether the medium is acid or alkaline, 
take a few c.c. of the medium and drop one or two 
drops of the phenolphthalein solution; if the medium is 
acid, no color develops; if alkaline-a pink color appears. 
Most of the media are acid because of the presence of 
meat acids. 
For the actual titration of acid medium the so-called 
"one-twentieth normal solution of sodium hydroxide" is 
used, which is expressed as follows: N/20 NaOH, while 
for the titration of the alkaline medium "one-twentieth 
of normal solution of hydrochloric acid" is used, which 
is expressed as follows: N/20 HC1. By a "normal" 
chemical solution we understand "the molecular weight 
of a substance, expressed in grams, dissolved in a liter 
(1,000 c.c.) of distilled water; in the case of sodium 
hydroxide the formula is NaOH, and its molecular weight 
is that of sodium (Na) which is 23 plus that of oxygen 
(O) which is 16 plus that of hydrogen (II) which is 
one; that is, 23-|-16-j-l=40; the molecular weight of 
sodium hydroxide is, therefore, 40; now, if we dissolve 
40 grams of it in 1,000 c. c. of distilled water, we will 
♦Any per cent solution of alcohol can be prepared from the stand- 
ard 95 per cent alcohol by taking' as many cubic centimeters of 95 
per cent alcohol as the percentage desired and filling it with water 
up to 95 c. c.; in this case, for example, take 50 c. c. of 95 per cent 
alcohol and add 45 c. c. of water. 96 
PRINCIPLES OF BACTERIOLOGY 
have a "normal" solution of sodium hydroxide; if we 
wish to make one-twentieth normal solution take 1/20 of 
40, i. e., 2 grams, and dissolve it in 1,000 c. c. of dis- 
tilled water; to make one-tenth normal solution take 1/10 
of 40, i. e., 4 grams of NaOH in 1,000 e. c. of water, etc. 
In the case of hydrochloric acid the formula is HC1; 
the molecular weight of hydrogen (H) is 1; that of 
chlorine (Cl) is 35.5; therefore, the molecular weight of 
HC1 is 36.5; now HC1 being a liquid, we measure it in 
cubic centimeters (c.c.) and not in grams; take 36.5 c.c. 
of hydrochloric acid and add distilled water up to 1,000 
c.c.; to make one-twentieth normal solution of hydro- 
chloric acid, take 1/20 of 36.5, i. e., 1.825 c.c. of hydro- 
chloric acid and add distilled water up to 1,000 c.c. 
Titration of Acid Medium.-Into an evaporating dish 
of 100 c.c. capacity pour 5 c.c. of the medium to be 
titrated, and add 45 c.c. of distilled water. Boil for 
three minutes over a free flame (to drive off the carbon 
dioxide). Add one c.c. of the phenolphthalein solution; 
the medium being acid, no color will be present, as ex- 
plained above. Now from a special tall tube called a 
"burette" (this is a tall, narrow tube, usually of 50 c.c. 
capacity graduated in tenths of a c.c., and provided with 
a stopcock to permit removal of small quantities of fluids), 
a few drops of the "twentieth normal solution of sodium 
hydroxide" (N/20 NaOH) are permitted to drop into the 
evaporating dish containing 5 c.c. of the medium and 45 
c.c. of distilled water; at first no color is present, but 
little by little a pink color will develop, which, however, 
will disappear upon stirring with a glass rod. When a 
permanent pink color-that is, one which will not disap- 
pear upon stirring-has been reached, we know that the 
neutral point (that is, neither acid nor alkaline) has been 
obtained. GENERAL BACTERIOLOGY 
97 
Titration of Alkaline Media.-Titration of alkaline 
media is carried out in the same manner as that of the 
acid medium, except that a N/20 HC1 (one-twentieth nor- 
mal solution of hydrochloric acid) is used. When 1 c.c. 
phenolphthalein solution is added, every drop of the 
hydrochloric acid solution added will cause a pink color, 
as explained above, but as more and more of the N/20 
HC1 is added the color will gradually fade away, and 
when the color has completely disappeared, the neutral 
point has been reached. 
This is, in other words, just the opposite of the acid 
titration: there we work from no color to a permanent 
pink, and here from pink to no color. 
The Calculation.-Let us suppose that in titrating an 
acid medium we had to use 2 c. c. of N/20 NaOH to 
make our medium in an evaporating dish (5 c.c. of 
medium, not 50 c.c., as 45 c.c. was distilled water). The 
calculation is then easy: 
If 5 c.c. of the medium requires 2 c.c. of N/20 NaOH 
for neutralization, then 100 c.c. of the medium requires 
20 times as much (because 100 c.c. is twenty times as 
great as 5 c. c.) ; that is, 2X20=40 c. c. of NaOH. 
If we had prepared 1,000 c.c. of the medium (a liter), 
this 1,000 c.c. of the medium will require 10 times as 
much of N/20 NaOH as 100 c.c. did; i. e., 40X10=400 
c.c. of N/20 NaOH. Thus, to our 1,000 c.c. of the medium 
we must add 400 c.c. to the N/20 NaOH to make it neu- 
tral ; but adding 400 c. c. will make our medium too 
bulky; for this reason instead of adding one-twentieth of 
NaOH we will add the normal solution of NaOH; how 
much? Since the normal solution of sodium hydroxide 
is twenty times as strong as the normal twentieth solu- 
tion, it is evident that we must use one-twentieth of the 98 
PRINCIPLES OF BACTERIOLOGY 
normal twentieth solution, i. e., instead of 400 c.c. of 
N/20 NaOH we will use 20 c.c. of N/l NaOH. 
The calculations of the alkaline titration do not differ 
in any way from the above. 
For ordinary purposes we use a medium which is not 
neutral but is about 1 per cent; i. e., requires one c.c. of 
N/l NaOH per 100 c.c. of the medium, or 10 c.c. per 1,000 
c.c. (liter). In the above calculation, therefore, we 
should, instead of adding 20 c.c. of N/l NaOH to the liter 
of medium, have added only 10 c.c. of it, thus leaving 
the medium still 10 c.c. of N/l NaOH short for complete 
neutralization; that is, leaving it 1 per cent acid. 
The acidity of the medium is expressed by the sign -|- 
and the alkalinity by that of -. 
The Colorimetric Titration of Media.-This-the mod- 
ern method of titrating culture media (as well as many 
other fluids)-is based on the so-called "Hydrogen-Ion 
Concentration," and it is necessary to explain the mean- 
ing of this expression before proceeding to describe its 
application to the titration of culture media. 
The acidity of any solution depends on its contents 
of hydrogen ions (H) and the alkalinity on hydroxyl 
ions (OH) : thus, hydrochloric acid (HC1) is acid because 
of its hydrogen (H) contents, while sodium hydroxide 
(NaOH) is alkaline because of its hydroxyl contents (OH). 
The inaccuracy of the old method of titrating culture 
media is due to the fact that in using the phenolphthalein 
as an indicator, and in using sodium hydroxide and hydro- 
chloric acid for the correction of either acidity or al- 
kalinity, we do not actually correct them, because 
phenolphthalein is not sensitive enough to determine 
the hydrogen-ion concentration (which is actual acid- 
ity) ; we all know that while, for example, hydrochloric GENERAL BACTERIOLOGY 
99 
acid and acetic acid are both acids, yet the acetic acid 
is much "weaker" (scientifically, it means that the hy- 
drogen-ion concentration of acetic acid is less than that 
of hydrochloric acid); in spite of this, when titrated 
with an alkaline solution, such as sodium hydroxide 
(NaOH), using litmus or phenolphthalein as an indica- 
tor, it will be found that both hydrochloric and acetic 
acids will require the same amount of the alkali to be- 
come neutral to either litmus or phenolphthalein indica- 
tors. On the other hand, if the hydrogen-ion concentra- 
tion of each acid were determined, it would be found 
that that of hydrochloric acid is very much greater 
than that of acetic acid, and thus would show us that 
hydrochloric acid is very much "more acid" than the 
acetic acid. 
The hydrogen-ion concentration is expressed by PH, 
attached to which is a number expressing the logarithm 
of the hydrogen-ion concentration, e.g., PH6, or PH6.2, 
or Ph8, and so forth. 
Another reason for the inaccuracy of the older method 
of titration is the fact that in our culture media there 
are such substances as peptones, protein, phosphates, 
etc., which are known as "buffer" solutions, because of 
their property to resist changes in reaction. 
Clark and Lubs who have done so much for the ap- 
plication of hydrogen-ion concentration to bacteriolog- 
ical work have suggested the various indicators which 
express the true acidity by changing their colors within 
a certain range. 
In the actual practice of colorimetric titration of media, 
a colorimetric set is purchased or made which consists of 
indicators, buffer solutions, and colorimetric scale. 
Into a thoroughly cleaned test tube, rinsed out with re- 100 
PRINCIPLES OF BACTERIOLOGY 
distilled water, 2 c.c. of medium are measured, and to 
this 8 c.c. of redistilled water are added; 10 drops of 
indicator are also added to the tube, thoroughly mixed, 
and the color is compared against the scale, and, if 
found to be too acid, NaOH is added drop by drop, 
until the proper color is reached; the amount of NaOH 
necessary to bring the entire medium to the desired Ph 
concentration is calculated, added, and the medium 
titration is completed. 
Tubing the Media.-After the media have been pre- 
pared they are poured into test tubes (about 10 c. c. 
into each) or into flasks (about 30 to 50 c.c. into each) ; 
this is usually done by pouring the medium into a fun- 
nel to the tip of which a rubber tubing had been fitted, 
with a pinchcock, and the tubes and flasks are so filled 
from this; a special apparatus has been devised (see 
Fig. 19) which consists of a large glass bulb with a 
glass outlet, a stopcock, and a gauge which permits to 
pour into the tubes or flasks equal amounts of the medium. 
Sterilization of Media.-The ordinary media which 
do not contain sugar, glycerin, gelatin or animal serum, 
are sterilized in the steam pressure sterilizer (autoclave) 
at fifteen pounds pressure for fifteen to thirty minutes; 
the media containing substances which are apt to be in- 
jured by the high temperature must be sterilized by the 
fractional method (see the section on Destruction of Bac- 
teria) ; that is, by exposure for twenty minutes in Ar- 
nold's sterilizer, on three successive days. 
The media containing animal serum must be sterilized 
in water-bath or hot-air sterilizer at the temperature of 
60° to 70° C. for half an hour on three successive days. 
Some media are best sterilized by passing through the 101 
GENERAL BACTERIOLOGY 
special bacterial filters (Berkefeld's or Chamberland's) 
which are made of unglazed porcelain or earthenware. 
Slanting of Media.-The solid culture media, after 
Fig. 19.-Tubing the media. 
they have been sterilized, usually are slanted, that is, the 
tubes are laid down on a desk or table so that the upper 
end of the test tube rests on a ledge or glass tubing, in 102 
PRINCIPLES OP BACTERIOLOGY 
order that the medium, when coagulated, will form a 
slant in the tube (Fig. 19). 
Actual Preparation of Various Culture Media 
1. Meat Extract Agar 
Agar is a Japanese seaweed. 
Meat extract agar is the most commonly used medium. 
Staphylococci, typhoid and colon bacilli grow well on 
this. Other bacteria require richer media. 
(a) Pour into an agate vessel 1,000 c.c. of distilled 
water, add 15 grams of shredded (not powdered) agar, 
10 grams of Witte's peptone, 5 grams of sodium chloride 
and 5 grams of Liebig's meat extract. 
(b) Heat until agar is completely dissolved (thirty to 
forty-five minutes). 
(c) Add enough water to make up for loss by evapora- 
tion. 
(d) Cool to 60° C. and add whites of two eggs. 
(e) Heat in Arnold's sterilizer for thirty minutes, 
stir and heat fifteen minutes. 
(f) Add water to make up for loss by evaporation. 
Filter through cotton. 
(g) Titrate to the desired reaction (usually about 0.2 
per cent acid, to phenolphthalein). 
(h) Tube and sterilize in autoclave for thirty minutes 
at 15 pounds pressure. Slant. 
2. Meat Infusion Agar 
Meat infusion agar is one of the richer media, suitable 
for culturing streptococci, etc. 
(a) In a two-liter Erlenmeyer flask pour 1,000 c.c. of GENERAL BACTERIOLOGY 
103 
water and put 1 lb. of finely chopped lean beef, and put 
in the ice box over night. 
(b) Boil for twenty to thirty minutes, make up loss 
of water, and filter through a cotton gauze filter (2 or 3 
layers of gauze lined with cotton; when no more fluid 
runs through, gather the edges of the gauze over the cot- 
ton and squeeze it dry). 
(c) Add 10 grams of peptone and 5 grams of sodium 
chloride and heat (not over 50° C.) until peptone is 
dissolved. 
(d) Add 15 grams of shredded agar and boil until 
agar is dissolved. Make up to 1,000 c.c. 
(e) Heat for thirty minutes in Arnold's sterilizer, stir, 
and reheat for fifteen minutes. 
(f) Titrate, adjust the reaction to 0.2 to 0.5 per cent 
acid, filter through cotton and tube. 
(g) Sterilize thirty minutes in the autoclave at 15 
pounds pressure. Slant: 
3. Meat Extract Broth 
Meat extract broth is a liquid medium corresponding 
to meat extract agar. 
(a) To 1,000 c.c. of water add 10 grams of peptone, 5 
grams of Liebig's meat extract, and 5 grams of sodium 
chloride. 
(b) Heat until dissolved, and add water up to 1,000 
c.c. 
(c) Titrate and filter through paper (if not clear, add 
whites of two eggs, heat for thirty minutes in Arnold's 
sterilizer, and then filter through cotton). 
(d) Tube and sterilize in autoclave thirty minutes at 15 
pounds pressure. 104 
PRINCIPLES OF BACTERIOLOGY 
4. Meat Infusion Broth 
Meat infusion broth corresponds to meat infusion agar. 
It is prepared exactly as the meat infusion agar 
(Medium No. 2) except that the agar is omitted, and 
sterilized like it (in the autoclave for thirty minutes at 
15 pounds pressure or in Arnold's sterilizer for thirty 
minutes on three successive days). 
5. Sugar-free Broth 
Sugar-free broth is used for making different sugar 
media. Prepare the broth just as the meat extract broth 
(No. 3) is prepared. 
After the step b, add 10 c. c. of a twenty-four-hour-old 
culture of colon bacillus. Then incubate at 37° C. for 
twenty-four hours. (The colon bacillus will destroy all 
sugar present.) Heat for thirty minutes in autoclave at 
15 pounds pressure. Add water up to 1,000 c.c., filter 
through paper and titrate. 
6. Sugar Broths 
After No. 5 lias been prepared (the sugar-free broth) 
add different sugars (dextrose, lactose, saccharose, etc.), 
in proportion of 1 per cent tube in fermentation tubes, 
and heat for twenty minutes (not more, as it will spoil 
the sugars) in Arnold's sterilizer on three successive days. 
(Instead of fermentation tubes one can use the following: 
tube in ordinary test tubes; when tubed, place into each 
test tube a very small tube (2x^4 inch) inverting it, so 
that its mouth rests against the bottom of the larger 
tube. During the sterilization the medium will be forced 
up into the smaller tube, and if fermentation will take 
place upon inoculation, the empty space where gas is 
collected in the small tube will show it. GENERAL BACTERIOLOGY 
105 
7. Enriched Media 
All of the meat infusion media (No. 2 and No. 4) can 
be enriched, by adding glucose 1 to 2 per cent, or a few 
drops of ascitic fluid or of defibrinated or whole blood, or 
glycerin. 
When ascitic fluid is added greatest aseptic precautions 
should be observed, as after its addition no further steril- 
ization by heat is permissible since it would coagulate the 
albuminous part of the ascitic fluid. An ordinary meat 
infusion agar is made as above described, tubed and 
sterilized. When ascitic fluid is to be added, melt the 
agar tubes by heating in water bath, cool to 45° C., then 
(with all aseptic precaution) add to each tube a few 
drops of ascitic fluid, flaming the test tubes before and 
after the addition; incubate all tubes at 37° C. for 
twenty-four hours to see if they are sterile. 
For defibrinated or whole blood, do just as for the as- 
citic fluid agar, except that a few drops of defibrinated or 
whole blood is added. To obtain defibrinated blood, 
bleed a rabbit from the carotid artery or jugular vein into 
a sterile flask with 10 to 20 glass beads, shake the blood 
thoroughly, add a few drops to each tube of melted and 
cooled agar, and incubate for twenty-four hours to test 
sterility. 
If sugar is added (usually glucose 1 to 2 per cent) 
sterilize for twenty minutes instead of thirty minutes in 
Arnold's sterilizer on three successive days. 
8. Gelatin 
Gelatin is used to determine the gelatin-liquefying 
properties of bacteria. 
(a) To 1,000 c.c. of water add 5 grams of Liebig's 
meat extract, 5 grams of sodium chloride, 10 grams of pep- 106 
PRINCIPLES OF BACTERIOLOGY 
tone and 100 grams of best French sheet gelatin ("Gold 
Seal" brand). 
(b) Dissolve by heating, add water up to 1,000 c.c. 
(c) Cool to 60° C., add whites of two eggs, stir and 
heat for thirty minutes in Arnold's sterilizer. 
(d) Add water up to 1,000 c.c. filter through cotton. 
Sterilize in Arnold's sterilizer for thirty minutes on 
three successive days. 
(e) Do not slant this medium, as it is inoculated by 
' ' stabbing. ' ' 
(a) Large potatoes are well washed and scrubbed with 
a nail brush. Peel and wash in running water. 
(b) Cylindrical pieces are removed with a large apple 
corer, and each cylinder is cut diagonally into two 
wedges. 
(c) Immerse the wedges overnight in 1 per cent solu- 
tion of sodium carbonate to correct the normal acidity. 
(d) Insert the wedges into test tubes (if desired they 
can be first soaked in 25 per cent glycerin-the glycerin 
potato medium). 
(e) Sterilize for thirty minutes in Arnold's sterilizer 
on three successive days, or autoclave for thirty minutes 
at 15 pounds pressure. 
9. Potato Media 
10. Litmus Milk 
Litmus milk medium is used for determining the power 
of bacteria to curdle the milk and produce acid (as 
shown by fading of the blue color and the tube turning 
red). 
(a) Fresh milk is heated in a flask in Arnold's steril- 
izer for fifteen minutes. 
(b) Put flask in ice box to allow the cream to separate. 
The cream is then syphoned off. GENERAL BACTERIOLOGY 
107 
(c) Neutralize the acidity if in excess of 1.5 per cent. 
(d) Add a few drops of a 5 per cent aqueous solution 
of litmus. 
(e) Sterilize in Arnold's sterilizer for thirty minutes 
on three successive days. 
11. Loeffler's Serum Medium 
Loeffler's serum medium is used for diphtheria bacil- 
lus. 
(a) Beef blood is collected in sterilized jars, allowed 
to clot, the clot is loosened by a glass rod, and jars are 
kept on ice overnight. 
(b) Clear serum is pipetted off to sterile flasks. 
(c) To three parts of the serum one part of 1 
per cent glucose meat infusion broth is added, and the 
whole is tubed. 
(d) It is then sterilized in a special sterilizer called 
"inspissator" at a temperature of 70° C. for two hours 
on six successive days. 
12. Endo's Medium 
Endo's medium is used for the differentiation of the 
typhoid and colon bacilli. 
1. Prepare ordinary meat extract agar, except that it 
should contain 25 to 30 grams of agar per 1,000 c.c. of 
media, instead of the usual 15 grams, and it should be 0.2 
per cent acid. Do not tube it, but keep it in flasks. When 
Endo's medium is wanted, melt the agar, add to it 1.8 
c. c. of filtered 10 per cent solution of basic fuchsin in 95 
per cent alcohol and 25 c.c. of freshly prepared 10 per 
cent aqueous solution of sodium sulphite crystals. Then 
add 10 grams of lactose (milk sugar) in 50 c.c. of sterile 
water. Sterilize for fifteen minutes in Arnold's sterilizer 
on three successive days. 108 
PRINCIPLES OF BACTERIOLOGY 
On such a medium the typhoid colonies are colorless, 
while those of colon bacilli are red. 
