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PATHOGENIC 



MICRO-OEGANISMS 



INCLUDING 



BACTERIA AND PROTOZOA 



A PRACTICAL MANUAL FOR STUDENTS, PHYSICIANS 

AND HEALTH OFFICERS 



BY 

WILLIAM HALLOCK PARK, M. D. 

PROFESSOR OP BACTBRIOLOGY AND HYGIBNB, UNIVBRSITT AND BBLLBVUB HOSPITAL MBDICAL 
COLLBGB, AND DIRECTOR OP THB RB8BARCH LABORATORY OP THB DBPARTMBNT 

OP HBALTH. NBW YORK CITY 

AND 

ANNA W. WILLIAMS, M. D. 

ASSISTANT DIRECTOR OP THB RESEARCH LABORATORY; PATHOLOGIST TO THB NBW YORK 

INFIRMARY POR WOMEN AND CHILDREN 



FOURTH EDITION. ENLARGED AND THOROUGHLY REVISED 

WITH 196 ENGRAVINGS AND 8 FULL-PAGE PLATES 




LEA & FEBIGER 

NEW YORK AND PHILADELPHIA 

1910 



• * ^ - 



_A 



Entered according to the Act of Congress, in the year 1910, by 

LEA & FEBIGER 
in the oflSce of the Librarian of Congress. All rights reserved 



• • • 



• ••• 



• • * 1 * * » 

• . • /; .•'• f 



I ^7 ID 



PEEFAC^E TO FOUETH EDITION. 



The small volume which made the first edition of this book was 
called Bacteriology in Medicine and Surgery. It was written to 
make available for others the practical knowledge which had been 
acquired in the work of the bacteriological laboratories of the City of 
New York and was intended more for medical practitioners than for 
medical students or laboratory workers. When the first editions had 
been exhausted the improvement in methods of cultivating and study- 
ing the protozoa had reached a point rendering it advantageous to in- 
clude the animal as well as the vegetable germs. This was done, and 
the title of the third edition was altered to conform with the text 
which had been broadened to cover the whole field of pathogenic 
microorganisms. 

The book in its later editions has come to be used in an ever- 
increasing degree by medical students, so that while its point of view 
has remained the same, namely, to dwell especially on the relations 
of microorganisms to disease in man, it has been thought wise to 
touch on other aspects; thus, in this fourth edition a chapter has 
been added upon the bacteria concerned in agriculture and in some 
of the important fermentations. The bringing out of this new edition 
has enabled Dr. Williams and myself to rewrite a number of portions 
of the book with which we were not satisfied. We have also rearranged 
this material and added a number of tables which we believe will be 
helpful to the student. The chapters on the colon-typhoid group of 
bacilli and on malaria are examples. 

Such subjects as the relation of bovine tuberculosis to that in man, 
the value of antimeningococcic serum, the use of bacterial vaccines, 
the etiology of anterior poliomyelitis and trachoma, and the prevention 
and cure of trypanosomiasis have been rewritten in the light of the new 
information which has been acquired since the writing of the preceding 
edition. 

The revision of the different portions of the book has been divided 
l)etween Dr. Williams and myself much as in the last edition. 
Dr. Williams has revised the portion of the book devoted to protozoa, 
while I have revised that on the pathogenic bacteria. We are greatly 
indebted to our associates in the laboratory for aid in many differ- 
ent ways. 

This new edition, like its immediate predecessors, is intended to 
answer the needs of the students and physicians, and to cover the 
whole subject of pathogenic microorganisms from their standpoint. 

W. H. P. 
New York, 1910. 



50<^oo 



CONTENTS. 



PART I. 

PRINCIPLES OF BACTERIOLOGY. 

PAGE 

CHAPTER I. 
Introductory — Historical Sketch 1 

CHAPTER II. 
General Characteristics of Bacteria — Classification 7 

CHAPTER III. 
Microscopic Methods 27 

CHAPTER IV. 
Effects of Surrounding Forces upon Bacteria 48 

CHAPTER V. 
The Materials and Methods Used in the Cultivation of Bacteria 59 

CHAPTER VI. 
Products of Bacterial Growth 81 

CHAPTER VII. 
The Soil Bacteria and their Functions — Air Bacteria — Bacteria in Industries . 95 

CHAPTER VIII. 
The Destruction of Bacteria by Chemicals — Practical Use of Disinfectants. . 103 

CHAPTER IX. 

Practical Disinfection and Sterilization (House, Person, Instruments, and 

Food) — Sterilization of Milk for Feeding Infants 113 

CHAPTER X. 
The Relation of Bacteria to Disease 131 

CHAPTER XI. 

The Antagonism Existing Between the Fluids and Cells of the Living Body 

and MicrodrganisiAs 144 

V 



vi CONTENTS, 

pagb 
CHAPTER XII. 

Nature of the Protective Defen^ses of the Body and their Manner of Action — 

Ehrlich's "Side Chain" and Other Theories 150 

CHAPTER XIII. 
The Nature of the Substances Concerned in Agglutination 163 

CHAI*TER XIV. 
Opsonins — Extract of Leucocytes — Bacterial Vaccines 172 

CHAPTER XV. 
The Use of Animals for Diagnostic and Test Purposes 185 

CHAPTER XVI. 

The Procuring and Handling of Material for Bacteriologic Examination from 

Those Suffering from Disease 188 

PART II. 

BACTERIA PATHOGENIC TO MAN INDIVIDUALLY 

CONSIDERED. 

CHAPTER XVII. 
The Bacillus and the Bacteriology of Diphtheria 195 

CH.\PTER XVIII. 
The Bacillus and the Bacteriology of Tetanus 232 

CHAPTER XIX. 
Intestinal Bacteria 245 

CHAPTER XX. 
The Colon-Typhoid Group of Bacilli 255 

CHAPTER XXI. 

The Dysentery Bacillus — The Paradysentery Bacilli (Mannite Fermenting 

Types) 274 

CHAPTER XXII. 
The Typhoid Bacillus 282 

CHAPTER XXIII. 
The Bacillus and the Bacteriology of Tuberculosis 310 

CHAPTER XXIV. 

Bacilli Showing Staining Reactions Similar to Those of the Tubercle Bacilli — 
Lustgarten's Bacillus — Smegma Bacillus — Leprosy Bacillus — Grass 
Bacilli 348 



CONTENTS, vil 

PAGB 

CHAPTER XXV. 
The Influenza and Pseudoinfluenaa Bacilli — ^The Koch- Weeks Bacillus 353 

CHAPTER XXVI. 
The Pyogenic Cocci , 361 

CHAPTER XXVII. 

The Diplococcus of Pneumonia (Pneumococcus, Streptococcus Pneumoniae, 

Micrococcus Lanceolatus) — The Pneumobacillus (Friedlander Bacillus) . . 381 

CHAPTER XXVIII. 

Meningococcus or Micrococcus (Intracellularis) Meningitidis, and the Re- 
lation of It and of Other Bacteria to Meningitis 392 

CHAPTER XXIX. 

The Gonococcus or Micrococcus Gonorrhceee — ^The Ducrey Bacillus of Soft 

Chancre 402 

CHAPTER XXX. 

Bacillus Pyocyaneus (Bacillus of Green and of Blue Pus) — Bacillus Proteus 

Vulgaris 412 

CHAPTER XXXI. 
Glanders Bacillus (Bacillus Mallei) 417 

CHAPTER XXXII. 
Microorganisms Belonging to the Hemorrhagic Septicemia Group 423 

CHAPTER XXXIII. 
The Anthrax Bacillus — The Pathogenic Anaerobes 429 

CHAPTER XXXIV. 
The Cholera Spirillum (Spirillum Cholerse Asiaticae) and Allied Varieties 443 

CHAPTER XXXV. 
Pathogenic Microorganisms Belonging to the Higher Bacteria 458 

CHAPTER XXXVI. 

The Pathogenic Moulds (Hyphomycetes, Eumycetes) and Yeasts (Blasto- 

mycetes) — Diseases Due to Microorganisms Not yet Identified 472 

CHAPTER XXXVII. 

The Bacteriologic Examination of Water, Air, and Soil — The Contamination 

and Purification of Water — The Disposal of Sewage 489 

CHAPTER XXXVIII. 
The Bacteriology of Milk and Its Relation to Disease 500 



viii CONTESTS. 



PART m. 

PROTOZOA. 