13. Russell's Double Sugar Medium 
Russell's double sugar medium is used for the differen- 
tiation of the typhoid, paratyphoid and colon bacilli. 
(a) Prepare ordinary meat extract agar (25 grams 
of agar per 1,000 c. c. of medium, 0.8 per cent acid) and 
keep it in flasks. When needed, melt it. 
(b) Add enough of sterile 5 per cent aqueous solu- 
tion of litmus (usually about 30 c.c.) to give the medium 
a distinct violet color. 
(c) Add 0.1 per cent glucose (grape sugar) and 1 per 
cent lactose (milk sugar) dissolved in 50 c.c. of water, 
tube and sterilize in Arnold's sterilizer for fifteen min- 
utes on three successive days. Slant. When inoculations 
are made smear the surface and also stab. 
On such a medium typhoid growth will show a par- 
tial red color formation and no gas bubbles. The para- 
typhoid A and B will show a partial red color formation 
and gas bubbles in the red area, while the colon growth 
will turn the entire tube red and gas bubbles all over. 
14. The Double Sugar-Lead Acetate-China Blue 
Medium 
This medium is the most complete differentiating me- 
dium, as it tells at a glance whether the growth is one of 
typhoid, paratyphoid A, paratyphoid B, colon or B. en- 
teritidis. 
(a) Prepare ordinary meat extract agar, 0.3 per cent 
acid, and flask it. 
(b) Heat some 2 per cent neutral lead acetate in sterile 
distilled water, at 100° C. for one hour in water-bath, 
5 c.c. of this is added to 100 c.c. of agar (which has been GENERAL BACTERIOLOGY 
109 
melted and cooled to 60° C.). Tube in Wassermami test 
tubes (4x1/2 inch). 
(b) Prepare 1 per cent solution of China blue in dis- 
tilled water. 
(c) 0.4 c. c. of normal sodium hydroxide is added to 10 
c.c. of 1 per cent solution of China blue, and the mixture 
is heated in water-bath for ten minutes at 100° C. (the 
blue color now changes to brown). 
(d) 1.2 c.c. of this mixture is added to 100 c.c. of 
agar. 
(e) 0.1 per cent glucose and 1 per cent lactose are also 
added, and the mixture is heated for ten minutes in 
water-bath at 100° C. Cool to 60° C., and pour on the 
first layer (step A) the same amount as the latter. 
The changes caused by the different bacteria on this 
medium are shown in the following table. 
Bacillus 
Gas 
Bottom Layer 
Top Layer 
Typhoid 
No 
Black 
Pale blue 
Paratyphoid A 
Yes 
No change 
Pale blue 
Paratyphoid B 
Yes 
Black 
Colorless 
Colon 
Yes 
Black 
Deep blue 
Enteritidis 
Yes 
No change 
Colorless 
15. Cornstarch Medium 
This little-known medium is the best medium for 
gonococcus cultivation, also for pneumococcus and other 
bacteria which grow with difficulty. On this medium the 
gonococcus, which dies on all other media in four to five 
days, will live over four weeks, so that where cultivation 
and frequent transfers are to be made this medium will 
be found of greatest help. 
(a) 500 grams of lean beef are added to 1,500 c.c. of 
water, and put in a flask on ice overnight. 110 
PRINCIPLES OF BACTERIOLOGY 
(b) Strain through gauze, heat slowly to boil, add 
15 to 17 grams of shredded agar; when dissolved, neu- 
tralize to 0.4 per cent acid (phenolphthalein). 
(c) Cool to 60° C. add three whole eggs (beaten with 
a little water), put back on fire, heat quickly to boil, 
then turn the flame down to a simmer for fifteen min- 
utes. 
(d) Filter through cotton and gauze (both previously 
sterilized), add 10 grams of Squibb's cornstarch per 1,000 
c.c. of medium, rub the starch with 60 c.c. of filtrate to 
a milky consistency, pour back into the rest of filtrate 
and shake well. (If lumps are present filter again and 
add more starch.) 
(e) Sterilize in Arnold's sterilizer, shaking every ten 
minutes. 
(f) Tube or flask. Autoclave fifteen minutes at 8 to 
10 pounds pressure. 
(g) Cool down to 42° C., tilt a few times (without 
wetting the stopper), slant overnight, then stand them 
up for a day. Good tubes show about % of an inch of 
water of condensation. 
16. Bile Medium. Used in Blood Cultures 
Take 900 c.c. of fresh ox bile, 100 c.c. of glycerin and 
20 grams of peptone. Put in small Erlenmeyer flasks 
(about 40 to 50 c.c. in each) and sterilize in Arnold's 
sterilizer for thirty minutes for three successive days. 
17. Media for Tubercle Bacillus 
The best media are those of Petroff; their preparation 
requires a good deal of skill, and the reader should con- 
sult Petroff's own description observing the most min- 
ute details. GENERAL BACTERIOLOGY 
111 
A very good medium is the glycerin potato. Meat in- 
fusion agar with about 10 to 15 per cent of whole blood 
(not defibrinated) is also excellent. 
One of the best media is that of Dorset: 
(a) Carefully break 16 eggs into a flask, shake it 
until the whites and yolks are well mixed. 
(c) Add 100 c.c. of distilled water, strain through a 
sterile cloth. 
(c) Pour 10 c.c. each into sterile test tubes, slant, and 
Fig. 20.-Two types of water-baths. 
put them into an inspissator, sterilizing at 72° C. for 
four hours on two days. 
(d) On third day raise the temperature to 76° C. 
(e) Sterilize in Arnold's sterilizer at 100° C. for 
fifteen minutes. 
Add two or three drops of sterile water to each tube 
before inoculation. 
One of the new media which is excellent is that of 
Williams and Burdick.* 
*Jour. Bacteriol., July, 1916, i. 112 
PRINCIPLES OF BACTERIOLOGY 
(a) Dilute the egg whites with 10 parts of distilled 
water, shake well; pass through a thin layer of cotton, 
heat to 100° C., filter through cotton. 
(b) Yolks are diluted with 10 parts of distilled water, 
and well stirred. Add 1 c.c. of normal sodium hydroxide 
solution to 100 c.c. of yolks emulsion. Heat to 100° C., 
and filter. 
(c) 500 grams of finely chopped lean veal are put into 
1,000 c.c. of wTater containing 15 per cent glycerin; put 
on ice for twenty-four hours, filter, and add 5 grams 
of sodium chloride, and heat to boiling. Filter and 
render 1 per cent alkaline (to phenolphthalein). 
(d) Put 300 c.c. of 10 per cent egg white solution 
(obtained in a) into a liter flask. Three hundred c. c. of 
the yolks solution are placed in another liter flask; 400 
c. c. of the veal infusion, to which 15 grams of powdered 
agar are added, are placed in a third liter flask. 
(e) Sterilize all three flasks in the autoclave at 15 
pounds pressure for fifteen minutes. While hot, pour 
into the meat infusion flask 1 c.c. of 1 per cent alcoholic 
solution of gentian violet. 
(f) Then pour the contents of this flask into the egg 
whites flask, and pour into it the contents of the yolks 
flask. Pour the whole back and forth until thoroughly 
mixed. Tube and slant for seventy-two hours at the 
room temperature. Flame the stoppers. 
No sterilization is permitted, and for this reason the 
entire procedure should be carried out under the strictest 
aseptic precautions. CHAPTER VI 
APPLIED BACTERIOLOGY 
Examination of Material from Patients. 
1. Examination of Smears.-Very frequently it is 
necessary to examine the discharge from some part of 
the body; in such case a drop of discharge is placed 
on a clean sterilized glass slide by means of a sterile 
swab or platinum loop, and is evenly spread (if the dis- 
charge is very "thick" it may be emulsified on the slide 
with a drop of sterile physiologic 0.8 per cent salt solu- 
tion). It is then dried in the air, and passed several 
times through a Bunsen burner flame. Stain by Gram's 
method. 
2. Making Ordinary Cultures.-"When cultures have 
to be made from discharge, one must be governed 
in his choice of media by the organism suspected; if 
staphylococcus is suspected use meat extract agar, but 
since streptococcus may also be present and since it does 
not grow so very well on meat extract media, it is best 
to use meat infusion agar or broth. If gonococcus is 
suspected use ascitic fluid salt-free agar, or meat in- 
fusion glucose or blood agar, or cornstarch agar. 
3. Anaerobic Cultures.-At times an anaerobic organ- 
ism may be suspected, and it is well then to make an 
anaerobic culture. This consists of making an ordinary 
culture and placing it in a specially constructed 
navy "anaerobic jar" and incubating it in the usual 
manner. 
113 114 
PRINCIPLES OF BACTERIOLOGY 
If such a jar is not on hand, a 2 per cent glucose meat 
infusion agar (not slanted) is inoculated by stabbing 
and about 3 to 5 c.c. of liquid paraffin or vaseline is 
poured on the top of the agar. 
J. II. Wright's Method.-Make a deep stab culture in 
glucose agar or gelatin, having boiled the medium be- 
fore inoculating. Then flame the cotton plug and press 
it down into the tube so that the top lies about three- 
Fig. 21A.-The incubator. 
fourths of an inch below the mouth of the test tube. 
Next fill in about one-fourth of an inch with pytogallic 
acid; then add 2 or 3 c.c of 10 per cent solution of 
sodium hydroxide, and quickly insert a rubber stopper. 
This is a very reliable and convenient method. 
4. Making Plates.-If a smear examination or the 
appearance of the culture suggests that more than one or- 
ganism is present and it is desirable to isolate them in pure 
culture, one must 1 'prepare plates": three tubes of agar 
are melted and cooled to 42° C. A platinum loopful of 115 
GENERAL BACTERIOLOGY 
pus or other infectious material is transferred to Tube 1 
and well mixed by rotating the tube; two loopfuls are 
transferred from Tube 1 to Tube 2, and five loopfuls 
from Tube 2 to Tube 3; in this way the bacterial dilu- 
tion is made progressively higher. The contents of each 
tube are then poured into each of three Petri dishes, 
which are numbered to correspond to tubes, and the 
plates are incubated. Next day the separate colonies are 
studied in plate No. 3 (containing the least number of 
colonies), and separate colonies are picked and inocu- 
lated on slants, if necessary. 
5. Examination of Peritoneal, Pleural and Pericardial 
Fluids.-These should be first centrifugalized and the 
sediment examined in a smear preparation. Cultures 
may be made either in a liquid medium by adding a 
few cubic centimeters of the material to a tube or a 
flask of the proper medium, or plated on agar. 
6. The examination of cerebrospinal fluid should be 
carried out in the same manner as above. 
7. Urine.-Catheterized specimen of urine should be 
secured, centrifugalized and smears and cultures made in 
the usual way. 
8. Feces is most frequently examined for typhoid 
bacillus: a small piece is emulsified in a test tube of 
sterile physiologic salt solution, and dilutions are made 
into three tubes, as in plating, the contents of each be- 
being poured in Petri dishes containing Endo's medium; 
next day a colorless colony is picked and transferred to 
Russell's double sugar agar tube; after twenty-four hours' 
incubation if the colony was one of typhoid bacillus, part 
of the tube will have turned red but no gas bubble will 
have formed. (See section on Russell's Medium under 
Culture Media.) 
9. Blood Culture.-Sterilize the elbow bend with two 116 
PRINCIPLES OF BACTERIOLOGY 
B 
NEEDLE 
A 
OUTER TUBE 
c 
THE. RUBBER 
TU0ING IN 
WHICH NEEDLE 
(A) IS FIXED 
D 
SEALED END 
E 
TUBE 
CONTAINING 
MEDIUM 
Fig. 21B.- Special tube for obtaining blood for Wassermann and similar 
tests. 117 
GENERAL BACTERIOLOGY 
applications of tincture of iodine, apply a tourniquet 
half way up the arm, to make the veins stand out promi- 
nently, and with a sterile all-glass Luer syringe with- 
draw 5 to 10 c.c. blood. 
If typhoid is suspected, pour about 5 c.c. of blood into 
a flask with bile-peptone-glycerin medium, incubate for 
12 hours, and then make transfers to ordinary broth 
(about 1 to 2 c.c. of bile medium) or make plates on 
Endo's medium. If septicemia or pneumonia are thought 
of, pour 1 to 2 c.c. of blood into each of 3 or 4 flasks 
with glucose meat infusion broth. 
A very simple and a very good method of obtaining 
pure blood cultures--a method that produces fewer con- 
taminations than any other-is the use of the special 
tubes which are now on the market (see Fig. 21-B) ; 
they are practically large Kei del vacuum tubes (which 
are used for obtaining blood for Wassermann and similar 
tests) containing various culture media; the outer tube 
(A) is taken off, after the arm is prepared in the usual 
way (scrubbed with alcohol) a tourniquet is placed above 
the elbow bend and the patient is told to keep the fist 
shut tight, in order to make the veins stand out, the 
needle (B) is thrust into the vein, the sealed end (D) 
of the tube containing medium (E) is broken with one's 
thumb and the index finger, within the rubber tubing 
(C), and then the vacuum in (E) causes the blood to 
flow through the needle (B) and the rubber tubing (C) 
into the medium in (E). 
As one can readily see, the blood is never exposed 
to the air contamination as it flows from the vein into 
the tube containing the medium; neither is the medium 
exposed to contamination. 
10. Sputum.-Sputum is usually examined for tubercle 
bacilli. A thick yellowish piece should be chosen and 
smeared on a glass slide in a usual way, dried and fixed 118 
PRINCIPLES OF BACTERIOLOGY 
by heat; it is then stained for tubercle bacillus as de- 
scribed in the section on Staining, or for pneumococci. 
11. Animal Inoculation.-Very often when no evi- 
dence of tuberculosis can be found on examination of 
smears or cultures, animal inoculation has to be resorted 
to. 
The fluid to be injected (peritoneal, pericardial, pleural 
or cerebrospinal fluid) is centrifugalized and the sedi- 
ment is mixed with a few cubic centimeters of sterile 
salt solution, and 1 c.c. is injected into a guinea pig in- 
traperitoneally. If sputum is used, a suitable (yellowish) 
piece is rubbed up with salt solution, instead of centrif- 
ugalization. Four weeks later the guinea pig is killed 
and examined for the evidences of tuberculosis. If the 
animal dies before the time specified, autopsy is immedi- 
ately made. 
If the guinea pig is x-rayed for thirty seconds before 
injection ten or fourteen days are a sufficiently long 
period to wait (as x-raying destroys the lymphoid tis- 
sue which protects the animal against tuberculosis, and 
it is thus rendered more susceptible). 
If pneumonia is suspected, one c.c. of blood or sputum 
is injected intraperitoneally into a white mouse, and 
cultures and smears are made from the peritoneal fluid. 
It should be always borne in mind that the final re- 
sults depend on the care with which all manipulations 
are made, as the opportunities for contamination are 
great. Scrupulous care should be exercised in flaming 
the cotton stoppers before and after opening tubes and 
flasks with culture media, the platinum needles and 
loops, etc. Certain procedures can not be explained, but 
have to be seen in order to be grasped; the pupil can, 
however, learn at the outset the fact that care in handling 
the materials is more essential than speed. SECTION II 
SPECIAL BACTERIOLOGY 
CHAPTER VII 
THE STAPHYLOCOCCUS GROUP 
I. Historical 
The staphylococcus group were first carefully studied 
in 1879 by Koch, Ogston, Pasteur and Rosenbach. They 
are so called because of their appearance in irregular 
clusters (from Greek, meaning bunch of grapes). 
II. Morphology and Staining 
Staphylococci are round or spherical, diameter about 
0.9 ft. They are usually arranged in clusters, but very 
young cultures may show diplococci (pairs). 
Staphylococci stain with all simple stains, and are 
Gram positive; they are nonmotile, have no spores, no 
capsules, no flagella. 
III. Cultural Characteristics 
Staphylococci grow readily on all plain media; they are 
aerobes and facultative anaerobes (that is, they may 
grow without oxygen). They form in 24 hours discrete, 
golden yellow or white colonies of various sizes. Gelatin 
is liquefied. Milk is coagulated and acids (lactic and 
butyric) are formed. 
119 120 
PRINCIPLES OF BACTERIOLOGY 
On sugar media acids are formed but gas is not pro- 
duced. Staphylococcus aureus produces yellow pig- 
ment, while no pigment is produced by Staphylococcus 
albus (Staphylococcus citreus-which produces green 
pigment-is of very feeble pathogenic power). 
IV. Destruction 
Staphylococcus is killed by heating to 58° C. for ten 
minutes; it is very resistant to low temperatures, and 
drying is well borne. 
Solution of bichloride of mercury 1:1000 destroys 
Fig. 22.-Staphylococcus. x 1100 diameters. (Park and Williams-Patho- 
genic Bacteria and Protozoa.) 
them in 10 minutes; tincture of iodine is excellent for 
destroying them. 
V. Disease-Producing Properties, Mode of Infection, 
Disinfection and Prophylaxis. 
Different strains vary in virulence. Ordinarily they 
cause local suppuration-abscesses, boils, carbuncles, etc. 
They also cause osteomyelitis (inflammation of the bone 
marrow) ; if they gain entrance to circulation they may 121 
SPECIAL BACTERIOLOGY 
cause septicemia, and abscesses in various organs. Child- 
birth fever is fairly frequently caused by them. For dis- 
infection see under Destruction. As to prophylaxis, sur- 
gical cleanliness is the only efficient means. The nurse 
should carefully destroy the soiled dressings by burning 
and disinfect her hands by washing them in 70 per cent 
alcohol or 1:1000 bichloride of mercury. 
VI. Infection and Immunity 
Staphylococci produce endotoxins, and in addition to 
this, hemolysin, which destroys the red blood cells, and 
Fig. 23.-Staphylococcus pyogenes aureus. Colony two days' old, seen 
upon an agar-agar plate, x 40 (Heim). (From McFarland-Pathogenic Bac- 
teria and Protozoa.') 
leucocidin which destroys the white blood cells (leuco- 
cytes) . 
During the infection agglutinins are produced, bac- 
teriolysins, and opsonins. Phagocytosis is important in 
staphylococcus infections. 
VII. The Varieties of Staphylococci 
(a) Staphylococcus pyogenes (pus-producing) aureus 
(yellow) produces yellow pigment. 122 
PRINCIPLES OF BACTERIOLOGY 
(b) Staphylococcus pyogenes albus (white) produces 
pigment. 
The aureus is the most frequently associated with 
infection. The albus causes the stitch abscesses seen 
after operations. 
Staphylococcus citreus produces green pigment ; it 
possesses very feeble pathogenic power. 
VIII. Bacteriologic Diagnosis 
Bacteriologic diagnosis is easy and rests on the fol- 
lowing : 
(a) Gram-positive cocci arranged in clusters. 
(b) Grow readily on simple (meat extract) media. 
(c) Liquefy gelatin, coagulate milk, produce acids, 
form no gas on sugar media. 
IX. Immunity Treatment 
None for acute infections. For chronic infections 
such as continuous abscess formation, persistent pus dis- 
charge, etc., a staphylococcus vaccine gives good results. 