PAGE 

CHAPTER XXXIX. 
General CharacteriBtics and Classification 519 

CHAPTER XL. 
Gymnamoebida — Mycetozoa v»- ^^2 

CHAPTER XLI. 
Flagellata 550 

CHAPTER XLII. 
Trypanosoma 557 

CHAPTER XLin. 
Spirochsta and Allies ^ 569 

CHAPTER XLIV. 
Bodo — ^Polymastigida — Ciliata — Sporozoa 584 

CHAPTER XLV. 
The Malarial Organisms — Babesia 596 

CHAPTER XLVI. 
Smallpox and Allied Diseases — Scarlet Fever — Measles 612 

CHAPTER XLVII. 
Rabies— Yellow Fever 622 

GLOSSARY 641 

INDEX 645 



PATHOGENIC MICEO-OEGANISMS. 



PART T. 



PRINCIPLES OF BACTERIOLOGY. 



CHAPTER I. 
INTRODUCTORY— HISTORICAL SKETCH. 

Although most of the more important discoveries in bacteriology 
which place it on the footing of a science are of comparatively recent 
date, the foundations of its study were laid over two centuries ago. 
From the eariiest times the history of bacteriology has been intimately 
associated with that of medicine. Indeed, it is only through the 
investigations into the life history of the vegetable and animal uni- 
cellular microorganisms that our present knowledge of the etiology, 
course, and prevention of the infectious diseases has been acquired. The 
prominent position which the study of microorganisms already holds 
toward medicine is, moreover, daily increasing in importance. Origi- 
nal discoveries are constantly adding to our knowledge of germ 
diseases, and the outlook is favorable for eventually obtaining, through 
serums, through attenuated cultures, or through the toxic substances 
produced by microorganisms themselves, means for immunizing 
against, if not curing, many of the specific infections. Even at present, 
bacterial products and protective serums are used successfully as pre- 
ventive or curative agents in several of the most prevalent infectious 
diseases. Our knowledge concerning other microorganisms has 
enabled us largely to limit their dissemination and so to prevent disease. 
An acquaintance, therefore, with the main facts concerning these 
microorganisms is most necessary to the education of the modem 
physician. 

But before entering into a detailed consideration of the subject it 
may be interesting and instructive to review very briefly a few of the 
important steps which led to the development of the science, and upon 
which its foundation rests, in which we shall see that the results ob- 
tained were gained only through long and laborious research and 

1 



2 PATHOGENIC MICRO-ORGANISMS. 

after many obstacles were met and overcome by accurate observation 
and experiment. 

Probably the first authentic observations of living microscopic 
organisms of which there is any record are those of Kircher, in 1659. 
This original investigator demonstrated the presence in putrid meat, 
milk, vinegar, cheese, etc., of "minute living worms," but did not 
describe their form or character. 

Not long after this, in 1675, Leeuwenhoeck observed in rain-water, 
putrid infusions, and in his own and other saliva and diarrhoeal evac- 
uations living, motile ''animalculae" of most minute dimensions, 
which he described and illustrated by drawings. Leeuwenhoeck 
practised the art of lens-grinding, in which he eventually became so 
proficient that he perfected a lens superior to any magnifying glass 
obtainable at that day, and with which he was enabled to see objects 
very much smaller than had ever been seen before. '* With the great- 
est astonishment," he writes, '*I observed distributed everywhere 
through the material which I was examining animalcules of the most 
microscopic size, which moved themselves about very energetically." 
The work of this observer is conspicuous for its purely objective char- 
acter and absence of speculation; and his descriptions and illustra- 
tions are done with remarkable clearness and accuracy, considering 
the imperfect optical instruments at his command. It was not until 
many years later, however, that any attempt was made to define 
the characters of these minute organisms and to classify them 
systematically. 

From the earliest investigations into the life history and properties 
of bacteria, microorganisms have been thought to play an important 
part in the causation of infectious diseases. Shortly after the first 
investigations into this subject the opinion was advanced that puer- 
peral fever, measles, smallpox, typhus, pleurisy, epilepsy, gout, and 
many other diseases were due to contagion. In fact, so widespread 
became the belief in a causal relation of these minute organisms to 
disease that it soon amounted to a veritable craze, and all forms and 
kinds of diseases were said to be produced in this way, upon no other 
foundation than that these organisms had been found in the mouth 
and intestinal contents of men and animals, and in water. 

Among those who were especially conspicuous at this time for their 
advanced views on the germ-theory of infectious diseases was Marcus 
Antonius Plenciz, a physician of Vienna. This acute observer, who 
published his views in 1762, maintained that not only were all infec- 
tious diseases caused by microorganisms, but that the infective mate- 
rial could be nothing else than a living organism. On these grounds 
he endeavored to explain the variations in the period of incubation of 
the different infectious diseases. He also insisted that there were 
special germs for each infectious disease by which the specific disease 
was produced. Plenciz believed, moreover, that these organisms were 
capable of multiplication in the body, and suggested the possibility 
of their being conveyed from place to place through the air. 



INTRODUCTORY^HISTORICAL SKETCH, 3 

These views, it is true, were largely speculative, and rested upon 
insufficient experiment, but they were so plausible, and the arguments 
put forward in their support were so logical and convincing, that they 
continued to gain ground, in spite of considerable opposition and 
ridicule, and in many instances the conclusions reached have since 
been proved to be correct. The mode of infection, its unlimited de- 
velopment among large numbers of individuals, and gradual spread 
over wide areas — the incubation, course of, and resulting immunity 
in recovery from infectious diseases — all pointed to a living organism 
as the probable cause. 

Among other distinguished men of the day whose observations 
exerted a most powerful influence upon the doctrine of infection, may 
be mentioned Henle. His writings (Pathological Investigaiions, 
1840, and Text-book of Rational Pathology, 1853), in which he de- 
scribed the relation of microorganisms to infectious diseases, and 
defined the character and action of bacteria upon certain phases and 
symptoms of these affections, are remarkable for their clearness and 
precision. 

But, meanwhile, the question which most interested these investi- 
gators into the cause of infectious diseases was: Whence are these 
microorganisms derived which were supposed to produce them? 
Were they the result of spontaneous generation due to vegetative 
changes in the substances in which the organisms were found, or 
were they reproduced from similar preexisting organisms — the so- 
called vitalistic theory? This question is intimately connected with 
the investigations into the origin and nature of fermentation and 
putrefaction. 

SpaUanzani in 1769 demonstrated that if putrescible infusions of 
organic matter were placed in hermetically sealed flasks and then 
boiled the liquids were sterilized; neither were living organisms found 
in the solutions, nor did they decompose; and the infusions remained 
unchanged ^or an indefinite period. 

The objection was raised to these experiments that the high tem- 
perature to which the liquids had been subjected so altered them that 
spontaneous generation could no longer take place. SpaUanzani met 
the objection by cracking one of the flasks and allowing air to enter, 
when living organisms and decomposition again appeared in the 
boiled infusions. 

Another objection raised by the believers in spontaneous generation 
was that, in excluding the oxygen of the air by hermetically sealing 
the flasks, the essential condition for the development of fermentation, 
which required free admission of this gas, was interfered with. This 
objection was then met by Schidze, in 1836, by causing the air ad- 
mitted to the boiled decomposable liquids to pass through strong 
sulphuric acid. Air thus robbed of its living organisms did not pro- 
duce decomposition. 

Schwann in 1839 obtained similar results in another way: he de- 
prived of microorganisms the air admitted to his boiled liquids by 



4 PATHOGENIC MICRO-ORGANISMS. 

passing it through a tube which was heated to a temperature high 
enough to destroy them. To this investigator is also due the credit 
of having discovered the specific cause — the yeast plant, or saccharo- 
myces cerevisiw — of alcoholic fermentation, the process by which 
sugar is decomposed into alcohol and carbonic acid. 

Again it was objected to these experiments that the heating of the 
air had perhaps brought about some chemical change which hindered 
the production of fermentation. Schroeder and von Dusch in 1854 
then showed that by a simple process of filtration, which has since 
proved of inestimable value in bacteriological work, the air can be 
mechanically freed from germs. By placing in the mouth of the 
flask containing the boiled solutions a loose plug of cotton, through 
which the air could freely circulate, it was found that all suspended 
microorganisms could be excluded, and that air passed through such 
a filter, whether hot or cold, did not cause fermentation of boiled 
infusions. 

Similar results were obtained by Hoffmann in 1860, and by Chev^ 
reul and Pasteur in 1861, without a cotton filter, by drawing out the 
neck of the flask to a fine tube and turning it downward, leaving the 
mouth open. In this case the force of gravity prevents the suspended 
bacteria from ascending, as there is no current of air to carry them 
upward through the tube into the flask containing the boiled 
infusion. 

TyndaU later showed (1876), by his well-known investigations 
upon the floating matters of the air, that the presence of living organ- 
isms in decomposing fluids was always to be explained either by the 
preexistence of similar living forms in the infusion or upon the walls 
of the vessel containing it, or by the infusion having been exposed to 
air which was contaminated with organisms. 

These facts have since been practically confirmed on a large scale 
in the preservation of food by the process of sterilization. Indeed, 
there is scarcely any biologic problem which has been so satisfac- 
torily solved or in which such uniform results have been obtained; 
but all through the experiments of the earlier investigators irregulari- 
ties were constantly appearing. Although in the large majority of cases 
it was found possible to keep boiled organic liquids sterile in flasks 
to which the oxygen of the air had free access, the question of spon- 
taneous generation still remained unsettled, inasmuch as occasionally, 
even under the most careful precautions, decomposition did occur 
in such boiled liquids. 

This fact was explained by Pasteur in 1860 by experiments show- 
ing that the temperature of boiling water was not sufficient to destroy 
all living organisms, and that, especially in alkaline liquids, a higher 
temperature was required to insure sterilization. He showed, how- 
ever, that at a temperature of 110° to 112° C, which he obtained by 
boiling under a pressure of one and one-half atmospheres, all living 
organisms were invariably killed. 

Pasteur at a later date (1865) demonstrated the fact that the organ- 



INTRODUCTORY— HISTORICAL SKETCH. 5 

isms which resist boiling temperature are, in fact, reproductive bodies, 
which are now known as spores. 

In 1876 the development of spores was carefully investigated and 
explained by Ferdinand Cohn. He, and a little later Koch, showed 
that certain rod-shaped organisms possess the power of passing into a 
resting or spore stage, and when in this stage they are much less sus- 
ceptible to the injurious action of higher temperatures than in their 
normal vegetative condition. 

Stimulated by the establishment of the fact, through Pasteur's 
investigations, that fermentation and putrefaction were due to the 
action of living organisms reproduced from similar preexisting forms, 
and that each form of fermentation was due to a special microorgan- 
ism, the study of the causal relation of microorganisms to disease was 
taken up with renewed vigor. Reference has already been made to 
the opinions and hypotheses of the earlier observers as to the microbic 
origin of infectious diseases. The first positive grounds, however, 
for this doctrine, founded upon actual experiment, were the investi- 
gations into the cause of certain infectious diseases in insects and 
plants. Thus, Bassi in 1837 demonstrated that a fatal infectious 
malady of the silkworm — p^brine — was due to a parasitic micro- 
organism. Pasteur later devoted several years' study to an exhaust- 
ive investigation into the same subject; and in like manner TiUdsse 
and Kiihne showed that certain specific affections in grains, in the po- 
tato, etc., were due to the invasion of parasites. 

Very soon after this it was demonstrated that microorganisms were 
probably the cause of certain infectious diseases in man and the 
higher animals. Davaine, a famous French physician, has the honor 
of having first demonstrated the causal relation of a microorganism to 
a specific infectious disease in man and animals. The anthrax bacillus 
was discovered in the blood of animals dying from this disease by 
Pollender in 1849 and by Davaine in 1850; but it was not until 1863 
that the last-named observer demonstrated by inoculation experi- 
ments that the bacillus was the cause of anthrax. 

The next discoveries made were those relating to wounds and the 
infections to which they are liable. Rindfleisch in 1866 and Wal- 
deyer and von Recklinghausen in 1871 were the first to draw atten- 
tion to the minute organisms occurring in the pysemic processes result- 
ing from infected wounds, and occasionally following typhoid fever. 
Further investigations were made in erysipelatous inflammations 
secondary to injury by Billroth, Fehleisen, and others, who agreed that 
in these conditions microorganisms could almost always be detected 
in the lymph channels of the subcutaneous tissues. 

The brilliant results obtained by Lister in 1863-1870, in the anti- 
septic treatment of wounds to prevent or inhibit the action of infec- 
tive organisms, exerted a powerful influence on the doctrine of bac- 
terial infection, causing it to be recognized far and wide and gradually 
lessening the number of its opponents. Lister's methods were sug- 
gested to him by Pasteur's investigations on putrefaction. 



6 PATHOGENIC MICRO-ORGANISMS. 

In 1877 Weigert and Ehrlich recommended the use of the aniline 
dyes as staining agents and thus made possible a more exact micro- 
scopic examination of microorganisms in cover-glass preparations. 

In the vear 1880 Pasteur published his discovery of the bacillus 
of fowl cholera and his investigations upon the attenuation of the 
virus of anthrax and of fowl cholera, and upon protective inoculation 
against these diseases. Laveran in the same year announced the dis- 
covery of parasitic bodies in the blood of persons sick with malarial 
fever, and thus stimulated investigations upon the immensely im- 
portant unicellular animal parasites. 

In 1881 Koch made bis fundamental researches upon pathogenic 
bacteria. He introduced solid culture media and the "plate method" 
for obtaining pure cultures, and showed how diflFerent organisms 
could be isolated, cultivated independently, and, by inoculation of pure 
cultures into susceptible animals, could be made, in many cases, to re- 
produce the specific disease of which they were the cause. To him 
more than to any other are due the methods which have enabled us 
to prove absolutely, in a broad sense, the permanence of bacterial 
varieties. It was in the course of this work that the Abbe system 
of substage condensing apparatus was first used in bacteriology. 

In 1882 Pasteur published his first communication upon rabies. 
The method of treatment devised by him is still in general use. A 
little later came the investigations of Loeffler and Roux upon the 
diphtheria bacillus and its toxins, and that of Kitasato upon tetanus. 
These researches paved the way for Behring's work on diphtheria 
antitoxin, which in its turn stimulated investigation upon the whole 
subject of immunity. The number of investigators rapidly increased 
as the importance of the earlier fundamental discoveries became ap- 
parent. Their additions to the science of bacteriology are considered 
in the pages of this book. 



CHAPTER II. 

GENERAL CHARACTERISTICS OF BACTERIA— CLASSIFICATION. 

Among the raicroSrganisms which have in common the ability to 
produce disease in animals and plants, the most important are the 
Bacteria. These minute organisms are usually classed as plants, 
but their structure is so simple and their biologic characteristics are so 
varied that their relationship to the vegetable kingdom is not clear- 
cut. In their possession of more or less rigid bodies, in the tendency 
of many to grow in filaments, and in the ability of some to use simple 
elements as food, they resemble plants; while in the motility of many, 
the non-possession by all of chlorophyll, and in the necessity of many 
for complex food, they resemble animals. 

There is a similar difficulty in definitely classifying the other groups of 
closely related microorganisms, namely, the protozoa, the yeasts, and the 
moulds, and it has been suggested that under the name Protista a third king- 
dom be formed consisting of all of these lowest microorganisms.^ 

Definition of Bacteria. — Bacteria may be defined as extremely 
minute simple unicellular microorganisms, which reproduce them- 
selves with exceeding rapidity, usually by transverse division, and 
grow without the aid of chlorophyll. They have no morphologic 
nucleus, but contain nuclear . material which is generally diflFused 
throughout the cell body in the form of larger or smaller granules. 

Natural Habitat. — There are such wonderful differences in the 
conditions of life and nutrition which suit the different varieties, that 
bacteria are found all over the known world. Wherever there is 
sufficient moisture, one form or another will find other conditions 
sufficient for multiplication. Thus, we meet with bacterial life be- 
tween 0° and 75° C. Some live only in the tissues of men, others 
in lower animals, a larger number may grow in both man and lower 
animals, others still grow only in plants, but by far the greater num- 
ber live in dead organic matter. For some, free oxygen is necessary 
to life, for others, it is a poison. 

Morphologic Oharacteristics of Bacteria.— The fact that each 
bacterial variety possible of cultivation may grow in distinctive 
ways upon so-called artificial culture media has been an immense 
aid in the differentiation of these microorganisms; for the indi- 
vidual cell of most varieties is so minute that even the highest 
magnification we have may show little if any morphologic diflfer- 

* A discussion of the relationship between plants and animals is given in Ray 
Lankester's '* Zoology, " Vol. I, Ist Fascicle. Introduction, 1907. London. 
For the relationship to Protozoa see section III. 

7 



8 PATHOGENIC MICRO-ORGANISMS. 

ence between organisms which produce distinctly different diseases, 
or between a pathogenic and a non-pathogenic form. There are, 
however, certain morphologic and biologic characteristics of the single 
cell which are pronounced, and we therefore study these before 
going on to the study of cultures, that is, of bacteria in masses. 