Vaccines are prepared as follows: 
A 24-hour-old agar culture of staphylococcus is ob- 
tained; about 10 c.c. of sterile salt solution are poured 
into the culture tube; the growth is gently scraped off 
with a platinum loop (care being taken not to scrape 
any particles of agar). The bacterial emulsion is poured 
into a killing tube (this is an ordinary test tube but with 
a long drawn out neck) in which 8 to 10 glass beads are 
placed, the tube is scaled off and shaken for an hour by 
hand (or for ten minutes in a special "vaccine shaker") ; 
the narrow tip is broken off and a few drops are poured 
into a sterile watch-glass, the tube is scaled again and SPECIAL BACTERIOLOGY 
123 
placed in a water-bath at 58° C. for one hour, to kill the 
bacteria; now we proceed to standardize the vaccine; i. e., 
to determine how many bacteria we have there per cubic 
centimeter. With a blood lancet prick a finger and draw 
blood into a capillary pipette for a distance of one inch 
(marked with red pencil) ; a little air is then allowed to 
enter and then the bacterial emulsion is drawn up to the 
same mark. We now have equal amounts of blood and bac- 
teria; these are thoroughly mixed by alternate drawing 
in and out of the contents of the pipette on a watch- 
glass. Then smears are made of this mixture on a glass 
slide in the same manner as blood smears; put a large 
drop of the mixture on a glass slide and with another 
glass slide resting on the drop make a quick rapid 
smear. Stain with Wright's stain (see section on 
Staining) ; count several fields, counting red blood cells, 
and bacteria. Let us suppose that we counted altogether 
300 red blood cells and 150 bacteria. That means there are 
twice as many red blood cells as bacteria. Since there are 
5,000,000 red blood cells in a cubic millimeter of normal 
blood, our vaccine (which contains half as many bac- 
teria as there are red blood cells) contains 2,500,000 bac- 
teria per cubic millimeter, or 2,500,000,000 per cubic 
centimeter. Now if we do not want our vaccine to be 
so concentrated, we can dilute it with salt solution to 
the desired concentration. 
The vaccine is now taken out of the water-bath, 
poured into a sterile graduated cylinder, enough salt 
solution is added to bring it to the desired concentration, 
and 0.25 per cent of tricresol is added, and the vaccine 
is allowed to stand at room temperature overnight; next 
morning aerobic and anaerobic cultures are made, and if, 
in forty-eight hours, no evidences of contamination are 124 
PRINCIPLES OF BACTERIOLOGY 
seen, the vaccine is poured into small ampoules and 
sealed. 
X. The Summary of Staphylococcus Group 
The staphylococcus is a common organism, found every- 
where, especially on human skin, causes localized sup- 
purations, at times septicemia, is Gram-positive, appears 
in clusters, has no spores, flagella or capsules, is non- 
motile, grows readily on all ordinary media, is aerobic 
and facultative anaerobic, produces endotoxin, hemolysin 
and leucocidin, the infected animal produces bac- 
teriolysins, opsonins, agglutinins; phagocytosis plays an 
important part in overcoming the infection. 
One of the most promising treatments for all infected 
wounds is that of Carrel-Dakin. 
It is somewhat complicated and should be thoroughly 
explained by one familiar with its use, and, for this 
reason, is omitted here; briefly stated its essential fea- 
ture is chloride of lime with sodium carbonate and bi- 
carbonate. 
When properly applied by Carrel-Dakin's method, it 
is the most efficient treatment of infected wounds yet 
devised. CHAPTER VIII 
THE STREPTOCOCCUS GROUP 
I. Historical 
The first studies on streptococci were made by Klebs, 
Koch, Pasteur and Ogsten. 
II. Morphology 
Streptococci are small spherical organisms, about 0.7 p 
in diameter. They usually are arranged in chains, 
whence their name (from Greek str ept os, meaning 
twisted). The chains are usually longer in the strains 
just isolated from animal tissues than in those grown on 
artificial culture media. They also appear to grow in 
longer chains in liquid than in solid media. 
The streptococci are nonmotile, have no flagella, no 
spores, no capsules, although there is a strain of strep- 
tococcus which regularly possesses capsules, the so-called 
streptococcus pyogenes (pus-producing) capsulatus, but 
today this organism is regarded as being one of the 
strains of pneumococcus and is called pneumococcus 
capsulatus (see section on Pneumococcus). 
Streptococcus is Gram-positive. 
III. Cultural Characteristics 
Streptococcus grows slowly on ordinary meat extract 
media, but luxuriantly on the "enriched" media, such 
as meat infusion media, especially when glucose or as- 
125 126 
PRINCIPLES OF BACTERIOLOGY 
citic fluid has been added. On blood agar plates 
the hemolytic streptococci colonies from a wide zone of 
hemolysis. Gelatin is not liquefied. Sugars are fer- 
mented and acid is produced. Milk is coagulated in 
three to four days. 
It is aerobic and facultatively anaerobic. It does not 
grow below 15° C. or above 42° C. 
Fig. 24.-Streptococcus pyogenes. Film preparation of a broth culture, 
x 1500. (Hewlett-Manual of Bacteriology.) 
IV. Destruction 
Heating at 60° C. for one hour will kill streptococci, 
5 per cent carbolic acid solution and 1:1000 bichloride 
of mercury kills them in a few min utes. They keep well 
on defibrinated blood in ice box. Drying destroys them 
in ten hours. SPECIAL BACTERIOLOGY 
127 
V. Disease Production, Mode of Infection, Disinfection 
and Prophylaxis 
Streptococci are widely distributed in nature, but 
usually in association with man, they are found in soil, 
water and milk. Some very severe epidemics of "strep- 
tococcus sore throat" have been caused by the use of 
infected milk. 
In human beings they cause both local suppurations 
like staphylococci and also septicemia ("blood poison- 
ing") ; infection develops very rapidly from twelve hours 
on, and if it becomes general, produces endocarditis 
(inflammation of the lining of the heart), meningitis 
(inflammation of the lining of the skull), childbirth 
fever, acute rheumatism, sore throat (tonsillitis), 
osteomyelitis (inflammation of the bone-marrow), pneu- 
monia ("septic" pneumonia) ; streptococci often accom- 
pany other infections, e.g., diphtheria. They invariably 
entej' through the skin or mucous membranes; the pre- 
vention is surgical cleanliness, disinfection, and fumi- 
gation. 
VI. Mechanism of Infection and Immunity 
Streptococci produce endotoxins, also hemolysin 
(which destroys red blood cells). 
Immune bodies produced in infected animals are ag- 
glutinins, bacteriolysins and opsonins; phagocytosis is 
important in combating the infection. 
VII. Bacteriologic Diagnosis 
Gram-positive cocci, usually arranged in chains, grow 
slowly or not at all on meat extract media, but very well 
on "rich" media, cause hemolysis on blood agar plates. 
If confused with pneumococci, differentiation may be 
difficult; see section on Pneumococcus. 128 
PRINCIPLES OF BACTERIOLOGY 
VIII. Immune Treatment 
In acute infections serums from animals immunized 
with streptococcus vaccine has been, at times, very help- 
ful. The vaccine is of not much benefit. In chronic in- 
fections the vaccine is, at times, very useful. The vac- 
cine is prepared as the staphylococcus vaccine is. 
IX. Classification of Streptococci 
1. Streptococcus Pyogenes Longus-causes suppura- 
tive and septicemic lesions, occurs in long chains. 
2. Streptococcus Pyogenes Brevis-short-chained. 
3'. Streptococcus Haemolyticus-produces hemolysis 
on blood agar cultures. 
4. Streptococcus Viridans or Mitis-produces no 
hemolysis and much milder lesions-carious teeth, etc. 
5. Streptococcus Anginosus--occurs in throats of 
scarlet fever patients. 
Rarely, if ever, pathogenic. 
6. Streptococcus Salivarius-found in the saliva. 
7. Streptococcus Fecalis-found in the intestines. 
8. Streptococcus Equinus-found in horses. 
(3) and (4) are the only two types now recognized 
in human infections. 
X. Summary of Important Characteristics 
Gram-positive cocci, arranged in chains, nonmotile, no 
flagella, no spores, no capsules, grow poorly on ordinary 
media, well on "rich" media, cause hemolysis on blood 
agar plates; cause either local suppuration or systemic 
infection; very dangerous organism, enters through the 
skin, produces an endotoxin and a hemolysin. CHAPTER IX 
THE PNEUMOCOCCUS GROUP (DIPLOCOCCUS 
PNEUMONITE, DIPLOCOCCUS LANCEO- 
LATUS-'1 LANCE-SHAPED ") 
I. Historical 
Pasteur isolated the organism from the saliva in 1881; 
at the same time, quite independently, it was discovered 
by the American bacteriologist, Sternberg (late Surgeon- 
General, United States Army). Fraenkel and WeichseL 
baum in 1886 definitely described it as the cause of 
pneumonia, in 90 per cent of cases. 
II. Distribution 
It is widely distributed, in close association with man. 
III. Morphology 
Through the brilliant work of Cole, Dochez, Avery and 
other workers at the Rockefeller Institute for Medical 
Research in New York, we now know that there are at 
least four different strains of pneumococci, which may 
be recognized by serologic methods (agglutination and 
precipitin reaction), but not by morphology. 
Pneumococcus is a lance-shaped, oval organism gen- 
erally occurring in pairs (in very young cultures not 
uncommonly it is seen in chains) of varying sizes; it 
is invariably encapsulated when freshly isolated from 
animal tissues, but readily loses the capsule on pro- 
129 130 
PRINCIPLES OF BACTERIOLOGY 
longed cultivation, which suggests that the capsule pro- 
tects the organism; it is nonmotile, has no flagella, no 
spores; is Gram-positive, (the Type III pneumococcus 
capsule is very large and may be seen without using the 
special capsule stain) the capsulated strain is always 
more virulent. (See Fig. 2.) 
IV. Cultural Characteristics 
Pneumococci do not grow on meat extract media; the 
best media are those which contain meat infusion and 
rabbit's defibrinated blood; on such media colonies are 
small, discrete and greenish. 
Gelatin is not liquefied, milk is coagulated and acid is 
produced. Among sugars fermented is inulin-this is 
very important in differentiating the pneumococcus from 
streptococcus because the latter does not ferment inulin; 
bile dissolves pneumococci, but does not dissolve strep- 
tococci-another important differentiating point, on 
blood plates it does not cause hemolysis. 
Pneumococcus grows equally well with or without 
oxygen. 
V. Destruction 
Pneumococcus does not grow below 25° C. or above 41° 
C. Heating to 52° C. for ten minutes kills them, as do 
most of the ordinary chemical disinfectants. Even on 
the most favorable media it dies within a few days. 
The best way to preserve pneumococci is to grow them 
on defibrinated rabbit's blood agar and keep the cul- 
tures in the ice box, or keep them in dried spleens of 
infected white mice, which should be kept in sealed tubes 
on ice; when culture is needed, spleen is rubbed with a 
little broth, the emulsion is injected into a white mouse, SPECIAL BACTERIOLOGY 
131 
and pneumococci are recovered from the fluid in the 
peritoneal cavity. 
VI. Disease Production, Mode of Infection, Disinfec- 
tion and Prophylaxis 
In addition to causing about 90 per cent of all cases 
of pneumonia, pneumococci may cause inflammation of 
bronchi, nose, throat, ear, meningitis, peritonitis (in 
young children I have seen two such cases), peri- and 
endo-carditis (inflammation of the sac and the lining of 
the heart), etc. Many normal persons harbor pneu- 
mococci in their mouth and throat. The pneumococci 
almost invariably enter the body through the respiratory 
tract. 
Disinfection and prophylaxis resolves itself into disin- 
fecting the mouth and throat, isolation of the patient, 
avoiding to have patient cough in one's face, etc.; fumi- 
gation of room should follow the removal of the pa- 
tient. Sputum must be carefully disinfected. 
VII. Mechanism of Infection and Immunity 
Pneumococcus produces an endotoxin; the antibodies 
formed by the infected animal are bacteriolysins, agglu- 
tinins, precipitin, and opsonins, phagocytosis always 
plays a part in recovery, and marked leucocytosis (in- 
crease of white blood cells up to 40,000 per cubic milli- 
meter) is very frequently seen. 
VIII. Bacteriologic Diagnosis 
The bacteriologic diagnosis is made on the Gram-posi- 
tive, capsulated diplococci (pairs), growing only on 
"rich media." If necessary white mice should be in- 
jected and autopsy will reveal encapsulated pneumococci. 132 
PRINCIPLES OF BACTERIOLOGY 
The organism which is most likely to be confused with 
pneumococcus is streptococcus, the following points 
should be borne in mind. 
Pneumococcus 
Almost always capsulated. 
Soluble in bile. 
Ferments inulin. 
No hemolysis on blood plates. 
Streptococcus 
Almost never capsulated. 
Insoluble in bile. 
Does not ferment inulin. 
Hemolysis present. 
Finally, agglutination test will not only enable one to 
make a definite diagnosis, but, in cases of pneumococ- 
cus, will identify the type of pneumococcus. 
IX. The Different Types or Strains of Pneumococci 
As has been mentioned above there are at least four 
types of pneumococcus: 
Type I causes pneumonia in 33 per cent of cases. 
Type II causes pneumonia in 31 per cent of cases. 
Type III causes pneumonia in 12 per cent of cases. 
Type IV causes pneumonia in 12 per cent of cases. 
Mortality caused by the different types also differs: 
Type I is fatal in 25 per cent of cases. 
Type II is fatal in 32 per cent of cases. 
Type III is fatal in 45 to 50 per cent of cases. 
Type IV is fatal in 16 per cent of cases. 
The above figures may vary to a slight extent and are 
those given by Bull in the Rockefeller Institute Mono- 
graph (1916) on Pneumonia (see also Bull. Arch. Inst. 
Med., 1914, xiv, 56). 
The agglutination test consists in mixing the isolated 
organisms with the serum of an animal injected with 
the different types, and observing where agglutination of SPECIAL BACTERIOLOGY 
133 
bacteria takes place. For complete technic the reader 
should consult the monograph on Acute Lobar Pneu- 
monia by Cole and his coworkers (The Monograph No. 
7 of the Rockefeller Institute for Medical Research). 
Recently Krumwiede and Valentine of New York has 
described a much simpler precipitin test. 
X. Immune Treatment 
The various vaccines and immune serums up to date 
are not of much value, but the serum prepared recently 
by Cole for Type I pneumococcus infection is very effi- 
cient and should be tried in all cases where the Type I 
has been identified. 
XI. Summary of Important Characteristics 
A Gram-positive, capsulated organism, occurs in pairs, 
grows only on "rich" media, causes no hemolysis on 
blood plates, but forms greenish colonies, ferments inulin, 
is bile soluble; there are four types or strains, the Type 
III being the most dangerous but occurring in the small- 
est number of cases; serum treatment is efficient for Type 
I; produces endotoxin. CHAPTER X 
THE MENINGOCOCCUS AND PARAMENINGOCOC- 
CUS GROUP (MICROCOCCUS INTRA- 
CELLULARIS MENINGITIDIS) 
Meningitis (the inflammation of the brain coverings) 
is caused in 70 per cent of cases by the meningococcus, 
in 20 per cent by pneumococcus, and in 10 per cent by 
staphylococci, streptococci, Bacillus tuberculosis, Bacil- 
lus influenzae, etc. 
I. Historical 
Meningococcus was first observed in 1884 by Marchia- 
fava and Celli, and thoroughly studied in 1887 by Weich- 
selbaum. 
II. Morphology 
Meningococci occurs within or without the pus cells 
when isolated from the cerebrospinal fluid; they usually 
occur in pairs, like pneumococci do; they vary in size; 
they are not motile, have no flagella, no spores, no cap- 
sules ; they stain readily with ordinary stains and are 
Gram-negative. 
III. Cultural Characteristics 
Meningococci do not grow well on ordinary meat ex- 
tract media, but grow easily on meat infusion media; 
milk is not coagulated, gelatin is not liquefied. Menin- 
gococcus is an aerobe. Growth does not take place 
below 25° C., or above 42° C. 
134 135 
SPECIAL BACTERIOLOGY 
IV. Destruction 
Sunlight and drying kills the meningococci within 
twenty-four hours; they are extremely sensitive to heat 
and cold and are most readily destroyed by highly di- 
luted solutions of the common disinfectants. 
V. Disease Production, Mode of Infection, Disinfec- 
tion and Prophylaxis 
Meningococcus causes epidemic cerebrospinal menin- 
gitis ; it is usually accepted that meningococci first lodge 
Fig. 25.-The meningococcus. Smear of cerebrospinal fluid, x 1000. (Hew- 
lett-Manual of Bacteriology.') 
in the nose, whence they enter the skull either directly 
(through the so-called cribriform plate of the ethmoid 
bone) or indirectly through the lymph vessels. 
The disease is transmitted directly from one human 
being to the other, as the low resistance of the meningo- 136 
PRINCIPLES OF BACTERIOLOGY 
coccus and its susceptibility to drying precludes its be- 
ing found at large in nature. Coughing, sneezing, the 
use of infected handkerchief, etc., is sufficient to in- 
fect. 
Patients should be isolated and quarantined, the nurse 
should change her outer clothing before mingling with 
other people, she should carefully disinfect her hands 
and hair and spray her nose with some silver prepara- 
tion (argyrol, 10 per cent) ; the same thing applies to 
the patient before he is discharged. 
VI. Mechanism of Infection and Immunity 
Bacteriolysin and agglutinin are produced: menin- 
gococcus produces endotoxin. 
VII. Bacteriologic Diagnosis 
The bacteriologic diagnosis is made on finding Gram- 
negative cocci, arranged in pairs, both within and with- 
out the pus cells, on the fact that the organisms grow 
only on meat infusion media, on the source of the organ- 
ism (the cerebrospinal fluid), and if necessary, on 
agglutination. 
VIII. Immune Treatment 
To Flexner, of the Rockefeller Institute for Medical 
Research of New York, belongs the credit of preparing 
the first successful antimeningitic serum (from the 
horse), which has reduced the mortality of this dreadful 
disease from 70 to 80 per cent to 15 to 20 per cent. 
IX. Summary of Important Characteristics 
A Gram-negative organism, occurring in pairs, grow- 
ing only on meat infusion media, no spores, no flagella, no SPECIAL BACTERIOLOGY 
137 
capsule; causes meningitis, produces endotoxin; pa- 
tients should be isolated and strictest attention should be 
given to disinfection; serum treatment is the only effi- 
cient treatment. 
The parameningococci or pseudo- (false) meningococci 
resemble the meningococci in everything except in not 
agglutinating in the same way (that is, they agglutinate 
in much lower dilutions). CHAPTER XI 
GONOCOCCUS 
The gonococcus, also called Diplococcus gonorrhee, 
causes gonorrhea, a highly contagious disease. 
I. Historical 
It was discovered by Neisser in 1879. 
II. Morphology 
It occurs in pairs, is Gram-negative, the pairs are 
characteristically flattened along the facing surfaces-■ 
"biscuit" shaped; the gonococci are always found within 
the pus cells (leucocytes). They are nonmotile, have no 
flagella, no spores, no capsides. 
III. Cultural Characteristics 
Gonococci grow only on ascitic fluid agar, cornstarch 
agar, and also glycerin-or sugar ascitic fluid agar. It is 
an aerobic organism, and does not grow below 30.5° C. 
The colonies are extremely delicate, grayish little spots. 
IV. Destruction 
Gonococci possess very slight resistance to heat and 
light, exposure to 42° C. for ten minutes kills them; they 
are, however, very resistant to drying. 
Most of the common disinfectants, even in high dilu- 
tions, kill the gonococci readily, especially the silver 
138 SPECIAL BACTERIOLOGY 
139 
salts, which fact explains the universal use of the various 
silver preparations in the treatment of this disease. 
V. Disease Production, Mode of Infection, Disinfec- 
tion and Prophylaxis 
At first the inflammation caused by gonococcus is 
strictly local (urethritis, inflammation of urethra), some- 
times, if the discharge is carried to the nose or eye, the 
Fig. 26.--The gonococcus. Smear of gonorrhea pus. x 1500. (Hewlett 
Manual of Bacteriology.) 
inflammation is produced in these parts. Gonorrhea is 
always produced by contact. 