The determination of morphologic characters for the description 
of bacteria should always be made from fully developed cultures; 
those which are too young may present immature forms, due to rapid 
multiplication, while in old cultures altered or degenerated forms may 
be observed. 

When grown upon different media, variations, especially in size, 
may generally be observed. Such differences should always be 
described, together with a note of the media upon which the organ- 
ism was developed and a statement as to whether each variation is 
a marked feature of the species under consideration. 

The conditions of temperature and of nutrition which favor growth 
are quite various for different species, so that no fixed temperature, 
medium, or age of growth can be regarded as applicable to al! species. 
Morphologic descriptions should always be accompanied by a defi- 
nite statement of the age of the growth, the medium from which it 
was obtained, and the temperature at which it was developed. 

The form and dimensions of bacterial cells at their stage of complete 
development must be distinguished from those which they possess 
just after or just before they have divided. As a spherical cell develops 
preparatory to its division into two cells it becomes elongated and 
appears as a short oval rod; at the moment of its division, on the con- 
trary, the transverse diameter of each of its two halves is greater than 
their long diameter. A short rod becomes in the same way, at the 
moment of its division, two cells, the long diameter of each of which 
may be even a trifle less than its short diameter, and thus they appear 
on superficial examination as spheres. 

8ise. — The dimensions of the adult individual vary greatly in the dif- 
ferent species as well as in members of the same species. The largest 
bacillus recorded is 50^1 to 60/(' long and4/( to S/i wide {B. ButschUi, 
see Fig. 15). One of the smallest forma known (B. influenza!) is 
0.5/1 X 0.2/1. The average size of the known pathogenic rod-shaped 
bacteria is 2/( x 0.5/(, while that of the pathogenic cocci is about 0.8/i 
in diameter. 

Some pathogenic organisms (supposed to be bacteria) are so small 
as to be invisible with any magnification which we now possess. We 
know of their existence only by the fact that they may be cultivated on 
ow.'R^:..! media, producing appearances of mass growth and that such 
ien inoculated into susceptible animals cause the charac- 
;ase (foot-and-mouth disease in cattle). These tiny organ- 
ass through the pores of the finest Berkefeld filter. 
I method for the examination of so-called ultramicroscopic 
has recently been devised, known as the dark-field illumi- 
licromi Hi meter, is .irAsa of an inch. 



GENERAL CHARACTERISTICS OF BACTERIA. 9 

nation (see p. 46). Micro-photography with ultraviolet light has also 
been employed, but so far very little has been learned by either of 
these means (see p. 47). 

Shape. — The basic forms of the single bacterial cells are threefold — 
the sphere, the rod, and the segment of a spiral. Although under 
different conditions the type form of any one species may vary con- 
siderably, yet these three main divisions under similar conditions are 
constant; and, so far as we know, it is never possible by any means to 
bring about changes in the organisms that will result in the permanent 
conversion of the morphology of the members of one group into that of 
another — that is, micrococci always, under suitable conditions, pro- 
duce micrococci, bacilli produce bacilli, and spirilla produce spirilla. 

As bacteria multiply the cells produced from the parent cell have 
a greater or less tendency to remain attached. This is on account of 
the slimy envelope which is more or less developed in all bacteria. 
In some varieties this tendency is extremely slight, in others it is 



Fig. 1 










Varieties of spherical forms: a, tendency to lancet-shape; 6, tendency to coffee-bean shape; 
c, in packets; a, in tetrads; e, in chains; /, in irregular masses. X 1000 diameters. (After 
Flugge-.) 

marked. This union may appear simply as an aggregation of sepa- 
rate bacteria or so close that the group appears as a single cell. Accord- 
ing to the method of the cell division and the tenacity with which the 
cells hold together, there are different groupings of bacteria, which 
aid us in their differentiation and identification. Thus, in cocci we 
get the bacterial cell dividing into one, two, or three planes (Fig. 1), 
while in bacilli and spirilla the divison is generally in only one plane 
(Figs. 2 and 12). 

1. Spherical Form, or Coccus (Fig. 1). — The size varies from 
about 0.3// as minimum diameter to 3/i as maximum. The single 
elements are at the moment of their complete development, so far as 
we can determine, practically spherical ; but when seen in the process 
of multiplication through division the form is seldom that of a true 
sphere. Here we have elongated or lancet-shaped forms, as frequently 
seen in the diplococcus of pneumonia, or the opposite, as in the diplo- 
coccus of gonorrhoea, where the cocci appear to be flattened against 
one another. Those cells which divide in one direction onlv and remain 



10 PATHOGENIC MICROORGANISMS. 

attached are found in pairs (diplococci) or in shorter or longer chains 
(streptococci). Those which divide in two directions, one at right 
angles to the other, form bunches of four (tetrads). Those which 
divide in three directions and cling together form packets in cubes 



f 4. s -, 






Various fornu ot bacilli : n. baeilli n 
dicuUr: b. bacilli with udn ivoUen < 
(After F1un«.) 




(sarcinfe). Those which divide in any axis form irregularly shaped, 
grape-like bunches (staphylococci). 

2. Rod Form, or BAaLLUs (Figs. 2, 3 and 4). — The type of this 
group is the cylinder. The length of the fully developed cell is always 
greater than its breadth. The size of the cells of different varieties varies 



Long slendfir bacilli. 



enormously: from a length of 30/t and a breadth of 4/i to a length 
of 0.2/1 and a breadth of O.l/i. The largest bacilli met with in dis- 
ease do not, however, usually develop over 3/i x l/i. Bacilli are roughly 
classed, according to their form, as slender when the ratio of the long 



GENERAL CHARACTERISTICS OF BACTERIA. 11 

to the transverse tliaiueter is from 1:4 to 1: 10, and as thick when 
the proportions of the long to the short diameter is approximately 1 : 2. 

The characteristic form of the bacillus has a straight axis, with 
uniform thickness throughout, and flat ends (Fig. 2 a and Fig. 5); 
but there are many exceptions to this typical form. Thus frequently 
the motile bacteria have rounded ends (Fig. 2); many of the more 
slender forms have the long axis, slightly bent; y^^ 5 

some few species, as for example the diph- 
theria bacilli (Fig. 2 b and Fig. 13), invariably 
produce many cells whose thickness is very 
unequal at different portions. Spore forma- 
tion also causes an irregularity of the cell out- 
line (Figs. 17 and 18). 

The bacilli except when they develop from 
spores or granules divide only in the plane 
perpendicular to their long axis. A classifi- 
cation, therefore, of bacilli according to their La-wet^c 
manner of grouping is much simpler than in 
the case of the cocci. We may thus have bacilli as isolated cells, as 
pairs (diplobacilli), or as longer or shorter chains (streptobaciUi). 

3. Spiral Form, on Spirillum. —The members of the third mor- 
phologic group are spiral in shape, or only segments of a spiral. 
Here, too, we have large and small, slender and thick spirals. The 
twisting of the long axis, which here hes in two planes, is the chief 
characteristic of this group of bacteria. Under normal conditions 
the twisting is uniform throughout the entire length of the cell. The 



K 1000 diamelen. 



spirilla, hke the baciUi, divide only in one direction. A single cell, 
a pair, or the union of two or more elements may thus present the 
appearance of a short segment of a spiral or a comma-shaped form, 
an S-shaped form, or a complete spiral or corkscrew-like form (Figs. 
6 and 7). 

The Highar 7onns of Bacteria (see end of Section II).— A group 
of organisms intermediate between bacteria and the moulds have been 



12 PATHOGENIC MICRO-ORGANISMS. 

called higher bacteria. They show increased complexity of structure 
and function (1) in forming irregularly segmented filaments composed of 
elements similar to those found in the tower forms and showing either 
true or false branching, (2) in developing certain portions of their 
substance into reproductive bodies from which the new individuals 
grow. 

The filaments seen sometimes among the lower forms have inde- 
pendent segments, which may easily separate and grow as tiny un- 
cellular forms, while in the higher forms, the filaments in their growth 
show a certain interdependence of their parts. For example, growth 
often occurs from only one end of the filament while the other becomes 
attached to some fixed object. 

The higher bacteria, therefore, show a close relationship to the 
fun^ which have a still more complicated development. On the other 
hand, in their formation of gonidia, or swarm spores, during repro- 
duction, they often present points of resemblance to the flagellata (see 
Protozoa). 