After the acute stage, gonococci frequently cause 
rheumatism, endocarditis (inflammation of the lining of 
the heart), and valvular heart disease. Babies born of 
mothers affected by gonorrhea, if not properly taken care 
of, develop blindness (gonorrheal ophthalmia of the new- 
born) ; it is, therefore, almost compulsory now to put 
one or two drops of a 2 per cent solution of silver nitrate 
into baby's eyes at birth, and this simple prevention 
known as Crede's treatment has practically eliminated 
this babies' scourge. 140 
PRINCIPLES OF BACTERIOLOGY 
The patients should be isolated, and everything 
touched by them should be most scrupulously disinfected; 
they should be warned of the danger of infecting their 
eyes, and the nurse should be extremely careful in dis- 
infecting her hands and had best put a drop of a 10 per 
cent solution of agyrol in her eyes once a day. 
VI. Mechanism of Infection and Immunity 
Gonococci produces endotoxins; the immune bodies 
concerned in recovery are bacteriolysins, opsonins (phag- 
ocytosis is very important). 
VII. Bacteriologic Diagnosis 
Bacteriologic diagnosis is made on finding a Gram- 
negative diplococcus, biscuit shaped, and within the pus 
cells. In chronic cases where no discharge is obtainable, 
complement-fixation test should be done. This test is 
very similar in its technic to the Wassermann test-is 
very reliable, but in patients who had been under a 
vaccine treatment, at least two months must elapse be- 
fore the test is made, since otherwise a false positive 
reaction may be obtained. 
VIII. Immune Treatment 
The gonococcus vaccine is used only in chronic cases, 
and in these gives very good results. 
Gram-negative, biscuit shaped, diplococci, found within 
pus cells, no spores, no flagella, no capsules, grow only on 
ascitic fluid agar, cornstarch agar, or other "rich" media, 
cause gonorrhea, sometimes rheumatism and heart disease, 
produce endotoxin; the vaccine treatment well worth 
trying in chronic cases. 
IX. Summary of Important Characteristics CHAPTER XII 
MICROCOCCUS CATARRHALIS AND GRAM- 
NEGATIVE COCCI 
I. Micrococcus Catarrhalis 
This is an organism found in inflammation of upper 
respiratory tract. Its significance is unimportant except 
that being a Gram-negative cliplococcus, it can not be 
differentiated in its appearance from gonococci and men- 
ingococci ; from gonococcus it differs by growing on sim- 
ple meat extract media. It is much more difficult to dif- 
ferentiate it from meningococcus, and yet it is especially 
important as both are usually found in the nose. It grows 
much better on simple media than the meningococcus, its 
colonies are coarse while those of meningococcus are very 
fine, and micrococcus will grow below 25° C., while the 
meningococcus will not. 
II. Gram-negative Cocci 
Gram-negative cocci producing green pigment can be 
separated from the gonococcus and meningococci by sugar 
fermentation and agglutination. 
141 CHAPTER XIII 
THE COLON-TYPHOID-DYSENTERY GROUP 
B. COLI COMMUNIS 
The reason all these bacteria are usually grouped to- 
gether although they produce quite different diseases, is 
that, morphologically (that is, by their appearance in 
stained preparations), they are almost indistinguishable, 
and in diagnosis our reliance is placed on cultural char- 
acteristics and agglutination. 
I. Historical 
The colon bacillus or, to give it its full name, Bacillus 
coli communis (which means the common bacillus of colon 
-the large intestine), was first described by Buchner in 
1885, and thoroughly studied by Escherich in 1887. 
II. Morphology 
Colon bacillus is a short, plump, rod-like organism, 
% /z long, usually occurring singly, is Gram-negative, 
has no spores, no capsules, is motile, and has flagella. 
(See Fig. 3.) 
III. Cultural Characteristics 
It is an aerobic organism, grows well on simplest media, 
i. e., meat extract agar and broth; it grows at any tem- 
perature between 20° C. and 40°C. It does not liquefy 
gelatin, forms indol on peptone media, coagulates milk 
and forms acid in it; as to sugar fermentation, it 
142 SPECIAL BACTERIOLOGY 
143 
produces both acid and gas on dextrose (glucose, or 
grape sugar), lactose (milk sugar), maltose (malt 
sugar), levulose (fruit sugar), galactose and mannite, 
but neither acid nor gas on saccharose (cane sugar). 
IV. Destruction 
Colon bacillus is readily destroyed by heating to 58° C. 
for ten minutes, cold affects it less, and ordinary disin- 
fectants readily kill it. Excessive alkaline reaction in- 
hibits its growth. 
Fig. 27.-Bacillus coli. Film preparation from a pure culture. x 1000. 
(Hewlett-Manual of Bacteriology.') 
V. Disease Production, Mode of Infection, Disinfec- 
tion and Prophylaxis 
The colon bacillus is normally found in the intestine of 
both man and animal, also occasionally found in soil, 
water and milk. Its presence in nature usually means 
contamination from animal or human sources. In the 
human intestine it is found in greatest numbers near the 
junction of the small and large intestine. 144 
PRINCIPLES OF BACTERIOLOGY 
Although the colon bacillus is a normal inhabitant of 
the human intestine and a useful one, too, since its pres- 
ence seems to inhibit the growth of many harmful putre- 
factive bacteria, yet at times it gives rise to various dis- 
eases; as a rule any enfeebled condition seems to make 
it easier for the colon bacillus to become pathogenic; 
among the various diseases caused by this organism are 
the various diarrheas-cholera infantum and cholera nos- 
tras, occasionally peritonitis (but usually accompanied by 
other organisms) ; very frequently it is found in inflam- 
matory conditions of the liver, gall bladder, and appen- 
dix; one of the most frequent conditions caused by the 
colon bacillus is the inflammation of the urinary bladder 
(cystitis) and pyelitis (inflammation of the basin of the 
kidney). 
It is often found in the abscesses within the abdominal 
and pelvic cavities. 
No special precautions are called for in handling the 
colon infections, beyond following the usual hygienic 
and sanitary rules. 
Bacillus coli communior differs from the colon bacillus 
in fermenting saccharose in addition to all other sugars 
with production of acid and gas. 
VI. Mechanism of Infection and Immunity 
The colon bacillus produces an endotoxin, and the anti- 
bodies produced by the animal during the colon infec- 
tions are the bacteriolysins, precipitins and agglutinins. 
VII. Bacteriologic Diagnosis 
The differentiation of the colon, typhoid, paratyphoid 
and dysentery bacilli will be discussed fully in the chap- 
ter on Typhoid Bacillus. SPECIAL BACTERIOLOGY 
145 
VIII. Immune Treatment 
Vaccine treatment of colon infection of the kidney and 
urinary bladder is very successful. 
IX. Summary of Important Characteristics 
A Gram-negative bacillus, no spores, no capsule, motile, 
has flagella, grows readily on common media, ferments 
with gas production all common sugars except sac- 
charose; is found normally in human and animal intes- 
tine, causes infections of the gall bladder, urinary blad- 
der and kidney, also found in several infections of the 
pelvis and abdominal cavity. CHAPTER XIV 
BACILLUS TYPHOSUS 
I. Historical 
Bacillus typhosus was first thoroughly studied by 
Eberth in 1880. Gaffky first isolated them in pure cul- 
ture in 1884. 
Fig. 28.-Bacillus typhosus. Film preparation of a pure culture, x 1500. 
(Hewlett-Manual of Bacteriology.) 
II. Morphology 
The morphology o>f Bacillus typhosus resembles that of 
the colon bacillus and other members of the group very 
closely, with the exception that it is more slender. Is 
146 SPECIAL BACTERIOLOGY 
147 
Gram-negative, is motile, has flagella, has no capsules or 
spores. 
III. Cultural Characteristics 
The cultural characteristics are the same as those of 
the colon bacillus except as regards some sugars and 
milk, as shown in the following table: 
Table of Reactions on Sugar Media and Milk 
by the Various Members of the Typhoid-Colon- 
Dysentery Group 
NAME OF 
BACTERIUM 
DEXTROSE 
(grape sugar) 
LEVULOSE 
(fruit sugar) 
W 
CG 
o 
o 
MANNITE 
MALTOSE 
(malt sugar) 
LACTOSE 
(milk sugar) 
SACCHAROSE 
(cane sugar) 
J 
s 
B. Fecalis 
Alcaligenes 
- 
- 
- 
- 
- 
No 
coagulation 
B, Dysenteriae 
(Shiga type) 
A 
A 
A 
No 
coagulation 
B. Dysenteriae 
("Y" type) 
A 
A 
A 
A 
No 
coagulation 
B. Dysenteriae 
(Flexner type) 
A 
A 
A 
A 
A 
- 
A 
No 
coagulation 
B. Typhosus 
A 
4 
A 
A 
A 
- 
No 
coagulation 
B. Para- 
typhosus 
AC 
AG 
AG 
AG 
AG 
- 
- 
No 
coagulation 
B. Coli 
Communis 
AC 
AG 
AG 
AG 
AG 
AG 
- 
Coagulation 
takes place 
B. Coli 
Communion 
AG 
AG 
AG 
AG 
AG 
AG 
AG 
Coagulation 
takes place 
(After Hiss and Zinsser.) 
A=Fermentation with acid production, A G=:Fermentation with 
both acid and gas production, --■ means no fermentation. 
IV. Destruction 
Bacillus typhosus is an aerobe and a facultative anae- 
robe; grows between 15° C. and 42° C., but, like all 
pathogenic bacteria, grows best at 37° C. Heating for 148 
PRINCIPLES OF BACTERIOLOGY 
ten minutes at 56° C. destroys it. On artificial culture 
it will remain for many months, if sufficient moisture is 
supplied. It will keep alive for many months on ice 
and in water. It is killed somewhat less readily by ordi- 
nary disinfectants than most of the bacteria, but 1:500 
solution of bichloride of mercury will kill it in a few 
minutes. 
V. Disease Production, Mode of Infection, Disinfec- 
tion and Prophylaxis 
Bacillus typhosus causes typhoid fever. During the first 
week or ten days the bacilli are found in the blood and 
"rose spots" (the rash of typhoid fever). Quite often 
the bacilli, after the patient recovers lodge in the gall 
bladder whence they are continually discharged into the 
intestine and thus appear in the feces, in this way while 
the patient is well and immune, he may become the so- 
called "bacillus carrier" and be a temporary or perma- 
nent source of danger to the community. For this reason 
all patients who have recovered from typhoid fever, 
should have their stools examined, and, if typhoid bacilli 
be found, should be warned to take special precautions 
about disinfecting the stools, etc. 
In about 25 per cent of cases the typhoid bacilli may 
be cultured from the urine. 
Very frequently the presence of this bacteria in the 
gall bladder causes its inflammation (cholecystitis) and 
the gallstones. 
Suppurations may be caused by the typhoid bacillus 
especially in the long bones and in the ribs or spine and, 
not infrequently, meningitis. 
Typhoid fever is always with us, but from time to time 
severe epidemics take place. 
Practically all cases of typhoid fever are contracted SPECIAL BACTERIOLOGY 
149 
through swallowing the bacilli either directly in food, 
water, ice, milk, vegetables, etc., or indirectly by soiling 
fingers with patient's excreta, or using the same utensils, 
glassware, etc. 
The prophylactic measures, therefore, consist of thor- 
ough disinfection of stools, urine, bed linen, etc., as de- 
scribed in detail in the chapter on Practical Disinfection; 
since typhoid fever is often a "water borne" dis- 
ease, communities should guard the purity of their 
water supply, most effectively by having a modern filtra- 
tion plant, and removing the source of contamination and 
sewage pollution; milk supply should be closely inspected 
and flies should be destroyed as they very often contami- 
nate our food-screening is necessary. 
During a typhoid epidemic drinking only boiled water 
is a wise precaution. The nurse attending a typhoid pa- 
tient should receive prophylactic typhoid vaccination, 
which is the best prevention. 
VI. Mechanism of Infection and Immunity 
Typhoid bacillus produces an endotoxin; the antibodies 
produced are bacteriolysins, precipitins and agglutinins, 
one attack usually renders a person immune to subse- 
quent attacks. 
As regards the agglutinins, a few words must be said 
here about this: 
The blood of most normal individuals has slight 
agglutinating property, but this is enormously increased 
during and following the diseases or by artificial immuni- 
zation (vaccine). From the fact that the typhoid, para- 
typhoid and the colon bacilli are all closely related to 
each other both morphologically (all being Gram-nega- 
tive, motile, flagella-possessing bacilli) and in their reac- 150 
PRINCIPLES OF BACTERIOLOGY 
tions on some sugars, it is not surprising to learn that 
the agglutinins produced by the blood in response to 
these bacteria are also evidently related to each other; 
that is to say, agglutinins caused by injection of typhoid 
bacilli will agglutinate, not only typhoid bacilli, but also 
those of the paratyphoid and colon bacilli although to a 
much lesser extent; those produced by the injection of 
the colon bacillus will cause agglutination-to a much 
lesser degree-of the typhoid and paratyphoid bacilli, etc. 
The expression "to a much lesser degree" means in a 
higher concentration; i. e., in lower dilution; for ex- 
ample, the agglutinins caused by injection of the typhoid 
bacilli will agglutinate the typhoid bacilli in the dilu- 
tion of the blood serum in salt solution of 1:10,000, while 
they will only agglutinate the colon and the paratyphoid 
bacilli in dilution of 1:200, etc. 
When the injection of an animal with one bacterium 
results in the production of agglutinins not only for that 
organism but also to a lesser extent for other bacteria, 
closely related to it (as when the blood serum of a 
rabbit injected with the typhoid will agglutinate 
not only the typhoid bacilli but also, to a lesser 
degree, i. e., in lower dilutions, the paratyphoid 
and colon bacilli)-such agglutinins clump or ag- 
glutinate the entire group, although to a varying 
extent. The specific agglutinins (that is, those 
against the bacteria injected) are called "major" 
agglutinins and those against other members of the 
groups are called '1 minor ' ' agglutinins; thus, if a rab- 
bit is injected with typhoid bacilli, he will produce the 
major or typhoid agglutinins, and minor or paratyphoid 
and colon agglutinins. 
This is shown in the accompanying table, which is 
copied from the actual work done in our laboratories. SPECIAL BACTERIOLOGY 
151 
(The rabbit was injected with typhoid bacilli, and his 
blood serum showed the following agglutination, in 
various dilutions of blood serum with salt solution.) 
Dilution 
1:100 
1:200 1:500 1:1000 1:3000 
Typhoid Bacillus 
Paratyphoid Bacillus 
± 
-1- ~-|- 
Colon Bacillus 
-g = Agglutination takes place. 
-^Agglutination does not take place. 
This table shows that the blood serum of a rabbit in- 
jected with the typhoid bacilli had developed (1) the 
major or typhoid agglutinins which agglutinated the 
typhoid bacilli in a dilution as high as 1:3000, and (2) 
minor paratyphoid agglutinin which agglutinated the 
paratyphoid bacilli in dilution up to 1:500, and (3) the 
minor colon agglutinin which agglutinated the colon 
bacilli in dilutions up to 1 TOO; this also shows that the 
paratyphoid bacillus are more closely related to the 
typhoid bacillus than the colon bacillus is. The technic 
of agglutinations is given in the next paragraph. 
VII. Bacteriologic Diagnosis 
Whether the culture is obtained from the blood or 
feces, plates must be made, as explained in the section on 
General Bacteriology. My method is to make the plates 
on Endo's medium (see section on Culture Media) where 
the colon bacillus colonies are red and the typhoid colonies 
are colorless; from such a plate a colorless colony is 
picked and transferred to a Russell's double sugar 
medium (see the section on Culture Media), inoculating 
both the surface and by stabbing'. By referring to the 
table of sugar fermentations it will be understood that 
if the culture is typhoid, only a part of the tube will 
turn from blue to red (because the typhoid bacilli 
act only on glucose, producing acid only), and no 152 
PRINCIPLES OF BACTERIOLOGY 
gas bubbles will be seen (because the typhoid does not 
form gas on glucose) ; if the culture is paratyphoid, again 
only part of a tube will turn to red but gas bubbles will 
also be seen in the red part only (because the paratyphoid 
bacilli form both acid and gas on glucose and do not 
ferment lactose); finally if the culture should be con- 
taminated with colon bacillus, the culture tube will be 
red and gas bubbles will be seen everywhere (because 
the colon bacillus produces both acid and gas on both 
the glucose and the lactose). If this is insufficient ag- 
glutination should be done as follows: 
Four rows of Wassermann test tubes (4 x inch) 
should be arranged, each row containing 10 test tubes; 
the first row is labeled "typhoid," the second "para- 
typhoid A," the third "paratyphoid B," and the fourth 
"colon." In the first tube of each row put 1.8 c.c. of 
sterile physiologic (0.8 per cent) salt solution, in all 
other tubes put 1 c.c. of salt solution; then into the first 
tube put 0.2 c.c. of the typhoid immune serum of known 
agglutinating properties (prepared by injecting the rab- 
bit three or four times with typhoid vaccine); now we 
have in this tube a 1:10 dilution of typhoid serum 
(1.8 c.c. salt solution and 0.2 c.c. of serum) ; take 
one c.c. out of this tube and put it into the 
next tube of the "typhoid" row; we had before 
in that second tube 1 c.c. of salt solution, now 
we have 2 c.c. of fluid, one c.c. being salt solution, 
the other c.c. being 1:10 dilution of the immune typhoid 
serum from the first tube, so that the second tube con- 
tains the serum in dilution 1:20; repeat this process until 
the last tube is reached, and throw away the last c.c.; 
all tubes now contain 1 c.c. of fluid, but the dilutions 
are all doubled, the amount of serum being halved as 
we go from one tube to another: 1st tube, 1:10; 2nd, SPECIAL BACTERIOLOGY 
153 
1:20; 3rd, 1:40; 4th, 1:80; 5th, 1:160; 6th, 1:320 ; 7th. 
1:640; 8th, 1:1280; 9th, 1:2560; 10th, 1:5120. Make 
similar dilutions in the other three rows, putting 0.2 c.c. 
of immune paratyphoid A serum in the paratyphoid 
A row, 0.2 c.c. of immune paratyphoid B serum in the 
paratyphoid B row, etc. Then take a platinum loop, 
flame it, and having added about 5 c.c. of salt solution 
to the culture tube containing the bacteria you want to 
identify, scrape off the growth, shake well by rotating 
the tube in your hands, and add to every tube in each 
row 5 drops of the bacterial emulsion (to make the en- 
tire procedure safe, heat the emulsion for 1 hour at 58° 
C.; this will kill the bacteria but will not interfere with 
the agglutination) ; incubate at 37.5° C. for one hour 
and then examine the tubes; the row which contains 
tubes showing agglutination in the highest dilution cor- 
responds to the infection of the patient, that is, if the 
typhoid row (which contains tubes with the typhoid 
immune serum) shows agglutination in a tube with 
higher dilution than in any other row-your culture is 
typhoid. 
Since, however, typhoid bacilli may be recovered from 
patient's blood only during the first ten days of the dis- 
ease, and the patient may be brought to the hospital 
later, the blood cultures may prove to be "negative," 
that is the bacilli will not be found. In such cases, pa- 
tient's blood is removed in a test tube (about 2 to 3 c.c.), 
and Widal test is made. This test is also an agglutination 
test but just the opposite to that made on the culture: 
there we worked on an unknown culture with known im- 
mune serums, while now we will work on an unknown 
serum with known cultures: four rows of 10 tubes in each 
prepared as before, the first tube in each row containing 
1.8 c.c. of salt solution, the others containing 1 c.c. of salt 
solution; the rows again are labeled as before "typhoid," 
' 'paratyphoid A, " " paratyphoid B, " and ' ' colon. ' ' Now 154 
PRINCIPLES OF BACTERIOLOGY 
in each first tube put 0.2 c.c. of the patient's serum, and 
continue the dilution along as described above, i. e., take 
1 c.c. from first tube to the second, from the second to the 
third, and so on, in each row; throw away the last c.c. 