Stnictltre of Bacterial Cells. — When examined living in a hanging 
drop (see p. 41) under the microscope bacteria appear usually as color- 
less refractive bodies with or without spores or other more highly refrac- 
j.,g g tive areas. It is only by the use of 

stains that we are able to see more of 
their structure. 

Capsule. — Special staining methods 
(see p. 33) show that many bacteria 
(some investigators say all) under 
certain conditions, possess a capmUe 
(Fig. 8 and Fig. 18, p. 34), a gelatinous 
envelope which is supposed to be 
formed from the outer layer of the cell 
membrane. Some bacteria easily de- 
velop a much thicker capsule than 
others. Such forms are known as 
capsule bacteria. These generally 
produce a slimy growth on cultivation 
(e. g., B. mwcojua). 
Capsules develop best in animal 
tissues. In cultures, with a few exceptions, they require for their 
development special albuminous culture media, such as milk, blood 
serum, bronchial mucus, etc. In ordinary nutrient media or on pota- 
toes the capsule may be visible in the first culture generations when 
Q the body, but usually it shows very indistinctly if at all. 
le is distinguished by a diminished power of staining with 
niline dyes, therefore, unless special staining methods are 
)acteria may appear to be lying in a clear unstained area, 
in dyes the inner portion of the capsule stains, giving the 
1 apparent greater diameter. The demonstration of the 
often of help in differentiating between different but 



Pne 
From 
earbo 


-Kicluin 


s'apuWm^.^ 


ined with 


weak 


Kid aim 


3l. MeI^G^ 




(Ks>« 


End Schm 







GENERAL CHARACTERISTICS OF BACTERIA. 13 

closely related bacteria; e, g,^ some forms of streptococcus and 
pneumococcus. 

Cell Membrane. — That all bacteria possess a cell membrane is shown 
(1) by special staining methods (e. g., flagella stains, see p. 35) and (2) 
by plasmolysis, demonstrated by placing the bacteria in a 1 per cent, 
solution of sodium chloride when the central portion (entoplasm ?) con- 
tracts and separates in places from the membrane (Fig. 9). In some 
bacteria the membrane is slightly developed, while in others (e. g., B. 
tuberculosis) it is well developed. It is different in composition from 
the membrane of higher plants in not possessing cellulose. In some~ 
forms, however, a similar carbohydrate, hemicellulose, has been demon- 
strated. In certain forms a substance related to chitin, found in the 
cyst walls of protozoa (Sec. Ill), has been found. Some observers 
consider the cell membrane merely a concentrated part of the cyto- 
plasm, similar to the ectoplasm of higher cells. That it is closely 
related to the living part of the cell is shown by the connection of the 
organs of locomotion (flagella) with it. 

Fia. 9 





^ 



Plsamolysis: a, ipiriUum undula; 6. bacillus solmsii; c. vibrio cholerse. The flacelU are well 

shown. (After A. Fischer.) 

The Cell Substance. — The nature and the structure of the cell sub- 
stance contained within the membrane (body of bacteria proper, ento- 
plasm) are still under discussion. The chief questions still unsolved 
relating to it may be summarized as follows: Is the bacterial cell similar 
to the higher cells in containing a definite nucleus surrounded by cyto- 
plasm, or, if it is a simpler structure, does it behave more like a 
nucleus or more like cytoplasm ? 

In attempting a solution the following views have been expressed, chiefly 
after study of some of the larger bacteria: 

1. Bacteria have a definite morphologic, more or less centrally situated 
nucleus (Feinberg, Nakamschi, Schottelieus, Swellengrebel, and others). 

2. Bacteria have no nucleus or differentiated nuclear material (Fischer, 
Migula, Massart, and others). 

3. The whole organism, except the membrane which is a delicate layer of 
cA'toplasm, is a nucleus (Biitschli, Lowit, Boni, and others). 

' 4. The nuclear material is in the form of distributed chromatin granules 
throughout the cytoplasm (Hertwig, Schaudinn, Guilliermond, Zettnow, and 
others). 

5. A variety of the fourth view is that bacteria possess both chief elements 
of a cell, namely, cytoplasm and karyoplasm, but that these are so finely 



14 PATHOGENIC MICRO-ORGANISMS, 

mixed that they cannot be morphologically differentiated (Weigert, Mitro- 
phanow, Gotschlich). 

6. The latest view advanced, which is a variation of the views 3, 4, and 5, is 
that the bacterial cell is a relatively simple body — a cytode in HaeckePs sense, 
or the plasson of Van Beneden — which possesses both chromatin and plastin, 
the relative amounts of these chief substances of a cell corresponding more 
to the amounts found in the nuclei of higher cells than in their cytoplasm 
(Rflii6ka, Ambroi). 

These last authors call attention to the fact that both nucleus and cytoplasm 
in the higher cells are composed of a mixture of chromatin and plastin and 
that the chief difference between the two mixtures is one of amount and 
not of kind. 

From our own studies of the structure of bacteria which have cor- 
robated the views expressed in Nos. 4 and 6 of the above summary, we 
are certain that bacteria possess both chief elements of a cell, namely, 
chromatin and plastin, and that according to the stage of growth and 
division (varying with species) the chromatin may be in the form of 
morphologic granules, or may be so finely divided and mixed with 
the plastin as to be indistinguishable from it. At least some of the 
so-called metachromatic granules (Figs. 12 and 14) of many bacteria are 
undoubtedly nuclear in character. These granules appear in unstained 
bacteria as light-refracting, in stained preparations as deeply stained 
areas. They have a great affinity for dyes, and so stain readily and 
give up the stain with some difficulty. With complex stains they show 
a greater affinity than the rest of the bacillus for certain constituents 
of the stain — e. g., with polychromic methylene blue they take up 
more of the azur, thus appearing red and indicating at the same 
time Iheir nuclear nature. In certain bacteria, such as the diphtheria 
bacilli, they are especially well marked in young, vigorous cultures. 
Here they have diagnostic value. 

Besides the metachromatic granules there are certain other granules 
which take up stains readily and others still which absorb stains with 
difficulty; some of these granules are of the nature of starch and some 
of fat or other food products. Certain saprophytic forms have sul- 
phur, others iron granules. 

Organs of Motility — ^The outer surface of spherical bacteria, is al- 
most always smooth and devoid of appendages; but that of the rods 
and spirals is frequently provided with fine, hair-like appendages, or 
flagella, which are organs of motility (Figs. 10 and 11). These flagella, 
either singly or in tufts, are sometimes distributed over the entire body 
of the cell, or they may only appear at one or both ends of the rod. 
The polar flagella appear on the bacteria shortly before division. The 
flagella are believed to be formed from the outer cell layer (ectoplasm) 
or possibly from the capsule, though they have been described by 
certain authors as arising in endoplasmic granules. They probablj 
have the property of protrusion and retraction. So far as we know, the 
flagella are the only means of locomotion possessed by the bacteria. 
They are not readily stained, special staining agents bdng required for 
this purpose (see p. 35). The envelope of the bacteria, which usually 



GENERAL CHARACTERISTICS OF BACTERIA. 15 

remains unstained vrith the ordinary dyes, then becomes colored and 
more distinctly visible than is commonly the case. Occ^ionally, how- 
ever, some portion of the envelope remains unstained, when the flagella 
present the appearance of being detached from the body of the bacteria 
by a narrow zone. In stained cultures of richly flagellated bacteria 
peculiar pleated masses sometimes are observed, consisting of flagella 
which have been detached and then matted together. Bacteria may 
lose their power of producing flagella for a series of generations. 
Whether this power be permanently lost or not we do not know. 

Bacteria are named according to the number and position of the 
flagella they possess as follows: Monotricha (a single flagellum at 
one pole; e.g., cholera spirillum); Ampkitricha (a flagellum at each 
pole; e.g., many spirilla); lophotricha (a tuft of Bagella at one pole;' 
e. g., SpiriUum undvJans) ; peritricha (flagella projecting from all parts 
of surface; e. g., B. alvei, B. typhosus, and others). 



Bncilli Bhowinc one polar flnieLlum. Bacilli shaiiiDC multiple Sagells. 

So far, in only a few bacteria (the largest spirilla) have flagella been demon- 
strated during life, and then only under special conditions (see K. Reichert 
for bibliograDhy). We have, however, an organism belonging to the B. alvei 
group, which showa its flagella very distinctly during lile when a small 
portion of the viscid growth in a liquefying Loffler's blood-serum tube is 
transferred to a hanging mass of agar (p. 42) and examined under high 
magnification. The flagella on this organism may also be seen with dark- 
field illumination. In a recent article Reichert claims that all motile 
bacteria show their flagella by this method. 

Physiologic Ohu-acteristics of Bacteria.— With the study of the 
organs of locomotion we pass naturally to the consideration of the 
essential physiologic activities of bacteria, namely, motility (irritabil- 
ity), growth, reproduction, and spore formation. 

B essentially a tuft, com- 



16 PATHOGENIC MICRO-ORGANISMS. 

Motility. — Many bacteria when examined under the microscope 
are seen to exhibit active movements in fluids. The movements are 
of a varying character, being described as rotary, undulatory, sinuous, 
etc. At one time they may be slow and sluggish, at another so rapid 
that any detailed observation is impossible. Some bacteria are very 
active in their movements, different individuals progressing rapidly 
in different directions, while with many it is diflScult to say positively 
whether there is any actual motility or whether the organism shows 
only molecular movements — so-called "Brownian" movements or 
pedesis — a dancing, trembling motion possessed by all finely divided 
organic particles. In order to decide definitely with regard to the 
motility of any bacterial preparation, it is well to make two hanging 
drops. To one, five per cent, of formalin is added, which of course 
kills the organism. If, now, the live culture shows motility, which is 
not shown by the killed culture, one may be certain that one is deal- 
ing with a motile culture. Very young cultures, of but three to four 
hours' development, in neutral nutrient bouillon should be examined 
at a temperature suitable for their best growth. Not all species of 
bacteria which have flagella exhibit at all times spontaneous move- 
ments; such movements may be absent in certain culture media and at 
too low or too high temperatures, or with an insufficient or excessive 
supply of oxygen; hence one should examine cultures under various 
conditions before deciding as to the non-motility of any organism. 

The highest speed of which an organism is capable has been approxi- 
mately estimated with some forms, and the actual figures show an 
actual slow rate of movement, though, comparatively, when the size 
of the organism is considered, the movement is rapid. Thus, the 
cholera spirillum may travel for a short time at the rate of 18 centi- 
meters per hour. 

Movement is influenced by many factors, such as chemicals (the 
oxygen in the air especially), heat, light, and electricity. The tactile 
property which enables microorganisms to take cognizance of various 
forces is known as taxis; when forces attract, the phenomenon is 
known as positive taxis and when they repel it is called negative taxis. 
Chemotoxis, or the effect of chemicals, is taken up in detail on 
page 58. 

Orowth and Beproduction. — Under favorable conditions bacteria 
grow rapidly to a certain size, more or less constant for each species, 
and then divide by fission into approximated equal halves. The 
average time required for this cycle is twenty to thirty minutes. 
Probably in all species the nuclear material divides first. This is 
certainly the case in the group to which the B. dipfUherice belongs 
where division of the nuclear granules may be observed in the living 
organism before the characteristic snapping of the cell body. 

According to our observations on the living cell of members of this group, 
division takes place at a point occupied by a metachromatic granule (Fig. 12). 
Before division of the cell body the metachromatic granule, which appears 
to contain nuclear substance, elongates and shows a darker line at or near 



GENERAL CHARACTERISTICS OF BACTERIA, 17 

its center. This seems to divide and form two lines, each of which has at a 
point near the surface a very tiny, refractive granule, staining deeply with 
chromatin stains. Between these two lines the cell body suddenly divides 
with a snap, like the opening of a jackknife, division beginning at the point 
between the two tiny granules, and the two new cells remain for a variable 
time attached at opposite points, thus giving the V-shaped forms. Kurth 
and Hill also called attention to division by snapping in members of the 
diptheria bacillus group, though neither recognized the relation between the 
position of the metachromatic granules and the point of di\ision. The tiny 
granules are probably similar to the cell-partition granules described by vari- 
ous observers. 

Fio. 12 




12 3 4 

Successive stages in division of B. diphtherise showing relation of line of division to metachromatic 
granule. Continuous observation of living bacillus drawn without camera lucida. (Williams.) 

It is very seldom that the favorable conditions mentioned above for 
the production of equal and rapid division obtain for any time, since 
even in pure cultures bacteria in their growth soon produce an en- 
vironment unfavorable for further multiplication. Several factors 
help to make this environment: First, the using up of suitable food 
and moisture; second, the disintegration of food substances into vari- 
ous injurious products, such as acids, alkalies, ferments; third, in 
mixed cultures the overgrowth of one or more varieties. As these 
unfavorable conditions are more or less constantly present, we seldom 
see such absolute symmetry in the growth and divison of bacteria as is 
usually described. In fact, except under ideally favorable conditions 
(e. g., rapid successive transfers from young cultures on the most 
favorable food medium), we can never see absolutely equal fission 
among bacteria; and in some species, notably the diphtheria group, 
division is extremely irregular even in our usual twenty-four cultures 
on favorable media. 

Involntioii and Degeneration Forms. — It follows, from the conditions 
considered above, that, as cultures grow older or when media unfav- 
orable to equal division are used, the bacteria may show extremely 
irregular forms, absolutely different from the young forms, such as 
long threads or filaments with irregular thickenings, coccus forms from 
bacilli and spirilla which have divided without increasing in length, 
bacillar forms from cocci which have grown without dividing, and 
apparently branched forms from many varieties of bacilli and spirilla. 
These have been called involution or degenerative forms. 

In our study of the so-called branched forms of the diphtheria bacillus we 
have observed the following interesting fact. Under certain conditions, 
marked apparent branching appears at a definite time in the age of the culture. 
The conditions are, slightly disturbed growth in pellicle on nutrient broth. 

2 



18 



PATHOGENIC MICRO-ORGANISMS. 



When such pellicles are examiDed every day they are found to contain, from 
the sixth to the twelfth day, varying chiefly with the amount of disturbance, 
many large intensely staining fonns with one to several apparent branches 
and many large metachromatic granules (Figs. 13 and 14). The facts that 
these forms were the only ones to show active growth and divison when ex- 




amined on a hanging mass of agar and that in such growth the metachro- 
matic granules seem to fuse (Fig. 14) before fission led us to suppose that 
these forms represent a primitive sexual process, a sort of autogamy. 
Schaudinn (Fig. 15) has shown a primitive conjugation (autogamy) and a 
relatJonship between the chromatin granules, or nuclear substance, nnd the 
spores in certain bacteria. 



ST and complete division 
ity of bacteria, there are 
parated from each other 
segmentation, the cells 



GENERAL CHARACTERISTICS OF BACTERIA. 19 

remaining together in masses, as the sarcinse, for example, which divide 
more or leas regularly in three directions. The indentations upon these 
masses or cubes, which Indicate the point of incomplete fission, give to 
these bundles of cells the appearance commonly ascribed to them — 
that of a bale of rags. As already said, incomplete division in two op- 
posite directions results in the formation of a group of forms as tetrads. 
Division irregularly in different directions without subsequent separa- 
tion of the daughter cells results in the production of clusters; similar 
clusters are also formed when transversely-dividing organisms remain 
partly attached and are pushed slightly from their position. The rod- 
shaped bacteria never divide longitudinally. 

Spore tormatlos must be distinguished from vegetative reproduction. 
This is the process by which the organisms are enabled to enter a stage 
in which they resist deleterious influences to a much higher degree 




Unatfuned ■parts in alighUy duModed Uoatajntd tporea 

bkcilli. (Tbe iporcs an the li^ht oval I 

■pBca Id the heavily itained bacilli.) 

than is possible for them to do in the growing or vegetative condition.^ 
It is true that in all non-spore bearing cultures a certain proportion of 
the bacteria are more resistant than the average. No marked differ- 
ence in protoplasm, however, has been noted in them other than the 
ability to stain more intensely and sometimes to show strong meta- 
chromatic areas. The difference between these and the less resistant 
forms is not great. Some have believed that this resistance is due to 
certain bodies called arthrosporas, which are abnormally large cells with, 
usually, a thickened cell wall and increased staining properties, formed 
as a rule in old cultures. Fullerton and others have described similar 
forms in some of the higher bacteria and consider them spores. See 
nocardis (streptothrix.) The true spores of the lower bacteria are 
definite bodies. These are strongly refractile and glistening in ap- 
pearance, oval or round in shape, and composed of concentrated proto- 
plasm developed within the cell and surrounded by a very dense envel- 
ope (Figs. 16 and 17). They are characterized by their power of resist- 
ing the injurious influences of heat, desiccation, and chemical disin- 



20 PATHOGENIC MICRO-ORGANISMS. 

fectants up to a certain limit. (See p. 103 for details.) Spores also 
stain with great difficulty. 