(that is, from the 10th tube). Now to each tube in the 
"typhoid" row add 0.5 c.c. of typhoid culture with salt 
solution after killing it with 2 per cent of formalin, to 
each tube of the paratyphoid A row add 0.5 c.c. of the 
paratyphoid A culture, and so on. Incubate for one 
hour at 37.5° C. and examine. The row containing the 
tube showing agglutination with the highest dilution 
gives you the nature of the patient's disease. 
Sometimes the so-called "microscopic Widal test" is 
made; this consists of making two or three dilutions 
(usually 1:20, 1:40, and 1:80) with patient's serum, and 
a drop of each is mixed with a drop of typhoid emulsion, 
and placed on a cover-glass which is then placed over a 
"hanging drop" slide, as described elsewhere, after one 
hour the slide is examined to see if motility has ceased 
and clumping has taken place. This is a convenient quick 
test, but not nearly so comprehensive as the preceding 
method. 
VIII. Immune Treatment 
Whether the use of the typhoid vaccine is to be 
adopted for the treatment of typhoid fever is rather doubt- 
ful ; but the use of such vaccine for the prevention of 
typhoid fever is not only an established fact but con- 
stitutes one of the most glorious achievements in bac- 
teriology and prevention of infectious diseases, and is 
one of the greatest medical services rendered the world 
by the English and American scientists. 
In England, the preventive use of vaccine was brought 
to perfection by Sir Wright, and in 1907-8, Major (now 
Colonel) Russell of the United States Army Medical SPECIAL BACTERIOLOGY 
155 
Corps, was sent to England to study the question. Upon 
his return the typhoid vaccine was adopted for the 
United States Army and Navy, and, subsequently, 
throughout the entire civilized world. 
All persons should be vaccinated with typhoid vaccine 
as it confers almost absolute immunity for at least three 
years. 
As it is now put up at the United States Army Med- 
ical School, it is a "triple" vaccine, containing the 
typhoid, the paratyphoid A and paratyphoid B bacilli, 
1,000 million of each to each c.c. 
The preparation of the vaccine is practically the same 
as that described for staphylococcus vaccine. 
IX. Summary of Important Characteristics 
The important characteristics are the same as those 
of colon, except that it produces acid only (no gas) on 
all common sugars except lactose and saccharose which 
are not fermented. Causes typhoid fever, often a "water- 
borne" disease; the preventive vaccination is of tremen- 
dous importance. 
The disease is of such practical importance to the 
nurse that no attempt is made to summarize this chap- 
ter, and the reader is advised to read carefully the en- 
tire chapter. CHAPTER XV 
THE B. PARATYPHOSUS A AND B. THE B. FECA- 
LIS ALCALIGENES. THE B. PROTEUS. 
DYSENTERY BACILLI 
B. Paratyphosus A and B 
The paratyphoid bacilli cause a disease which simu- 
lates typhoid fever so closely that no differentiation is 
possible from the symptoms (unless it be the milder form 
of the disease when caused by the paratyphoid bacilli), 
and the diagnosis is usually made from the cultural 
study and agglutination (see the table of Sugar Fer- 
mentations and the Bacteriologic Diagnosis of Typhoid 
Fever). Of the two types of the paratyphoid bacilli, B 
is of much more importance than A. 
B. Fecalis Alcaligenes 
This organism is of little pathogenic importance and 
is only interesting because of ease with which it may be 
confused with bacillus typhosus. 
It is frequently found in normal intestine and feces. 
It is differentiated from typhoid, colon, and paratyphoid 
bacilli by the fact that it does not ferment any of the 
common sugars. 
Bacillus Proteus 
Bacillus proteus possesses slight pathogenic proper- 
ties, but occasionally it may cause infections of the blad- 
der and abscesses. 
156 SPECIAL BACTERIOLOGY 
157 
It is very frequently encountered in the laboratory 
work and may be confused with typhoid bacillus, but 
cultural studies and agglutination make its differentia- 
tion easy. 
The Dysentery Bacilli 
Dysentery is an infectious disease characterized by 
severe diarrhea. 
Fig. 29.-Dysentery bacilli, x 1000. (Park and Williams-Pathogenic Bac- 
teria and Protozoa.) 
The first to discover the organism was a Japanese bac- 
teriologist, Shiga (1898). 
There are three organisms-the Shiga type, the "Y" 
(Hiss-Russell) type, and the Flexner type. All three 
types morphologically resemble each other and the rest 
of the typhoid-colon-dysentery group. 
The differentiation and the diagnosis is made on cul- 
tural (sugar fermentation) characteristics and aggluti- 
nation, as described in Chapters XIII, XIV and XV. CHAPTER XVI 
BACILLUS MUCOSUS CAPSULATUS (FRIEDLAND- 
ER'S OR PNEUMO-BACILLUS) 
This organism first described by Friedlander in 1882 
causes about 5 per cent of all cases of pneumonia; such 
pneumonia is extremely dangerous. 
It is occasionally found in ulceration of the mouth, 
nasal catarrh, ozena (fetid nasal catarrh), empyema 
(pus in the pleural cavity), and spinal fluid. The author 
once recovered it from a gall bladder infection and 
from blood in subsequent septicemia. 
Friedlander's bacillus is short and plump, has no 
spores, no flagella, is nonmotile, has very large capsule. 
It is Gram-negative, and grows well on all ordinary meat 
extract media, producing peculiar, "slimy" colonies, does 
not liquefy gelatin (culture assumes a characteristic nail- 
like appearance). 
All sugars, except lactose are fermented with the for- 
mation of gas. 
158 CHAPTER XVII 
THE TETANUS BACILLUS (LOCKJAW) 
I. Historical 
The Bacillus tetani was discovered by Nicolaier in 
1885 and first studied and cultured by Kitasato in 1889. 
II. Morphology 
The bacillus is a slender organism, possesses spores, no 
capsules; the vegetative forms are motile and have 
flagella. Spores are developed after twenty-four to 
forty-eight hours of incubation. The spore-bearing forms 
resemble a drumstick, since the spore is usually at the 
end of the bacillus. (See Fig. 4.) 
The bacillus is Gram-positive. 
III. Cultural Characteristics 
Bacillus tetani is a strict anaerobe; it grows readily 
on meat infusion media. Gelatin is slowly liquefied, 
milk is not coagulated. The addition to media of 1.5 
per cent of glucose results in a more rapid and abun- 
dant growth. The "cooked meat medium" has been 
warmly recommended by Robertson (Jour. Path, and 
Bacteriol., Jan., 1916, xx, No. 3). 
IV. Destruction 
The vegetative (non-spore-bearing) form are readily 
destroyed by ordinary disinfectants and heat, but the 
159 160 
PRINCIPLES OF BACTERIOLOGY 
spore-bearing forms resist dry heat at 80° C. for one 
hour and even live steam for five minutes, and it takes 
twelve to fifteen hours for a 5 per cent solution of car- 
bolic acid to kill them. 
Protected from sunlight the tetanus spores will live 
for years. 
V. Disease Production, Mode of Infection, Disinfec- 
tion and Prophylaxis 
The tetanus bacilli are found in superficial layers of 
soil, especially near stables and manured fields. 
Bacillus tetani produce a disease called lockjaw, char- 
acterized by convulsions and closing of the jaws. Deep 
lacerated wounds where there is much tissue destruction, 
as, for example, gun-shot wounds, are especially favor- 
able for the development of the tetanus. 
The true prophylaxis consists in administering anti- 
tetanic serum in all traumatic wounds, especially gun- 
shot, or where the patient had stepped on a nail, espe- 
cially near the tilled soil. 
VI. Mechanism of Infection and Immunity 
The tetanus bacillus produces an exotoxin, which has 
a special affinity for the nervous system. It is one of 
the most poisonous substances known, one-millionth of a 
gram being sufficient to kill a mouse. 
The common fowl is extremely resistant to tetanus, 
while the man and horse are unusually susceptible. 
The animal injected with nonfatal doses of tetanus 
produces an antitoxin as the only antibody. SPECIAL BACTERIOLOGY 
161 
VII. Bacteriologic Diagnosis 
Bactcriologic diagnosis is easy and consists in finding 
a Gram-positive, "drumstick" like spore-bearing bacil- 
lus; culturally it is an anaerobe, growing well on meat 
infusion media. 
VIII. Immune Treatment 
Immune treatment is both prophylactic and curative, 
and consists in administering the antitetanic serum, in- 
travenously or intraspinally. The serum is prepared by 
injecting a horse with gradually increasing doses of the 
tetanus bacilli or broth filtrate, and obtaining the horse's 
blood, separating the blood serum and testing its anti- 
toxic (that is, exotoxin-neutralizing properties). 
IX. Summary of Important Characteristics 
B. tetani causes lockjaw, is a Gram-positive, spore-bear- 
ing, motile bacillus, having the appearance of a 
drumstick (in spore form), anaerobe, produces exotoxin; 
antitetanic serum is a perfect preventive and a good 
curative agent, if not given too late. CHAPTER XVIII 
THE ANTHRAX BACILLUS. BACILLUS AERO- 
GENES CAPSULATUS. BACILLUS OF 
MALIGNANT EDEMA 
The Anthrax Bacillus 
Anthrax occurs both in animal and man. It is an 
ancient disease well known from the earliest days of 
history, and is especially common in Russia. Historically 
it is of especial interest as it was the first infectious 
disease proved to be caused by a specific organism. The 
organism was discovered by Pollender in 1849, and thor- 
oughly studied by Davaine in 1863 (see Fig. 31), but 
it was not until 1877 that Pasteur and Koch proved it to 
be the cause of anthrax. 
The bacillus is a very long rod, 5 to 10 /* long. It 
occurs singly in blood, but grown on artificial cultures, 
is found in long threads. It has spores and is Gram- 
positive. A capsule is seen occasionally (never when 
grown on artificial media). The anthrax is an aerobic 
organism, facultatively anaerobic, and grows well on or- 
dinary culture media. It is nonmotile, has no flagella. 
Spore formation ceases below 18° C. and above 42° C. 
Because of its spores the anthrax bacillus is very re- 
sistant to heat and ordinary chemicals, it takes 10 min- 
utes' exposure to live steam to kill them. 
In man three varieties of infection are produced: (1) 
162 SPECIAL BACTERIOLOGY 
163 
skin, (2) lungs (''the wool sorters' disease"), and (3) 
gastrointestinal. No other disease shows so many bacilli 
in blood as the anthrax infections. It produces an endo- 
toxin. Pasteur's vaccine is a wonderfully efficient treat- 
ment of the infection in the cattle (the cultures for the 
Fig. 30.-Bacillus welchii. Film preparation of a milk culture, x 1000. 
(Hewlett-Mamial of Bacteriology.) 
vaccines are grown at 42° to 43° C., so that no spore- 
forming bacteria are produced). 
Bacillus Aerogenes Capsulatus 
Bacillus aerogenes capsulatus was discovered by Welch 
of Johns Hopkins University, in 1892. 
The organism is 3 to 6 /z long, usually occurs single, 
has spores, is nonmotile, has no flagella, has a capsule, is 
Gram-positive. 
This bacillus is a strict anaerobe and grows on all 164 
PRINCIPLES OF BACTERIOLOGY 
simple media. Gelatin is slowly liquefied. Grape sugar, 
milk and cane sugar are all fermented. 
The infection usually occurs after injury, especially 
after open fractures. In the present war this infection 
has been of great importance, and it seems that gunshot 
wounds offer great opportunity for such infections, es- 
pecially where there is well manured soil. Spores have 
been repeatedly found on the uniforms of soldiers. 
Fig. 31.-The anthrax bacilli showing spores. (Mallory and Wright-Patho- 
locjical Technic.) 
A new ray of hope is offered by the brilliant work of 
our own bacteriologist, Bull, of the Rockefeller Institute, 
who has succeeded in preparing an antitoxin (the organ- 
ism producing an exotoxin) which has proved highly 
successful in animal experimentation. 
Bacillus of Malignant Edema 
The bacillus of malignant edema was discovered by 
Pasteur in 1877. SPECIAL BACTERIOLOGY 
165 
This is a slender long rod, motile, with numerous 
flagella, has spores, and is Gram-negative. It is a strict 
anaerobe, grows readily on simple media. It produces 
acute inflammation and (edema) swelling which ex- 
tends deeply into the tissues, and is very dangerous. CHAPTER XIX 
BACILLUS BOTULINUS. BACILLUS MALLEI. 
BACILLUS PYOCYANEUS 
Bacillus Botulinus 
Bacillus botulinus causes about 25 per cent of all meat 
poisoning, a very dangerous disease. It was discovered 
by Van Ermengem in 1896. It is a large rod, 4 to 6 
motile, has flagella, no spores, no capsules, is Gram-posi- 
tive, is a strict anaerobe, grows well on meat infusion 
media. It produces an exotoxin. 
Bacillus Mallei 
Bacillus mallei causes glanders. It is a small bacillus, 
is nonmotile, has no flagella, no spores, no capsules, is 
Gram-negative, stains with methylene blue, it is not un- 
like the diphtheria bacillus, stained parts alternate with 
faint parts, grows well on meat infusion media. 
It produces an endotoxin. 
In diagnosing the disease, the mallein test is used: a 
small amount of the endotoxin is injected in the skin, 
and the presence of infection, a severe reaction takes 
place. 
Bacillus Pyocyaneus 
Bacillus pyocyaneus causes green suppurations which 
are fortunately rare, for while they are not dangerous, 
they seriously interfere with healing if in wounds. Oc- 
casionally kidney infections are caused by this organism 
(the author saw three cases within six months). 
166 SPECIAL BACTERIOLOGY 
167 
The organism was discovered by Gessard in 1882. It 
is a short rod, Gram-negative, motile, has flagella, no 
spores, no capsules. It is an aerobic organism, faculta- 
tively anaerobic, grows well on simple media on which 
within twenty-four hours, a green pigment is formed. 
It produces both an exotoxin and endotoxin. As to the 
treatment a new method has been evolved during the 
present war which is by far better than anything so 
far used; this consists of applying a 1 per cent solution 
of acetic acid to infected areas; in three cases treated by 
this method at the Charity Hospital all three cases showed 
complete disappearance of green discharge within 
twenty-four to forty-eight hours. CHAPTER XX 
BACILLUS DIPHTHERIA 
I. Historical 
Klebs in 1883 and Loeffler in 1884 were first to dis- 
cover and describe the organism. The toxin was first 
obtained from cultures by Roux and Yersin in 1888, 
while the antitoxin was discovered by Von Behring and 
Kitasato in 1890. 
II. Morphology 
The organisms are slightly curved rods, 4 to 5 
they are not uniform in thickness, usually being club- 
shaped. The best stain for the diphtheria bacilli is 
Loeffler's "alkaline methylene blue," the bacilli show 
a characteristic lack of uniformity in staining so that 
the stained preparations show lighter parts alternating 
with darker, shaded parts, the bacilli possessing a char- 
acteristic "beaded" appearance. It is a Gram-positive 
organism. It has no spores, capsules, or flagella. 
III. Cultural Characteristics 
The organism is an aerobic one, growing best at 37.5° 
C., although it may grow between 20° C., and 42° C. 
The best medium to grow the bacilli in, is Loeffler's beef 
blood serum (see Chapter on The Culture Media Mak- 
ing). On this medium the organism show small, gray- 
ish white, glistening colonies. 
168 SPECIAL BACTERIOLOGY 
169 
IV. Resistance 
The organism is killed by ten minutes' exposure to 
58° C. Low temperatures are well borne, and it may 
resist drying for several weeks. Chemical disinfectants 
easily destroy the organism (but not in the throats of 
carriers). 
V. Disease Production, Mode of Infection and 
Prophylaxis 
Diphtheria is usually confined to the throat, larynx, 
and nose, but it may attack other mucous membranes for 
which the bacillus seems to have a special predilection. 
Fig. 32.-One of the very characteristic forms of diphtheria bacilli from 
blood-serum cultures, showing clubbed ends and irregular stain. x 1100 
diameters. Stain, methylene blue. (Park and Williams-Pathogenic Bac- 
teria and Protozoa.) 
Diphtheria is transmitted by coming in contact with 
those suffering with disease or carriers of virulent 
bacilli. All persons infected should immediately receive 
an injection of diphtheria antitoxin; the patient should 
be isolated, quarantined, and the strictest precautions 
should be taken in disinfecting utensils, linen, etc. 
Severe epidemics are frequent, usually in institutions, 
such as schools, asylums, etc. 170 
PRINCIPLES OF BACTERIOLOGY 
A very important question today is that of diphtheria 
bacilli carriers. It is known that many persons may har- 
bor the diphtheria bacilli in their throats and yet show 
no signs of the disease; in spite of this, however, they 
are always a source of danger to others; and therefore, 
the patient should not be allowed to mingle with others 
until at least two cultures made from their throats have 
been found negative. 
The Schick test is based on the principle that persons 
whose natural content of antitoxin is low, i. e., insuffi- 
cient to protect them from diphtheria, can be identified 
by injecting into their skin a small amount of diphtheria 
toxin when in such persons a marked reaction will take 
place at the site of injection, while in persons whose con- 
tent of antitoxin is normal, no such reaction will take 
place. In practice the amount of diphtheria toxin in- 
jected is 1/50 of the amount sufficient to kill, in four days, 
a guinea pig weighing 250 grams. 
Prevention of diphtheria means isolation and quaran- 
tine of the patient and nurse, strictest disinfection of all 
utensils, bedding, etc., and injection of diphtheria anti- 
toxin to all those who had come in contact with the 
patient. Schick's test will show who needs such injec- 
tions. 
When Schick's test is positive, showing that the pa- 
tient does not possess the normal amount of diphtheria 
antitoxin, instead of giving the patients some anti- 
serum, today they are given an injection of the toxin 
and the antitoxin mixture, which is more efficient, be- 
cause the toxin (carefully neutralized by antitoxin) acts 
as a prolonged and slow stimulant to the patient's or- 
ganism to produce his own antitoxin. SPECIAL BACTERIOLOGY 
171 
VI. Mechanism of Infection and Immunity 
Diphtheria bacillus, like the tetanus, produces an exo- 
toxin ; it was in connection with this exotoxin that most 
of the experimental work on immunity was done by Ehr- 
lich and his pupils. The antibody produced in infected 
animals is, of course, an antitoxin. The antitoxin is pro- 
duced on a large scale, by injecting horses with gradually 
increasing doses of toxin, until according to Weigert's 
law, many more antibodies (antitoxin) are produced than 
are really necessary to protect the horse. The blood 
Fig. 33.-Pseudodiphtheria bacilli. (B. hofmanni.) (Park and Williams- 
Pathogenic Bacteria and Protozoa.') 
serum is removed, standardized (that is, its strength is 
determined), and it is then ready for use. It is evi- 
dent that immunity conferred by the injection of anti- 
toxin serum is acquired artificial, passive immunity. 
VII. Bacteriologic Diagnosis 
Bacteriologic diagnosis rests on peculiar beaded ap- 
pearance of a bacillus, which is often club-shaped, Gram- 
positive; cultural appearance on Loeffler's beef blood 
serum is diagnostic: small, round, glistening, grayish 
white colonies. 172 
PRINCIPLES OF BACTERIOLOGY 
VIII. Immunity Treatment 
Together with the smallpox vaccination, the preven- 
tion and treatment of diphtheria with the antitoxin serum 
(initiated by Behring, Roux and Yersin) is, without 
doubt, the greatest accomplishment achieved by medicine. 
No one today realizes what diphtheria meant in former 
days: death was inevitable; during an epidemic, mourn- 
ing crepe would be hung out on one house after another, 
an eloquent and silent witness of the dreadful scourge. 
Today a death from diphtheria is a scarlet spot on a 
physician's escutcheon, there is absolutely no excuse for 
death from diphtheria today, for, unlike the tetanus anti- 
toxin serum which is a much better prevention than a 
cure, the diphtheria serum is an extremely efficient cure, 
as well as preventive; the earlier the administration of 
the serum is begun the better the results. 
IX. Summary of Important Characteristics 
A slender Gram-positive bacillus, usually club-shaped, 
presenting when stained, a beaded appearance, growing 
in beef blood serum in small, glistening, grayish colonies, 
nonmotile, causes diphtheria; possesses an affinity for 
mucous membranes, produces an exotoxin, the body re- 
cured by the antitoxin serum. 
The pseudodiphtheria bacillus (Hoffmann's bacillus) is 
shorter and thicker, the beads or bands in stained prep- 
arations seldom exceed two or three. It grows more 
abundantly and colonies are whiter than those of the 
diphtheria bacilli. CHAPTER XXI 
BACILLUS TUBERCULOSIS 
Tuberculosis, or consumption, is an infection which 
occurs most often in the lungs, but no organ is safe; the 
bones, lymph glands, intestine, peritoneum, genital or- 
gans, skin, all frequently fall victims of the "white 
plague. ' ' 
I. Historical 
Koch, in 1882, was first to isolate and cultivate the 
bacillus. 
II. Morphology 
The bacilli are slender rods, 2 to 4 in length. They 
are nonmotile, have no flagella, no spores, no capsules, 
but are said to have a cell membrane which enables them 
to resist drying. It does not stain wuth ordinary dyes, 
and a special method is used for the purpose (see chapter 
on Staining in General Bacteriology) ; because, once 
stained, it resists the action of strong acids, the tubercle 
bacillus is called "acid-fast." It is Gram-positive. 
III. Cultural Characteristics 
The tubercle bacillus is very difficult to cultivate. 
Special media (described in chapter on Culture Media) 
are necessary for successful cultivation. Growth takes 
place from the tenth day on; when well developed the 
growth has a characteristic "bread crumb" wrinkled dry 
173 174 
PRINCIPLES OF BACTERIOLOGY 
appearance. The tubercle bacillus is an aerobic organ- 
ism. 
Dry heat (100° C.) will be resisted for one hour. The 
tubercle bacilli are resistant to cold and very resistant 
to drying; in sputum the bacillus remains alive for two 
or three months. A 5 per cent carbolic acid solution 
kills the bacilli in a few minutes, sunlight kills them in a 
few hours. 
IV. Destruction 
Fig. 34.-Tubercle bacilli, human, x 1000 diameters. (Park and Williams- 
Pathogenic Bacteria and Protozoa.) 
V. Disease Production, Mode of Infection, Disinfec- 
tion and Prophylaxis 
Disease is produced both by inhalation (of infected 
dust) and ingestion (of infected meat, milk, etc.). 
Formation of small nodules, the so-called "tubercle," 
is the characteristic feature of the disease; this may be 
followed by ulceration and cavity formation. Unsani- 
tary environment is the best ally the disease has; 
crowded, dark rooms, lack of fresh air, sunlight, nour- 
ishing food-is the favorite playground of tuberculosis. 
It is not a hereditary disease; the reason that so many 
children of tuberculous parents develop the disease is 175 
SPECIAL BACTERIOLOGY 
not that they were born of tuberculous parents, but be- 
cause they live with them. Prevention means isolation 
of the patient, disinfection of sputum and other excreta 
and linen, etc., fumigation of the room, etc. See special 
chapter on Disinfection and Fumigation. 
VI. Mechanism of Infection and Immunity 
The tubercle bacillus produces an endotoxin (tuber- 
culin). Tuberculin, prepared in several ways, is used 
for diagnosis of the infection; no cattle are to be killed 
for food unless they have been tested with tuberculin. 
Also all cows producing milk for sale should be tested. 
In human beings the tuberculin test is applied into or 
under the skin, and in infected individuals. Such injec- 
tion causes a marked reaction at the site of injection. 
This test is called von Pirquet's test. The antibodies 
produced by infected animals and human beings are 
essentially bacteriolysins. 
VII. Bacteriologic Diagnosis 
Bacteriologic diagnosis rests on finding the bacilli 
stained by the special method (described in chapter on 
Staining). If this fails, sputum or urine may be in- 
jected intraperitoneally into a guinea pig and if the 
animal does not die in three or four weeks, it is killed, and 
evidences of tuberculosis are looked for at the site of 
injection, in the lungs, liver and spleen. Tuberculin test 
and, recently, complement-fixation test (similar to Was- 
sermann test for syphilis) are of great value. 
VIII. Immune Treatment. 
No such treatment is very successful, although vari- 
ous authors have claimed good results obtained in some 
cases treated with small doses of tuberculin. 176 
PRINCIPLES OF BACTERIOLOGY 
IX. Summary of Important Characteristics 
A slender rod, stained by special method, resists drying 
and heating better than most of the bacteria, requires 
special methods of cultivation. Tuberculosis is the most 
widely spread disease, not only among human beings, but 
also among cattle (bovine tuberculosis), birds (avian 
tuberculosis), fish, etc. Any organ in the body may be 
involved, but the lungs are the most frequently affected 
organ. Very contagious disease, favored by unhygienic 
surroundings, requires strictest disinfection; diagnosis 
rests on finding the bacilli in sputum, urine, tissue, or 
on animal inoculation, tuberculin test and complement- 
fixation test. Produces an endotoxin, no immunity treat- 
ment is successful. CHAPTER XXII 
THE BACILLUS OF LEPROSY. THE SMEGMA 
BACILLUS 
The Bacillus of Leprosy 
Bacillus lepras is the organism that causes the hor- 
rible (until recently incurable) disease called leprosy. 
It resembles the tubercle bacillus very closely except 
Fig. 35.-Leprosy. Section of skin, x 1500. (Hewlett-Manual of Bac- 
teriology.') 
that it is not quite as slender. The disease is almost 
never met with in this country, and the bacteriology of 
the affection is, therefore unimportant. The disease is 
less contagious than most other bacterial diseases, and 
those who observe ordinary precautions of cleanliness 
seldom, if ever, contract it. The differentiation between 
the tubercle and leprosy bacilli is practically impossible. 
177 178 
PRINCIPLES OF BACTERIOLOGY 
The Smegma Bacillus 
The smegma bacillus is a harmless (saprophytic) or- 
ganism, which occurs around the genital organs of both 
male and female, and is important because it may be 
readily confused with the tubercle bacilli. The best 
method of differentiation between the two is that of 
Pappenheim. 
Fig. 36.-The smegma bacillus. Smear preparation of smegma, x 1500. 
(Hewlett-Manual of Bacteriology.') 
1. Make smears and fix by heat in the usual manner. 
2. Stain with Zeihl's carbol-fuchsin solution (steam- 
ing) for two minutes. 
3. Pour off the stain, and without washing, pour the 
following mixture: 
4. Wash in water. Smegma bacilli will be decolorized, 
but the tubercle bacilli will be stained bright red. 
Rosolic acid 1 gram 
Absolute alcohol 100°C. 
Methylene blue added to saturation. 
Glycerine (added after other ingredients 
have been mixed) 20°C. 
This mixture is poured on and off several times. CHAPTER XXIII 
THE INFLUENZA GROUP. THE BACILLUS PER- 
TUSSIS. BACILLUS PESTIS. THE CHOLERA 
GROUP. THE BACILLUS OF DUCREY 
The Influenza Group 
Until the last epidemic, or rather, pandemic of influ- 
enza, it was customary to regard the Pfeiffer's Bacillus 
as the cause of influenza, but today, in the light of the 
enormous amount of work done since the last epidemic, 
it seems that the great mortality (almost always due to 
bronchopneumonia) during epidemics is due to some or- 
ganisms in addition to B. influenzae, most probably strep- 
tococci and pneumococci. In fact, there seems to be 
quite a discrepancy of opinions as to just what the etio- 
logical (causative) agent is. 
1. That it is a special streptococcus (Tunnecliff, Rose- 
now). 
2. That it is the influenza bacillus (considerable doubt 
now exists as to this, chiefly because of negative results 
of agglutination tests of Park and Williams). 
3. That it is some filterable virus. No definite con- 
clusions can be formed as yet. 
Pfeiffer's Bacillus influenza? is an extremely small 
organism (0.5 micron) noncapsulated, possessing no 
spores and no flagella. They do not stain very well 
with the ordinary aniline dyes, and the best stain is 
fuchsine (10 per cent) or Loeffler's methylene blue-al- 
lowed to act about 5-7 minutes. 
The best culture medium for the cultivation of the 
179 180 
PRINCIPLES OF BACTERIOLOGY 
influenza bacilli is either the blood agar, Avery's sodium 
oleate blood medium (Jour. Am. Med. Assn., 1918, vol. 
71) or Park and Williams special influenza medium. 
The cultures are very sensitive to changes in the tem- 
perature and are very short-lived, requiring transfers 
every 3 or 4 days. The endotoxins of the Pfeiffer's 
bacillus are very powerful. 
Between the epidemics this organism is found in the 
upper respiratory tract, and apparently bears no rela- 
tionship to the epidemic disease. The best explanation 
for the recurrence of the epidemics is probably the short 
duration of the immunity conferred by an attack of the 
disease. 
The Bacillus Pertussis 
The Bacillus pertussis causes whooping cough, and was 
discovered by Bordet and Gengou in 1900. It is a very 
small, short bacillus, almost resembling a coccus, Gram- 
negative, nonmotile, has no flagella, no spores or cap- 
sules. It grows on defibrinated blood agar potato. 
Patients with whooping cough should be isolated. The 
complement-fixation test is used in the diagnosis, but is 
not yet perfected; the bacillus produces an endotoxin. 
The immunity treatment-the pertussis vaccine-is of 
great value in shortening the disease and hastening re- 
covery. 
Bacillus Pestis 
Bacillus pestis which causes the most fatal of all dis- 
eases-the bubonic plague-was discovered by Kitasato 
and Yersin in 1894. It is difficult to grasp the havoc 
wrought by this scourge; in the reign of Justinian one- 
half of the population was wiped out by the plague. 
Known as "Black Death," it swept the entire world in 
the fourteenth century, killing 25,000,000 people. It is SPECIAL BACTERIOLOGY 
181 
Fig. 37.-Influenza bacilli, x 1100 diameters. (Park and Williams-Patho- 
genic Bacteria and Protozoa.') 
Fig. 38.-Bacillus pestis. Smear preparation of sputum, x 1000. (Hewlett- 
Manual of Bacteriology.') 
always present in India, exacting* as its toll several thou- 
sand lives yearly. Bacillus pestis is a thick, short bacil- 
lus, Gram-negative, has no spores, no capsules, no flagella, 
and is not motile. It grows well on meat infusion media. 
It is aerobic, and may remain alive for months and years 182 
PRINCIPLES OF BACTERIOLOGY 
in the dark, if sufficient moisture is present. It lives for 
two weeks in the pus and sputum of patients. Complete 
drying kills the bacilli in two to three days, live steam 
in a few minutes. The disease is usually acquired by 
entrance of bacilli either through the skin or by the 
respiratory tract, in the latter case the disease is not 
unlike pneumonia. The disease is extremely dangerous 
causing great mortality. 
The bacilli are usually transmitted by rats and ground 
squirrels. One attack of plague usually confers im- 
Fig. 39.-Spirillum cholerae. Film preparation of a pure culture, x 1500. 
(Hewlett-Manual of Bacteriology.') 
munity. Both the vaccine and the serum treatments 
have been used for prevention with encouraging results. 
The Cholera Group 
Cholera, which at times becomes epidemic and invades 
large territories, is but little known in this country. It 
is a very dangerous disease, characterized by persistent 
diarrhea and collapse. The "comma bacillus," so-called SPECIAL BACTERIOLOGY 
183 
because of its shape, or the Spirillum cholera was dis- 
covered by Koch in 1883. It is a small curved rod, ac- 
tively motile, has one flagellum, no spores or capsules, 
is Gram-negative and grows readily on meat extract 
media. It is an aerobic organism, and can be quickly 
killed by ordinary antiseptics. The disease is contracted 
by eating contaminated food or drinking impure water. 
It is essentially a "water-borne" disease, as has been 
shown in the famous epidemic of Hamburg (1892) where 
a terrible epidemic of the disease raged, while the town 
of Altona (as close to Hamburg as Brooklyn is to New 
York) which had a separate water supply, was scarcely 
touched. The prevention during an epidemic means a 
thorough filtration of water or the use of boiled water 
and the patient should be handled in the same manner 
as a typhoid case. 
The cholera spirillum produces an exotoxin and the 
infected animal elaborates bacteriolysins; it was in con- 
nection with the cholera spirillum that Pfeiffer has 
demonstrated bacteriolysins. One attack usually con- 
fers a permanent immunity. No successful immune treat- 
ment has as yet been developed. 
The Bacillus of Ducrey 
Bacillus of Ducrey is the organism which produces 
chancroid, an acute inflammatory lesion on the genital 
organs. It is acquired by direct contact. The bacillus 
is very small, nonmotile, has no flagella, no spores, no 
capsules. It is Gram-negative and grows on blood agar. CHAPTER XXIV 
THE SPIROCHETAL DISEASES 
Spirochetes or treponemata belong to a class which is 
half way between the true bacteria and protozoa (the 
lowest order of animal). They are corkscrew-like, wavy 
threads. Several very important diseases are caused by 
the various spirochetes. (See Fig. 40.) 
I. Syphilis 
Syphilis, probably, is the most disseminated disease, 
transmitted by direct contact, sexual or asexual. It is 
caused by Spirocheta pallida, discovered by Schaudinn 
and Hoffmann in 1905. The spirochetes must be stained 
by a special method, not to be attempted by the begin- 
ner, and can be grown anaerobically on ascitic fluid agar 
to which a piece of sterile rabbit kidney has been added. 
Noguchi, of the Rockefeller Institute, is the discoverer 
of this method. 
One of the simplest methods of staining is to place a 
drop of material from a syphilitic lesion on a glass slide 
and mix it with a drop of India ink, then make a smear 
as if a blood smear is to be made. Examined with the 
oil-immersion lens the spirochetes appear unstained 
against a black background. 
Within a year of the discovery of the Spirocheta pal- 
lida, Wassermann, Neisser and Bruck devised a special 
test known as the "Wassermann test," which shows the 
presence of the disease, even though no symptoms or 
signs are detected. 
184 SPECIAL BACTERIOLOGY 
185 
A year later Ehrlich discovered the famous Salvarsan 
(606) which, combined with mercury, insures the cure 
in practically all cases. Truly, this has been a wonder- 
ful age! 
The moot question of whether syphilis is an hereditary 
disease seems to have been definitely settled, thanks to 
the Wassermann test. Practically all authorities agree 
that syphilis is not hereditary in the sense that certain 
characteristics are transmitted by the germ-cells; what 
really happens is this: the spirochetes exist in the pla- 
centa and the fetal organs, carried there (in a vast 
majority of cases) by maternal blood, so that infection 
clearly takes place in the uterus (an intrauterine infec- 
tion) ; therefore, syphilis is a congenital, and not an 
hereditary, infection. The old statements that in some 
cases a mother may be nonsyphilitic while the child is 
syphilitic, and that in other cases the mother may be 
syphilitic while the child is not (the former known as 
Colles' law, and the latter as Profeta's law), have been 
shown, by positive Wassermann tests, to be false; the 
mothers of syphilitic children and, conversely, the chil- 
dren of syphilitic mothers may be only apparently 
healthy, but in reality are syphilitic. 
The Wassermann Test.-The exact technic will not 
be given here, as the test should be learned in a sero- 
logical laboratory by actual work under a careful super- 
vision of some competent instructor; but the beginner 
should be acquainted with the general principles of 
the text. Pead the chapters on Immunity before you 
read the following. 
If a person has syphilis, he must have the syphilitic 
antibody (amboceptor) ; that is what we wish to deter- 
mine in the Wassermann test. We know that ordinarily 
in the body the antigen (the infecting bacterium), the 186 
PRINCIPLES OF BACTERIOLOGY 
amboceptor (the antibody) and the complement (the 
substances always found in the blood) unite together; 
if, therefore, we would take in a test tube, the comple- 
ment (for this we use a guinea pig's blood), the antigen 
(the preparation from the bacteria causing the given 
disease--in this case Spirocheta pallida), and the pa- 
tient's blood serum, then, the antigen and the comple- 
ment would unite with the amboceptor if the patient has 
syphilis and his blood serum had the amboceptor; but 
even if it were so how would we know it? Such a 
union of the three substances in a test tube would show 
no change visible to the naked eye. This phenomenon of 
fixation of the complement by the union of the ambocep- 
tor and the antigen is known as the Bordet-Gengou 
phenomenon. 
For this reason, Wassermann, Neisser, and Bruck 
took advantage of the following: they injected the rab- 
bit with a sheep's blood until the rabbit's serum con- 
tained the amboceptor against the sheep's red blood 
cells; now if into a test tube we place the sheep's red 
blood cells (the antigen), a guinea pig's serum (the 
complement) and the rabbit's serum (the amboceptor), 
and these three substances unite, in this case-, there is 
a, change visible to the naked eye, namely, the turbid 
contents of the tube become clear and the red blood cells 
of the sheep disappear-this is called hemolysis, and is 
due to the rabbit's serum having produced the ambocep- 
tor against the sheep's red blood cells. 
Now, in the Wassermann test, we put into a test tube 
the syphilitic antigen (a preparation from Spirocheta 
pallida*), the complement (a guinea pig's serum) and the 
*In actual practice it is not necessary to use a preparation from 
spirochetes as it has been found that the fatty (lipoid) substances 
(oleates, lecithins and cholesterol) on which the spirochetal antigen 
apparently depended for its efficiency-are more abundant in extracts 
of normal organs, e. g., the human hearts. The best antigen today 
is probably the so-called Kolmer's antigen which is prepared from 
normal human or beef hearts. SPECIAL BACTERIOLOGY 
187 
patient's blood serum which may or may not contain 
the syphilitic serum depending on whether or not the 
patient has syphilis. We incubate this at 37° C. for 
thirty minutes to permit the three substances to unite. 
Then add to the tube the rabbit's serum and the 
sheep's red blood cells, and again incubate for thirty 
minutes, at the end of which we take out the tube and 
examine it. If the patient has syphilis, his blood serum 
must have the syphilitic amboceptor, so that at the first 
incubation, this amboceptor and the syphilitic antigen 
must have united together, so that when we added the 
rabbit's serum and the sheep's red blood cells all com- 
plement had been bound up by the syphilitic system 
(the antigen and the amboceptor) and none was avail- 
able for the hemolytic system (the rabbit's serum and 
the sheep's red blood cells) and consequently these 
two could not unite with the complement and the con- 
tents of the tube did not become clear, that is, hemolysis 
did not take place; if on the other hand, the patient 
did not have syphilis, and his serum did not have the 
syphilitic amboceptor, then, after the first incubation, 
the complement was free, and, upon the addition of the 
hemolytic system (the rabbit's serum and the sheep's 
red blood cells), the complement united with the hemo- 
lytic antigen (sheep's red blood cells) and the hemo- 
lytic amboceptor (the rabbit's serum), hemolysis took 
place, and the contents of the tube became clear. 
As one can now readily see, the test uses two systems: 
the syphilitic antigen and amboceptor (if present) and 
the hemolytic antigen and amboceptor; each system 
equally capable of uniting with the complement. 
Graphically it may be represented as follows: 188 
PRINCIPLES OF BACTERIOLOGY 
Syph. ambo. 1. 
(patient's serum) 
4. hemolytic ambo. 
' (rabbit's serum). 
3 
Complement 
Syph. antigen 2. 
(preparation of 
Spirocheta pallida). 
5. hemolytic antigen 
(sheep's red blood 
cells). 
If patient has syphilis, his serum has (1) and then 
12 3 combine during the first incubation, (4) and (5) 
have no available complement (3) which is now held 
Fig. 40.-Various types of spirochetae. (MacNeal-Pathogenic Organisms.) 
bound in 1 2 3 combination, no hemolysis takes place, 
and the test is positive. If the patient has no syphilis, 
his serum has not 1, and (4) and (5) combine with 
the complement (3) into the 3 4 5 combination, hemolysis 189 
SPECIAL BACTERIOLOGY 
takes place, and the Wassermann test is negative. It 
is now clear why the test is also called a complement- 
fixation test. 