The production of endospores in the different species of bacteria, 
though not identical in every instance, is very similar. The conditions 
under which they are produced in nature are supposed to be similar to 
those observed in artificial cultures, but they may not always be simi- 
lar, hence we must not consider a bacterium a non-spore bearer because 
it has not been seen to form spores in the laboratory. Usually the 
formation of spores in any species is best observed in a streak culture 
on nutrient agar or potato, which should be kept at the temperature 
nearest the optimum for the growth of the organism to be examined. 
At the end of twelve, eighteen, twenty-four, thirty, thirty-six hours, 
etc., specimens of the culture are observed, first unstained in a hang- 
ing drop or on an agar mass, and then, if round or oval, highly re- 
fractile bodies are seen, stained for spores. Each bacillus, as a rule, 
produces but one spore, and more than two have never been observed. 

Motile bacteria usually come to a state of rest or immobility pre- 
vious to spore formation. Several species first become elongated. 
The anthrax bacillus does this, and a description of the method of its 
production of spores may serve as an illustration of the process in 
other bacteria. In the beginning, the protoplasm of the elongated 
filaments is homogeneous, but after a time it becomes turbid and 
finely granular. These fine granules are then replaced by a smaller 
number of coarser granules, the so-called sporogenous granules, sup- 
posed to be chiefly nuclear in nature, which by coalescence finally 
amalgamate into a spherical or oval refractive body. This is the spore. 
As soon as the process is completed there may appear between each two 
spores a delicate partition wall. For a time the spores are retained in a 
linear position by the cell membranes of the bacilli, but these are 
later dissolved or broken up and the spores are set free. Not all the 
cells that make the effort to form spores, as shown by the spherical 
bodies contained in them, bring these to maturity; indeed, many 
varieties, under certain cultural conditions, lose altogether their prop- 
erty of forming spores. The following are the most important spore 
types: (a) the spore lying in the interior of single, short, undistended 
cells; (6) the spores lying in the interior of a chain of undistended cells; 
(c) the spore lying at the extremity of a cell much enlarged at that 
end — the so-called "head spore" or plectridium, e, g,, the tetanus 
bacillus (Fig. 17); and (d) the spore lying in the interior of a cell very 
much distended in its central portion, giving it a spindle shape or Clos- 
tridium, e.g., Bacilhis btUyricus. 

According to Schaudinn and others, in certain spore bearing bacteria the 
spore formation is part of a sexual-like process (see under Reproduction). 

The germination of spores takes place as follows: By the absorp- 
tion of water they become swollen and pale in color, losing their 
shining, refractive appearance. Later, a little protuberance is seen 
upon one side (equatorial germination) or at one extremity of the 



GENERAL CHARACTERISTICS OF BACTERIA. 21 

spore (polar germination) and this rapidly grows out to form a rod 
which consists of soft-growing protoplasm enveloped in a membrane, 
which is formed of the endosporium or inner layer of the cellular 
envelope of the spore. The outer envelope, or exosporium, is cast off 
and may be seen in the vicinity of the newly formed rod. 

The chief spore formers among the pathogenic bacteria are the an- 
aerobes (tetanus, malignant, oedema, intestinal bacteria). Only one 
distinctly pathogenic aerobe produces spores — the anthrax bacillus. 

Reprodnctioii Among the Higher Bacteria. — These forms increase in 
length for a time and then, at the free ends, or at intervals"along the 
filaments, they produce small rounded cells, called gonidia or spores 
from which new individuals are formed. The terminal spores may be 
flagellated after their separation from the parent filament. 

The flagellated forms frequently resemble certain flagellata among 
the protozoa. 

OHEMIOAL COMPOSITION OF BACTERIA. 

Qualitatively considered, the bodies of bacteria consist largely of 
water, salts (chiefly phosphorus, potassium, chlorine, calcium and 
sulphur), fats, and albuminous substances. There are also present, in 
smaller quantities, extractive substances soluble in alcohol and in 
ether. Special varieties contain unusual substances, as wax and 
hemicellulose in tubercle bacilli. Bacteria possess the capacity in a 
high degree of accommodating their chemical composition to the 
variety of soil in which they are growing. The same variety of 
bacteria thus varies greatly in the quantitative estimation of its chem- 
ical constituents. Each variety, furthermore, yields proteid sub- 
stances peculiar to itself, as shown in the effects produced by animal 
inoculation. At present we know but little concerning the differentia- 
tion of these specific substances. This subject will be taken up in 
detail under bacterial toxins, etc. According to Cramer, many bacteria 
contain amyloid substances which give a blue reaction with iodine. 
True cellulose has not been found in bacteria, but large quantities of a 
gelatinous carbohydrate similar to hemicellulose have been obtained. 
Nuclein is found frequently. The nuclein bases — xanthin, guanin, 
and adenin — have been obtained in considerable amounts. There 
is a group of bacteria which contain large amounts of sulphur — viz., 
the Begglaioa — and another group, the Cladothru:, is capable of 
separating ferric oxide from water containing iron. 

Some light has been thrown upon the chemical composition of 
bacteria, quantitatively, by the studies of Cramer, though so far 
only a few species have been thoroughly investigated. The per- 
centage of water contained in bacteria grown on solid culture media, 
as well as the amount of residue and ash, depends largely on the 
composition of the media. Thus, Bacillus prodigiosus when grown 
on potato contains 21.5 per cent, of dry residue and 2.7 percent, 
of ash; when cultivated on turnips it contains 12.6 per cent, of dry 



22 PATHOGENIC MICRO-ORGANISMS. 

residue and 1 . 3 per cent, of ash. Besides the concentration of the 
culture, its temperature and age also influence the amount of residue 
and ash produced. The residue varies, moreover, qualitatively in 
the same species under the influence of the culture media employed. 
Thus, it appears that an additional quantity of peptone in the culture 
media tends to increase the percentage of nitrogenous matter in the 
bacillus, while the addition of glucose decreases it. 

Microchemical Reactions. — ^To a certain degree the chemical com- 
position of the individual bacterium may be studied both in the living 
and in the dead organism by the addition of the testing substances 
to a hanging drop or to a spread of such organism and the examina- 
tion of it under the microscope. 

Of special importance in this regard is the resistance which bac- 
teria possess to diluted alkaUes. Inasmuch as the majority of animal 
tissues are dissolved when treated with alkahes, this method has been 
adopted for rendering visible unstained bacteria in tissues. As a rule, 
bacteria are stained yellowish with iodine solution, a few only in con- 
sequence of their starchy constituents being stained blue. (See also 
Principles of Staining Bacteria, p. 30.) 

CLASSIFICATION OF BACTERIA. 

The position of the bacteria at the lower end of plant Ufe and their 
relationship to the next higher plants may be seen in the following 
table: 

Thallophyta (lower plants with no distinction between root and stem and leaf). 



Forms with chlorophyll Fonns without chlorophylL 

(algae, etc.). I 



Multicellular; spores in differentiated Unicellular; siK>res freauently absent, 

spore-bearing organs. (The true spore-bearing cells little or not 

fungi, or moulds.) at all differentiated. 



[ 



The bacteria The yeasts 

(schisomycetes). (blastomyoetes). 

Bacteria themselves have been classified in many different ways 
by different observers. As a rule, the genera are based upon morpho- 
logic characters and the species upon biochemic, physiologic, or patho- 
genic properties. While the form, size, and method of division are the 
most permanent characteristics of bacteria, and should be naturally 
utilized for classification, nevertheless, in this basis of division, because 
of the minute size of the organisms and of our consequent inability to 
detect important morphologic differences, there are decided difficulties. 
Then, too, though the form and size of the different varieties are fairly 
constant under the same conditions, under diverse conditions as we 
have already noticed, they may be quite different. Another serious 



CLASSIFICATION OF BACTERIA. 23 

drawback for our purposes is that these morphologic characteristics 
give no indication whatever of the relations of the bacteria to disease 
and fermentation — the chief characteristics which give them their im- 
portance to human beings. The properties of bacteria which are 
fairiy constant under uniform conditions and which have been more or 
less used in systems of classification are those of spore and capsule 
formation, motility (flagella formation), reaction to staining reagents, 
relation to temperature, to oxygen, and to other food material, and, 
finally, their relation to fermentation and disease. 

But any one of these properties under certain conditions may so 
vary that, taking it as a basis for classification, an organism could 
be dropped from the group with which it had been classified and be 
placed in an entirely different group. 

Thus, the power to produce spores or flagella may be held in abey- 
ance for a time or, in the case of the former, be totally lost; the rela- 
tions to oxygen may be gradually altered, so that an anaerobic species 
grows in the presence of oxygen; parasitic bacteria may be so cultivated 
as to become saprophytic varieties, and those which have no power 
to grow in the living body may acquire pathogenic properties. 