Precipitation Tests.-Several tests have been offered 
for the diagnosis of syphilis based upon the formation 
of specific precipitins when a proper antigen is mixed 
with patient's serum and incubated; the precipitin par- 
ticles (flakes and granules) can be readily seen with the 
naked eye. These tests are, of course, much simpler 
than the Wassermann test, and, if equally accurate, 
would be extremely valuable. The best known tests of 
this group are the Sachs-Georgi, the Meineeke, the Dold 
and the Kahn tests, which differ from each other both 
in the technic and the preparation of antigen. 
All these precipitin tests are very promising, and as 
time goes by, it appears, judging from both the review 
of the literature and personal experience, that the Kahn 
test is destined to stay as a very reliable and a very 
simple test for syphilis, checking up very favorably with 
the Wassermann test. 
II. Relapsing Fever 
Relapsing fever, rarely seen in this country, is caused 
by Obermeier's spirochete (1873). This is a much 
longer organism than that of syphilis. It can be culti- 
vated by Noguchi's method, like the Spirocheta pallida 
of syphilis. The disease consists of recurrent attacks of 
fever. 
III. Vincent's Angina 
Vincent's angina, a variety of "sore throat" is prob- 
ably caused by Vincent's spirillum, which is a small 
spirochete. The disease is a mild one and yields readily 
to local treatment. 
Yaws, or frambesia, is a disease which occurs in trop- 
ical countries and resembles syphilis. It is caused by 
Treponema pertenue. 
IV. Yaws, or Frambesia CHAPTER XXV 
MALARIA. THE TYPHUS FEVER 
Malaria 
The organism causing malaria was discovered by 
Laveran in 1880. There are three forms of malaria: 
tertian, when chills recur every forty-eight hours; quar- 
tan, when they recur every seventy-two hours; and 
festivo-autumnal-more or less irregular. Each of these 
varieties is caused by a different parasite, the tertian by 
Plasmodium vivax, the quartan by Plasmodium malarise, 
and the aestivo-autumnal by Plasmodium falciparum. 
The gestivo-autumnal fever is the most dangerous of all 
forms, but is seldom met with in the temperate climate. 
The parasites are transmitted by mosquitoes, whose bite 
carries them into the human body where they pass 
through regular phases of development within the red 
blood cells, multiplying and breaking into numerous 
young forms, which are liberated into other red blood 
cells. When a mosquito bites a malarial patient he car- 
ries these young forms, which undergo the second cycle 
(sexual) of development within the stomach of the mos- 
quito, then new forms reach the salivary glands of the 
mosquito and are transmitted to human beings with the 
bite. 
In order to demonstrate the malarial parasite in the 
human blood, a blood smear is made on a glass slide or a 
cover-slip and is stained with Wright's stain just as an 
ordinary blood preparation is made. The appearance of 
the parasites vary according to the variety and the stage 
190 SPECIAL BACTERIOLOGY 
191 
of the development. The prevention of malaria requires 
the destruction of swamps and marshes-the breeding 
places of mosquitos, the screening of the houses and the 
administration of quinine, which is a specific for ma- 
laria. 
The Typhus Fever 
Typhus fever, which has nothing to do with typhoid 
fever, is very seldom met with in this country, but is 
very frequent in some parts of Europe-Russia, 
Roumania, Servia, etc. The disease is characterized by 
severe fever, rash and intoxication symptoms, and is a 
very dangerous malady. The organism causing it was 
thought to be one discovered in 1914, by an American 
bacteriologist, Plolz, but it now seems doubtful. No 
definite work has yet been completed with regard to im- 
mune treatment, but it is well known that the prevention 
depends on cleanliness, as the bacilli very often are 
carried by body lice. CHAPTER XXVI 
THE HIGHER BACTERIA. THE YEASTS. 
THE MOLDS 
The Higher Bacteria 
The higher bacteria are of more complex structure 
than the ordinary bacteria and cause a number of dis- 
eases. 
I. Streptothrix 
The streptothrix causes an infection of skin and, oc- 
casionally, of other organs. It usually appears in 
stained preparations (with Loeffler's alkaline methylene 
blue) as rods and branching thread-like filaments. 
Streptothrix can be cultivated on sterile fresh kidney 
tissue of rabbits. 
II. Actinomycosis 
Actinomycosis is the 1 'lumpy jaw" of cattle, and 
not infrequently seen in human beings, in whom it af- 
fects not only the jaw, but the skin, lungs and the intes- 
tinal tract as well. In pus the parasites appear as small 
granular bodies, grayish or yellowish. Microscopically 
they appear as rosette-like matter with characteristic, 
club-shaped bodies. 
The Yeasts 
Of these, several cause disease in man, especially the 
so-called Saccharomyces hominis. Another yeast infec- 
192 SPECIAL BACTERIOLOGY 
193 
tion is the disease of the skin called blastomycosis and 
sporotrichosis. 
The Molds 
The molds cause a number of skin diseases and a dis- 
ease of the mouth (most common in children) known as 
thrush. Of the skin diseases caused by molds the com- 
mon ones are ringworm, favus, pityriasis versicolor, etc. SECTION III 
CHAPTER XXVII 
FILTERABLE VIRUSES. DISEASES OF 
UNKNOWN CAUSATION 
There are certain infectious diseases from which no 
one as yet has been able to isolate any individual micro- 
organism ; if infective material from such diseases be 
passed through certain stone-filters it will be found that 
the filtrates contain certain poisonous material which is 
capable of reproducing the disease in all or in its most 
important characteristics if inoculated into a susceptible 
animal. When such filtrate is examined microscopically, 
in a vast majority of cases nothing (that is, no particu- 
late matter) can be seen, either because the organisms 
contained in the filtrate are ultramicroscopic and, there- 
fore, are not to be seen with our ordinary microscopes, 
or possess staining properties and refraction too faint 
to be demonstrated by our present methods. 
Whatever the pathogenic materials contained in the 
filtrates may be, it is customary to refer to them as 
'4 filterable viruses. ' ' 
Park and Williams ("Pathogenic Microorganisms," 
Lea and Febiger, 1920) divide diseases caused by filter- 
able viruses into three groups. (Only those occurring 
in human beings are mentioned here.) 
Group 1. Diseases produced by filterable agents of 
194 DISEASES OF UNKNOWN CAUSATION 
195 
unknown morphology. Here belong rabies (hydro- 
phobia), yellow fever (although as mentioned below, in 
the article on yellow fever, the pathogenic microorgan- 
ism causing this disease seems to have been definitely 
isolated by Noguchi who called it Leptospira icteroides), 
and trench fever (in this case the virus seems to be 
transmitted by the louse). 
Group 2. Diseases probably produced by filterable 
agents shown to be visible. Here belong epidemic polio- 
myelitis (infantile paralysis), encephalitis lethargica and 
mumps. 
Group 3. Disease produced by viruses of questionable 
filterability. Here belongs the remainder of the diseases 
mentioned and described in this chapter, such as Small- 
pox, Measles, Scarlet Fever, Trachoma, etc. 
Smallpox, one of the most virulent of all infectious 
diseases, used to be a veritable scourge, but today is 
practically subdued, thanks to the epoch-marking dis- 
covery of Jenner, who, in 1798, introduced vaccination 
for smallpox and thus conferred an everlasting obliga- 
tion upon mankind. The vaccine is prepared from the 
infectious material taken from the calves which have 
been inoculated with the material from human smallpox. 
Smallpox 
Measles 
Measles is essentially a disease of childhood. Nothing 
definite is known about the organism causing it. 
No specific organism has been discovered for mumps. 
Mumps 
Scarlet Fever 
Scarlet fever is also essentially a disease of childhood. 
Several investigations have claimed to have discovered 196 
principles of bacteriology 
the specific organism, the last one being Mallory, of 
Boston, but so far no definite conclusions have been 
drawn. 
Trachoma 
Trachoma, a dangerous disease of the lining of the 
eye (conjunctiva), is very common among the Indians 
and the poorer classes of some European countries. No 
specific organism has been discovered. 
In all these diseases strictest isolation and quarantine 
is necessary, as well as strict disinfection and fumiga- 
tion. 
Infantile Paralysis 
Infantile paralysis is called " acute anterior poliomye- 
litis. It is an infectious disease which has become a very 
serious menace, as some alarming epidemics have oc- 
curred within the last ten years. 
As yet no definite statement is possible as to the 
microorganisms causing this disease; Rosenow claims to 
have isolated an aerobic streptococcus which he regards 
as the causative agent, while Noguchi and Flexner an- 
nounced in 191^1 that they had obtained "globoid 
bodies" (grown on Noguchi's medium for the cultiva- 
tion of the spirochetes), and that monkeys inoculated 
with certain strains of this organism may die with 
typical lesions of the disease. 
Yellow Fever 
Yellow fever is an acute infectious disease, rare in this 
country, but prevalent in the tropics. It is transmitted 
by the variety of mosquito called Stegomyia fasciata 
(now called Aedes Calopus). The organism causing the 
disease is in all probability the Leptospira Icteroides dis- 
covered by Noguchi in 1918. The bulk of the work on DISEASES OF UNKNOWN CAUSATION 
197 
yellow fever is a glorious chapter in American bacteri- 
ology. Reed, Carrol, Lazear and Agramonte, all of the 
United States Army, have solved the mystery of the 
transmission of yellow fever, all three having sacrificed 
themselves to the altar of science. The brilliant work 
of eradicating yellow fever in Panama is due to the then 
Commission Surgeon-General of the United States Army, 
Gorgas, and is probably one of the most splendid 
achievements in the world's history. 
Dengue 
Dengue is an acute infectious disease characterized by 
chills, fever, bone-ache, etc.; the organism causing it is 
unknown. 
Rocky Mountain Spotted Fever 
Rocky Mountain spotted fever, a disease propagated by 
tick bites, is restricted to the region of the Rockies. The 
organism is unknown. 
Foot and Mouth Disease 
Foot and mouth disease occurs chiefly in cattle, sheep, 
and goats. It appears as a blistery eruption in the 
mouth and on the skin between the hoofs. The specific 
organism causing it is unknown. 
Hydrophobia, or Rabies 
Hydrophobia is a fatal infection of practically all 
mammalia. Although the organism is unknown, Pas- 
teur's brilliant work has given us an absolute defense 
against it, if applied early. 
The infectious agent is transmitted in the saliva of 
the mad animal. By special staining methods (the best 198 
PRINCIPLES OF BACTERIOLOGY 
is that of Lentz) we can demonstrate in the brain of 
mad animals the so-called "Negri bodies," which are 
characteristic of the disease. 
The Pasteur treatment consists of administering a vac- 
cine prepared from the cord of rabbits inoculated with 
the spinal infectious material. It absolutely prevents 
the disease if administered early. Whenever one is bit 
by an animal (usually a dog), the latter should be killed 
and its brain should be examined; the Pasteur treatment 
should be at once instituted. CHAPTER XXVIII 
BACTERIA IN SOIL, AIR, WATER, AND MILK 
The nurse who can give relief and comfort to those 
already diseased, and at the same time can protect her- 
self and others from becoming infected, is in a true 
sense, capable. To do this, it is important that she not 
only have a knowledge of bacteria after they have gained 
access to the body, but also that she be informed as to 
the means by which these organisms live and are detected 
in other environments. 
The surroundings that are the most vital in sustaining 
life in the human body, and are often the haven for 
liarmful bacteria are soil, water, air, and milk. 
Microorganisms of the Soil 
The number and kind of bacteria found in the soil 
are relative, depending largely upon its fertility, cli- 
matic conditions, and its relation to plant and animal 
life; as we are interested only in the pathogenic micro- 
organisms we can exclude the great list of bacteria and 
molds, which, while not important in this study, are ab- 
solutely indispensable in plant life. 
Pathogenic bacteria found in the soil, those giving the 
most trouble are: the B. aerogenes capsulatus (gas 
bacilli), tetanus (lockjaw), typhoid bacilli, and spirilla 
of Asiatic cholera. The first two apparently thrive in 
the soil, the latter, unless under very favorable condi- 
tions, lie dormant and, in a short time disappear. It is 
cpicstionable if any pathogenic bacteria find the soil 
199 200 
PRINCIPLES OF BACTERIOLOGY 
their natural habitat or rather, if they are not carried 
there by dead organic matter and excrement. For full 
detail of these organisms, see special chapter given to 
each. 
Microorganisms of the Water 
Nothing is more important to a community than that 
it is supplied with pure water. Not only that it is clear 
and palatable, but it must be free from pathogenic bacilli. 
The eye is no judge in this matter, for water that 
may look clear and pure will often contain many harm- 
ful bacteria, while cloudiness and odors may be due 
to recent disturbances, to harmless plants, etc. 
Most waters contain many bacteria, but the presence 
of pathogenic bacteria is due to pollution either by sewer- 
age or imperfect drainage. Where the water supply is 
obtained from a well, great care must be taken in select- 
ing a site that is free from the drainage of stables, privy 
vaults, etc. In the city where the source of supply is 
from lakes and rivers, constant watch must be kept to 
see that the sewage and industrial wastes do not render 
it unfit for use. Pools and swamps are often a menace 
to public health, for while this water may not be taken 
into the body for drink, they furnish a breeding place 
for insects that often carry disease germs. 
A large body of water tends to be self-purifying. 
When contamination occurs by the natural processes, i. 
e., sedimentation, oxidation, and the continual action of 
sunlight, these harmful bodies are destroyed. Sedi- 
mentation is the gravitating of the particles to the bot- 
tom, carrying with them impurities and bacteria. The 
action of oxygen and sun rays destroys many bacteria, 
especially those on the surface. 
Water-borne Disease.-When pollution occurs, the dis- 
ease germs that are usually found are the typhoid-colon BACTERIA IN SOIL, AIR, WATER, AND MILK 
201 
group, dysentery and cholera. Most other harmful bac- 
teria are short-lived in water, but those mentioned thrive 
there, and have often caused severe epidemics. 
Should water become contaminated with these germs, 
it may be purified by boiling and distillation. Where a 
large body of water must be purified, science has devised 
the filters and the action of chemicals. With the use of 
these agents from 95 to 98 per cent of the bacteria is 
destroyed. 
Many cities now filter their water supply. The water 
is pooled in large reservoirs. The bottom of these reser- 
voirs is composed of a layer of coarse and a layer of fine 
gravel, on top of this is a strata of coarse and fine sand. 
The water percolates through these at a certain rate, 
as it does so the impurities and bacteria are deposited. 
After a certain time the deposit must be removed. 
There are many kinds of domestic filters, although only 
a few can be recommended for permanent use. Many, 
particularly the cheaper kind, are actually harmful. It 
is impossible to clean them properly, so that the bac- 
teria they hold back breed there. The better and more 
expensive types, to be efficient, need constant care and 
should be cleaned and sterilized at regular intervals. 
When the germ content of water is unknown, during 
certain seasons and epidemics, the use of boiled water 
for drinking purposes should be encouraged. All bac- 
teria are destroyed by this process. The flat, insipid 
taste resulting may be partially overcome by shaking the 
water in open vessels, aerating it. 
Distillation is the process of driving steam into a cool 
jar where it condenses; the water resulting is not only 
free from bacteria but all minerals. It is used largely 
in the laboratory, but is not recommended for drinking 
purposes. When taken into the body in large quanti- 202 
PRINCIPLES OF BACTERIOLOGY 
ties, by osmosis it tends to extract the salts and minerals 
from the tissues. 
Most communities have a regular bacteriologic ex- 
amination made of the water supply; knowing the num- 
ber of bacteria present in the water under normal con- 
ditions, any deviation from this standard must mean 
danger. 
A sample of water to be tested may be taken from the 
faucet, first allowing the water to run for thirty min- 
utes. When it is not possible to obtain a specimen in 
this way it should be collected in sterile vessels, kept at 
a cool, even temperature and hastened to the laboratory. 
The number of bacteria in the water is determined by 
placing 1 c.c. of the sample in a sterile Petri dish, add to 
it a tubeful of melted agar or gelatin, mix and let stand 
for three or four days in a dark, moist atmosphere, at 
37° C. If the number of colonies are small, they may 
be counted very easily. The result may be taken as the 
number of individual bacteria contained in a quantity 
of water measured. If the number of colonies are large 
the original specimen of water must be diluted with 
sterile water or a large number of colonies may be 
counted with the aid of some mechanical device. The 
Wolffhuegel plate is generally used, although any black 
surface ruled in squares with white lines may be Used. 
A certain number of these are counted, then the aver- 
age is obtained. 
The typhoid and cholera bacilli are difficult to isolate 
from contaminated water, or rather it is difficult to dif- 
ferentiate the typhoid from the colon bacillus, unless 
cultural tests are made (see chapter on Typhoid-Colon 
Group). The presence of colon bacilli usually indicates 
the close proximity of sewage pollution. BACTERIA IN SOIL, AIR, WATER, AND MILK 
203 
Bacteria of the Air 
In the early days of surgery, it was thought that in- 
fection was caused mainly through the air. "Working 
upon this principle antiseptic sprays were used. Later 
sterilizing instruments, gloves, clothing, etc., and keep- 
ing the air free from dust as much as possible was in- 
stituted. Sprays were then discontinued. 
All infection does not come through the air, yet it is 
the means of spreading some diseases. Microorganisms 
being so small and light are by any draft or sweeping, 
disturbed, and suspended in the air. In coming to rest, 
if they chance upon suitable media, they thrive, and 
infection occurs. 
The air in a crowded room is filled with bacteria, like- 
wise the air in the open contains more organisms on a 
dry, windy day, than in moist weather. To lessen con- 
tamination from the air, laboratories and operating 
room should be free from drafts. As an added precau- 
tion, to prevent the exhaled breath from blowing organ- 
isms in the air, many surgeons breathe through gauze 
mouthpieces. When bacteria are found in the air they 
usually exist as spores. Living tubercle bacilli have 
been found in the air after sweeping the room where 
tuberculous patients live. Anthrax bacilli have been 
isolated from the air which surrounds the stable of an 
animal suffering from that disease. 
Smallpox, measles, influenza, scarlet fever, and other 
diseases of unknown origin are sometimes called air- 
borne diseases. This term should not be taken literally, 
as "water-borne" diseases is, for the organisms causing 
these illnesses do not live in the air, but are only sus- 
pended or carried by it. They are picked up from 
dry septum or pustules, or sent into the air by sneezing 
or coughing. 204 
PRINCIPLES OF BACTERIOLOGY 
Sedgwick-Tusher's aerobioscope is the most accurate 
instrument used in studying organisms of the air. A 
fair result may be obtained by exposing a Petri dish 
containing culture media. Opening it for one to two 
minutes should be sufficient. It would be interesting to 
do this before and after sweeping a room. 
Bacteria of Milk 
Milk, having great nourishing qualities, is one of our 
chief articles of food. On account of its great food value 
it furnishes a favorable medium for the growth of bac- 
teria. 
By careful handling, the bacteria count of milk may 
be kept down. This is accomplished by submitting the 
cows to the tuberculin test, to see that they are free from 
disease, by sanitary stables free from flies and dust, by 
clean utensils and milkers, and keeping the milk cool 
until consumed. On account of the expense, such meth- 
ods are not always employed, and a form of sterilization 
is used. Pasteurization is generally used to keep milk 
pure. The milk is heated in a water-bath to 160° F., for 
twenty minutes. There are several domestic pasteur- 
izers. They are especially useful in infant feeding. Boil- 
ing for ten minutes is another method for destroying the 
bacteria, but as boiled milk has a rather unpleasant odor 
and taste, it is not generally used. 
Pathogenic organisms in milk usually come from un- 
healthy cows, infecting it with tuberculosis, foot and 
mouth disease, septic sore throat, etc. Many cases of 
sore throat are traced directly to streptococci in milk. 
The method used to determine the number of bacteria 
in milk is similar to that used in water (see chapter 
under that heading). CHAPTER XXIX 
GENERAL CARE OF THE LABORATORY 
Cleanliness should be the watchword. The laboratory 
should be cleaned at regular intervals; the floors and 
working desks should be cleaned once a week with a 5 
per cent carbolic acid solution; all infected culture 
should be properly disinfected. In case of an accident- 
such as breaking a culture tube-a towel soaked in car- 
bolic acid should be applied for an hour to the infected 
area, before the broken glass is removed and the place is 
cleaned. The desk should always be "cleared" at the 
end of the day's work. The workers should wear gowns 
while in the laboratory and should change them before 
leaving. They should carefully disinfect the hands after 
working with infectious material. 
All stock cultures should be transferred at regular in- 
tervals varying according to their nature; staphylococci, 
typhoid-colon-dysentery group and other vigorous or- 
ganisms may be transferred every month, but had better 
be kept in the ice box, as should be pneumococci, which, 
however, must be transferred more frequently. Animals, 
usually rabbits and guinea pigs, should be kept in clean 
places, fed oats, cabbage, and water, and the cages should 
be cleaned daily. 
205 CHAPTER XXX 
LIST OF QUESTIONS 
1. Name the most important discoveries in bacteriol- 
ogy. 
2. Name five important discoveries made by Ameri- 
can bacteriologists. 
3. What are bacteria? What is their structure? Their 
shape ? 
4. What is a flagellum? A spore? A capsule? 
5. How are bacteria reproduced? Their chemical 
composition? Their food requirements? 
6. What is a parasite ? Saprophyte ? Symbiosis ? An- 
tagonism? Give examples of each. 
7. How does temperature affect the bacteria? Mois- 
ture ? Drying ? 
8. Is capsule formation constant in the same species? 
Significance of capsule? Of spores? 
9. What do bacteria do? Discuss fully, mentioning 
their various activities. 
10. Of what use are bacteria in nature and in animal 
body? 
11. What is the effect of electricity, radium, x-rays, 
sunlight, and heat on bacteria? 
12. How should sputum, urine, and feces be disin- 
fected? The room? The patient at recovery? The 
nurse? Apply this to a case of typhoid fever. 
13. What are the different methods of destruction of 
bacteria? Their various purposes? 
14. What is aerobe, anaerobe, either obligatory or fac- 
ultative ? 
206 207 
LIST OF QUESTIONS 
15. How are bacteria studied? What are the most 
commonly used stains? What is Gram's method of stain- 
ing? 
16. Name ten Gram-negative and ten Gram-positive 
bacteria. 
17. What is a culture medium? What is an "ordi- 
nary" medium and what is an "enriched" medium. Name 
three of each, stating for what organism they may be 
used. 
18. What is a "hanging drop" preparation and what 
is it used for? 
19. What are some of the usual bacteriologic exam- 
inations? What is examined and for what infection? 
20. Name three anaerobes and explain how they may 
be cultivated. 
21. What is infection? Immunity? Its varieties? 
22. What is necessary for an infection? What is viru- 
lence ? Antigen ? Antibody ? 
23. What is Ehrlich's theory of immunity? Metchni- 
koff's? 
24. How do bacteria produce an infection? What is 
exotoxin, endotoxin, antitoxin? Blood serum? 
25. What is a vaccine? In connection with what dis- 
eases is it used? How is it prepared? 
26. What is an amboceptor? Agglutinin? Precipitin? 
Bacteriolysin? Hemolysin? Hemolysis? Phagocytosis? 
Opsonin? Leucocytosis? Complement? Weigert's law? 
27. How is antitoxin serum prepared? In what dis- 
eases is it used? Widal test? 
28. What is a Wassermann test? Anaphylaxis? 
29. For what is blood most often examined? Sputum? 
Urine? 
30. What diseases are produced by staphylococci and 
streptococci? How do they differ from each other mor- 208 
PRINCIPLES OF BACTERIOLOGY 
phologically, culturally, and in disease-producing prop- 
erties ? 
31. What bacteria cause pneumonia? Describe briefly 
pneumococci. What is the latest work on pneumococci? 
32. Describe briefly gonococci and meningococci. Of 
what value is the immunity treatment in infections with 
staphylococci, streptococci, pneumococci, and meningo- 
cocci ? 
33. What are the members of the typhoid group ? How 
do they resemble each other and how can they be differ- 
entiated ? 
34. Describe the tetanus bacillus, gas bacillus. 
35. Describe the tubercle bacillus. What other bac- 
teria belong to the "acid-fast" group? 
36. Describe the diphtheria bacillus. 
37. Describe the organism causing influenza, whooping 
cough. Describe plague bacilli. 
38. Describe the anthrax bacillus. 
39. Describe the cholera spirillum. 
40. What diseases are caused by spirochetes ? Describe 
the most important one. 
41. What diseases are caused by yeasts and molds ? 
42. Describe malaria and its parasite. 
43. What diseases are caused by "higher bacteria"? 
44. What are the 11 diseases of unknown causation ' ' ? 
45. What is Pasteur treatment? 
46. Why is the knowledge of bacteriology important 
to the nurse? 
47. What bacteria are of importance in air? Soil? 
Water? Milk? BIBLIOGRAPHY 
The books on bacteriology by the following authors are ex- 
cellent for reference: 
Chester: Determinative Bacteriology, Macmillan Co., New York. 
Hiss and Zinsser: Bacteriology, D. Appleton & Co., New York. 
Hewlett: Manual of Bacteriology, C. V. Mosby Company, St. Louis, 
Mo. 
Jordan: General Bacteriology, W. B. Saunders Co., Philadel- 
phia, Pa. 
Kendall: Bacteriology, Lea & Febiger Co., Philadelphia, Pa. 
MacNeal: Pathogenic Microorganisms, P. Blakiston's Son & Co., 
Philadelphia, Pa. 
Muir and Eitchie: Bacteriology, Macmillan Co., New York. 
Park and Williams: Pathogenic Microorganisms, Lea & Febi- 
ger Co., Philadelphia, Pa. 
Stitt: Bacteriology, Blood Work, Parasitology, P. Blakiston's 
Son & Co., Philadelphia, Pa. 
The following is a list of the best journals devoted to bac- 
teriology: 
Journal of Bacteriology, published bimonthly by Williams and 
Wilkins Co., Baltimore, Md. 
Journal of Immunology, published bimonthly by Williams and 
Wilkins Co., Baltimore, Md. 
Abstracts of Bacteriology, published bimonthly by Williams 
and Wilkins Co., Baltimore, Md. 
Journal of Infectious Diseases, published monthly by Me- 
morial Institute for Infectious Diseases, Chicago, Ill. 
Journal of Experimental Medicine, published monthly by the 
Eockefeller Institute for Medical Eesearch, New York. 
Journal of Laboratory and Clinical Medicine, published monthly 
by C. V. Mosby Company, St. Louis, Mo. 
Journal of Medical Eesearch, published monthly, 240 Longwood! 
Ave., Boston, Mass. 
209 INDEX 
A 
Abscess (see Staphylococcus, 
Streptococcus, Pyocy- 
aneus, etc.), 54 
Acid, carbolic (see Disinfect- 
ants) 
Acid-fast bacteria, definition, 
90 
bacillus tuberculosis, 173 
leprae, 177 
smegmas, 178 
Actinomycosis, 192 
Aerobes, 32 
facultative, 32 
obligatory, 32 
Aerogenes capsulatus, bacillus, 
163 
Aestivo-autumn al malaria par- 
asite, 190 
Agar media, 102 
Agglutination, 152 
macroscopic, technic, 152 
microscopic, technic, 152 
Agglutinins, 63 
Air, bacteria in, 203 
Anaerobes, 32 
facultative, 32 
obligatory, 32 
Anaphylaxis, 76 
Angina, Vincent's, spirochete 
of, 189 
Anilin gentian violet stain, 85 
Animal inoculation, 118 
Antagonism, 33 
Anthrax, 162 
Antibodies, 60 
Antidiphtheritic serum, 172 
Antiseptics, 45 
Antitoxins, 62 
in diphtheria, 172 
in tetanus, 161 
in gas bacillus infection, 164 
Arnold steam sterilizer, 43 
Asepsis, 40 
Autoclave, 44 
B 
Bacilli carriers, 39 
Bacillus (see individual names), 
24 
Bacteria, 24 
cell division of, 29 
cell membrane of, 25 
chemical composition of, 30 
destruction of, 40 
by drying, light, electric- 
ity, radium, x-ray, 
heat, 41 
discovery of, 19 
distribution of, 38 
effect of chemical agents on, 
45 
effect of disinfectants on, 40 
effect of physical agents on, 
41 
forms of, 24 
gram-negative, 87 
gram-positive, 87 
granules in, 25 
in air, 203 
in milk, 204 
in soil, 199 
in water, 200 
methods of studying, 82 
motility of, 82 * 
study of, 82 
nutrition of, 31 
pathogenic, 33 
reproduction of, 29 
size of, 24 
spore-formation in, 28 
staining of, 85 
structure of, 25 
Bacterial toxins, 54 
Bacteriolysis, 63 
Bateriophage, 74 
211 212 
INDEX 
Beef broth, preparation of, 103 
Berkefeld filter, 101 
Bile medium, 110 
Blood, constituents of, 61 
culture, 116 
plasma, 61 
serum, 61 
stain for, 91 
Blood transfusion tests, 64 
Blood typing, 64 
Bordet-Gengou bacillus, 179 
Botulinus bacillus, 166 
Broth, 103, 104 
Brownian movement, 28 
Bubonic plague, 180 
C 
Capsule of bacteria, 26 
Capsules staining, 88 
Carbol-fushsin, 86 
Carrel-Dakin solution, 51 
Carriers, 39 
Cerebrospinal meningitis, epi- 
demic, 134 
fluid, examination of, 115 
Chemotaxis, negative, 68 
positive, 68 
Cholera, 182 
Coccus, 24 
Colon-typhoid bacilli group, 142 
Comma bacillus, 182 
Complement, 61, 73 
fixation of, 74 
fixation tests: 
for syphilis, 185 
for tuberculosis, 175 
for whooping cough, 180 
Contagion, 19 
Cornstarch medium, 109 
Credo's prophylactic treatment 
for gonorrheal oph- 
thalmia, 139 
Culture media, 92 
agar, 103 
meat extract, 102 
meat infusion, 102 
broth, 103 
meat extract, 103 
meat infusion, 104 
blood serum, 107 
Culture media-Cont 'd 
cornstarch, 109 
sugar broth, 104 
sugar-free broth, 105 
gelatin, 105 
glycerin-potato, 112 
litmus milk, 106 
Pctroff's for tubercle ba- 
cillus, 110 
potato, 106 
tubercle bacillus, 110 
Cultures, anaerobic, 112 
plate, technic of making, 114 
D 
d'Herelle's phenomenon, 74 
Dakin's solution, 51 
Dengue, 197 
Diphtheria, 168 
antitoxins, 171 
Disinfectants, 40 
Disinfection, 40 
by Dakin's solution, 51 
of cloth, 48 
of feces, 48 
of rooms, 49 
of skin, 48 
of sputum, 48 
of surgical instruments, 49 
of the bath, 48 
of thermometer, 47 
of urine, 48 
Dorset's medium, 111 
Ducrey bacillus, 183 
Dysentery, 157 
E 
Ehrlich's receptor theory of 
immunity, 68 
Electricity, 41 
Endocarditis, acute, 127 
Endoplasm, 25 
Endo's medium, 107 
Endct oxins, 55 
Enzymes produced by bacteria, 
36 
Epidemic cerebrospinal men- 
ingitis, 134 
Estivo-autumhal| fever, 190 
Exotoxins, 54 213 
INDEX 
F 
Facultative aerobes, 32 
anaerobes, 32 
Favus, 193 
Fecalis alcaligenes bacillus, 156 
Feces, examination of, 115 
Fermentation, 36 
Ferments, 36 
Filter plants, 199 
Filterable virus, 194 
Filtration of water, 200 
Flagella, 28 
staining, 88 
Flexner's serum, 136 
type of dysentery bacillus, 
157 
Fluid, cerebrospinal, examina- 
tion of, 115 
pericardial, 115 
peritoneal, 115 
pleural, 115 
Food supply of bacteria, 31 
Foot-and-mouth disease, 197 
Formaldehyde, 47 
Formalin, 47 
Frambesia, 189 
Free receptors, 70 
Friedlander's pneumobacillus, 
158 
Fumigation, 40 
Fungus, 192 
G 
Gas bacillus, 163 
Gelatin agar, 105 
Generation, spontaneous, 20 
Glanders, 166 
Glassware, sterilization, 42 
Glycerin-potato medium, 111 
Gonococcus, 138 
Gonorrhea, 139 
Gram negative, 87 
positive, 87 
Gram's iodine, 85 
method of staining, 85 
Granules, 25 
Group agglutination, 150 
agglutinins, 150 
Growth of bacteria, 34 
influence of antiseptics on, 
41 
of electricity on, 35 
of environment on, 32 
of food supply on, 30 
of light on, 35 
of moisture on, 35 
of oxygen on, 31 
of temperature on, 34 
Gruber-Widal reaction in ty 
phoid fever, 151 
H 
Hanging drop, 82 
Haptophore atom group, 69 
Hiss-Russel type of dysentery 
bacillus, 157 
Hoffman's pseudodiphtheria 
bacillus, 172 
Hydrophobia, 197 
I 
Immune bodies, 60 
Immunity (see individual), 56, 
60 
acquired, 58 
active, 59 
artificial, 59 
mechanism of, 62 
natural, 57, 59 
passive, 59 
theories of, 66 
Ehrlich's receptor, 68 
Metchnikoff's, 67 
Infantile paralysis, 196 
Infection, 52 
requirements for, 52 
Infectious diseases of unknown 
causation, 194 
Influenza, 179 
Inoculation, animal, 118 
Instruments, sterilization of, 49 
L 
Leprosy, 177 
Leucocytosis, 67 
Leucocytosis and infection, 78 
Leucopenia, 67 214 
INDEX 
Light, influence of, on growth 
of bacteria, 35 
Litmus milk, 106 
Lockjaw, 159 
Loeffler's methylene blue stain, 
84 
blood serum for isolating 
diphtheria bacillus, 107 
Luetin test for syphilis, 77 
M 
Macrophages, 67 
Malaria, 190 
Malarial organisms, 190 
stain for, 90 
Malignant edema, 164 
Mallei bacillus, 166 
Mallein test in diagnosis of 
glanders, 77 
Measles, 195 
Meat extract agar, 102 
broth, 103 
infusion agar, 102 
broth, 104 
Media, 92 
enriched, 105 
preparation of, 102 
sterilization of, 100 
titration of, 96 
Meningococcus, 134 
Micrococcus catarrhalis, 158 
Microphages, 67 
Milk as culture medium, 107 
bacteria in, 204 
Molds, 193 
Motility of bacteria, 28 
study of, 82 
Mucosus capsulatus group of 
bacilli, 158 
Mumps, 195 
N 
Negri bodies, 198 
Nutrition of bacteria, 31 
O 
Obermeier's spirochete, 189 
Obligatory aerobes, 32 
anaerobes, 32 
Ophthalmia, gonorrheal, 139 
Opsonins, 63. 
p 
Pappenheim's stain, 178 
Paralysis, infantile, 196 
Parameningococcus, 137 
Parasites, 32 
Paratyphoid bacillus, 156 
Pasteur treatment, 198 
Pasteurization of milk, 204 
Pathogenic bacteria, 32 
Pericardial fluid, examination 
of, 115 
Peritoneal fluid, examination 
of, 115 
Pertussis, 180 
Petroff's medium, 110 
Phagocytes, 67 
Phagocytosis, 66 
Physical agents, effect of, on 
bacteria, 41 
Pigment production by bac- 
teria, 37 
Plague, 180 
Plasmodium falciparum, 190 
malariae, 190 
vivax, 190 
Plate cultures, 114 
Pleural fluid, examination of, 
115 
Pneumobacillus, 158 
Pneumococcus, 129 
Poliomyelitis, 196 
Potato media, 106 
Precipitation tests, for syphilis, 
189 
Precipitins, 63 
Proteus, bacillus, 156 
Protozoa, 19 
Pseudodiphtheria bacilli, 172 
Pseudomeningocoeci, 137 
Pyemia, 54 
Pyocyaneus, 166 
Q 
Quartan malarial parasite, 190 
R 
Rabies, 197 
treatment of, 198 
Receptor theory of immunity, 
Ehrlich's, 68 INDEX 
215 
Receptors, cell, 72 
of first order, 72 
of second order, 72 
of third order, 72 
Relapsing fever, 189 
Reproduction, bacterial, 29 
Ringworm, 193 
Rocky mountain spotted fever, 
197 
Rose spots in typhoid fever, 
148 
Russel's double sugar medium, 
108 
S 
Saccharomyces hominis, 192 
Saprophytes, 32 
Scarlet fever, 195 
Schick test, 170 
Septicemia, 54 
Serum disease, 77 
Shiga bacillus, 157 
Side-chain theory of immunity, 
Ehrlich's, 68 
Smallpox, 195 
Smear, examination of, 113 
Smegma bacillus, 178 
Soil, bacteria in, 199 
Solution, normal, 98 
Spirillum, 24 
choleras, 182 
of Vincent's angina, 189 
Spirocheta of relapsing fever, 
189 
of Vincent's angina, 189 
pallida, 187 
partenuis, 189 
Spores, formation of, 28 
stain for, 88 
Sporotrichosis, 193 
Sputum, examination of, for 
tubercle bacillus, 117 
Stained bacteria, examination 
of, 84 
Staining, 85 
acid-proof bacilli, 89 
anilin gentian violet method, 
86 
blood, 90 
capsules, 88 
flagella, 88 
Staining-Cont'd 
Gram method, 85 
Loeffler's methylene-blue 
method, 88 
spores, 88 
Treponema pallidum, 184 
tubercle bacillus, 89 
Staphylococcus, 119 
Sterilization, 41 
Streptococcus, 125 
Streptothrix, 192 
Sugar-free broth, 104 
Syphilis, 187 
Syphilis precipitation tests for, 
189 
T 
Temperature, influence of, on 
growth of bacteria, 32 
Tertian malaria parasite, 190 
Test: 
luetin, 77 
mallein, 77 
tuberculin, in diagnosis of 
tuberculosis of cattle, 
175 
von Pirquet's cutaneous, for 
tuberculosis, 175 
Wassermann's, for diagnosis 
of syphilis, 185 
Tetanus, 159 
Thrush, 193 
Tinea versicolor, fungus of, 
193 
Titration of media, 95 
Toxins, 54 
Toxophore group, 71 
Trachoma, 196 
Treatment, Pasteur, 198 
Tuberculin, 175 
test, 77 
Tubercle bacillus, stain for, 89 
Tuberculosis, 173 
Typhoid bacillus, 146 
vaccination, 154 
Typhus fever, 191 
U 
Urine, examination of, 115 216 
INDEX 
V 
Vaccination, 195 
Vaccine, 122 
Vincent's angina, 189 
Virulence of bacteria, 53 
Vitality, 57 
von Pirquet's method of cu- 
taneous diagnosis of 
tuberculosis, 175 
W 
Wassermann's reaction for di- 
agnosis of syphilis, 
185 
Water, bacillus typhosus in, 
200 
bacteria in, 200 
Welch's bacillus, 163 
Whooping cough, 180 
Widal-Gruber reaction in ty- 
phoid fever, 152 
Williams and Burdick's me 
dium, 112 
Wright's stain, 90 
Wool-sorter's disease, 163 
Y 
Y type of dysentery bacillus. 
157 
Yaws, 189 
Yeasts, 192 
Yellow fever, 196