The possibiUty of making any thoroughly satisfactory classification 
is rendered still more difficult by the fact that many necessarily im- 
perfect attempts have already been made, so that there is a great deal 
of confusion, which is steadily increased as new varieties are found 
or old ones reinvestigated and classified differently in the different 
systems. 

As one of the .more successful attempts to classify bacteria, the 
system devised by Migula is here given, simply as an example. The 
morphology of bacteria is used as the basis of the division : 

FAMILIES. 

I. Cells globose in a free state^ not elongating in 

any direction before division into 1 , 2, or 3 planes . . 1 . Coccacese. 
II. Cells cylindrical, longer or shorter, and only dividing 
in one plane, and elongating* to about twice the 
normal length before the division. 
a. Cells straight^ rod-shaped, without sheath, non- 
motile, or motile by means of fiagella 2. Bacteriacese. 

6. Cells curved, without sheath 3. SpirillacesB. 

c. Cells enclosed in a sheath 4. Cnlamydobacter- 

iacesB. 

QENERA. 

1. Coccacece. 
Cells without organs of motion. 

a. Division in one plane 1. Streptococcus. 

b. Division in two planes 2. Micrococcus. 

e. Division in three planes 3. Sarcina. 

Cells with organs of motion. 

a. Division in two planes 4. Planococcus. 

b. Division in three planes 5. Planosarcina. 

2. BacteriacecB, 

Cells without organs of motion 1. Bacterium. 

Cells with organs of motion (flagella). 

a. Flagella distributed over the whole body 2. Bacillus. 

b. Flagella polar 3. Pseudomonas. 



24 PATHOGENIC MICRO-ORGANISMS. 

3. Spirillacece . 
Cells rigid, not snake-like or flexuous. 

a. Cells without organs of motion 1. Spirosoma. 

6. Cells with organs of motion (flagella). 

1. Cells with 1, very rarely 2 to 3 polar flagella . . 2. Microspira. 

2. Cells with polar flagella-tufts 3. Spirillum. 

Cells flexuous 4. Spirochseta. 

4. Chlamydobaclitiacece (higher bacteria, also known as Trichomycetes). 
Cell contents without granules of sulphur. 
a. Cell threads unbranched. 

I. Cell division always only in one plane 1. Streptothrix. 

II. Cell divsion in three planes previous to the formation 
of gonidia. 

1 . Cells surrounded by a very delicate, scarcely visible 

sheath (marine) 2. Phragmidiothrix. 

2. Sheath clearly visible (in fresh water) 3. Crenothrix. 

6. Cell threads branched 4. Cladothrix. 

Cell contents containing sulphur granules 5. Thiothrix. 

The above table makes changes in the designation of some of the 
most common bacteria, as in the restoration of the old title bacterium 
and the assigning it to all of the non-motile, rod-shaped organisms, 
thus altering the name of some of the most common pathogenic bacteria 
from bacillus to bacterium. Other changes are seen in the spirilla, 
and the classification of the higher bacteria is quite different from that 
now accepted (see end of Sec. II). Any such scheme is at times arbi- 
trary in placing some varieties under one generic division and others 
closely allied in another. It has also the objection, already noted, that 
it is only one of several classifications already in use, and until an au- 
thoritative body agrees on some one, it seems unwise in such a volume 
as this to change the usually employed names for others which are, per- 
haps, intrinsically better. Another important reason for waiting is 
that with the increase of our knowledge we are constantly changing the 
position of different bacteria. Thus, such a well-known germ as the 
tubercle bacillus is now found to produce, under certain conditions, 
long, thread-like branching forms; so that it ceases to be under the 
classification of Migula, either a bacillus or bacterium. We shall, there- 
fore, simply use in this book the older, less scientific nomenclature, of 
classing all rod forms as bacilli and all spiral forms as spirilla, and con- 
sider together, in so far as is practicable, certain groups of bacteria 
whose members are closely allied to each other in some one or more 
important directions. 

It is well to call attention, however, to the fact that in naming bacte- 
rial species the binomial law of nomenclature has been frequently 
violated. Such names as Bacillxis coli communis should not be ac- 
cepted; the name Bacillus coli is suflBcient as well as correct. 

Permanence of Bacterial Species. — When we come to study special 

varieties or groups of bacteria, such as the bacilli which produce typhoid 
fever, diptheria, and tuberculosis, it is of great importance for us to 
determine, if possible, to what extent the peculiar characteristics which 
each of these groups of bacteria possess are permanent in the gener- 
ations which develop from them. 



CLASSIFICATION OF BACTERIA. 25 

We cannot believe that the multitude of bacterial varieties which 
now exist have always existed. The probability is very strong that 
with succeeding generations and changing conditions new bacterial 
varieties have developed with new characteristics. 

From time to time the changing conditions under which life pro- 
gresses probably expose certain animals to the invasion of varieties 
which never before have gained access to them. If the bacteria find 
some means of transmission to other animals equally susceptible, a 
parasitic species becomes established which at first, perhaps, finds 
conditions only occasionally favorable to it. Thus in some such way 
a multitude of bacterial groups have arisen, some of which accustom 
themselves to the conditions present in living plants, others to those 
in fishes, others to those in birds, and others still to those in man and 
the higher animals. 

These are, however, theories. What has been actually observed in 
the few years during which bacteria have been studied? In this 
short time the pathogenic species as observed in disease have remained 
practically unaltered. The diptheria bacilli are the same to-day 
as when Loeffler discovered them in 1884, and the disease itself is 
evidently the same as history shows it to have been before the time of 
Christ. The same permanence of disease type is true for tuberculosis, 
smallpox, hydrophobia, leprosy, etc. Under practically unchanged 
conditions, therefore, such as exist in the bodies of men, bacteria 
which have once become established as parasites, continue to re- 
produce new generations which retain their peculiar (specific) charac- 
teristics. It is true that among the countless organisms developed 
some fail to hold the parasitic characteristics. These either continue 
as saprophytes or cease to exist. Whether new disease varieties are 
coming into existence from time to time is, of course, a possibility, but 
not a certainty. The one thing we can probably safely assert is that 
it is very unlikely that any saprophji;ic variety now existing can de- 
velop into the now recognized varieties of pathogenic bacteria. It is 
difficult to conceive that any such variety should develop parasitic ten- 
dencies under exactly the same circumstances as those varieties which 
now produce disease. 

The fact that the chief pathogenic varieties of bacteria which 
excite disease in man seem to have retained for centuries their charac- 
teristics, in no way proves that when placed under different conditions 
they would remain stable. As already stated, certain characteristics 
of some bacteria can be radically altered by changed conditions, such 
as being grown outside the natural host, either in the test-tube or in an 
unaccustomed host. When these new surroundings are unfavorable, 
the organisms, while retaining their morphology, may lose their power 
of developing and producing specific poisons in the original host. Such 
attertuation may also occur in certain organisms when retained for a 
long time in an apparently immune host, as is seen in the streptococci 
and pneumococci of the throat or in the colon bacilli of the intestines. 

The recovery of poison production is often brought about by developing 



26 PATHOGENIC MICRO-ORGANISMS. 

the microorganism for a considerable length of time under the condi- 
tions best suited for it. The recovery of the ability to grow in the body 
of any animal species is brought about by causing the germ to develop 
in a series of animals of the same species whose resistance has been 
overcome by reducing their vitality through poisons, heat, cold, etc., or 
by giving enormous doses of bacteria to produce the first infection. 
Mother method is to accustom the microorganism to the animal's body 
by letting it remain surrounded by the animal fluids but protected from 
phagocytes in a pervious capsule in the peritoneal cavity or by growing 
it in unheated fresh serum or blood media. 

The above examples of variations may be classed under those known 
as fluctuating variations. . True mutations or discontinuous variations 
among bacteria have been very seldom observed. 

Bibliography. 

Ambroi. Entwickelungszyklus des B. nitri n. sp., etc. Centralbl. f. Bakt., 
etc., I. Abt., orie., 1909, 51, 193 (with bibliography on structure and develop- 
ment of bacteria). 

Meyer. Flora, 1908, 95. 

Migula. System der Bakterien, Jena, 1897. 

Schatulinn. Beitrftge zur Kenntnis der Bakterien, etc. Arch. f. Protistenk, 
1902, I, 306, and 1903, II, 416. 

RUiicka. Cytologic der sporenbildenden Bakterien, etc. Centralbl. f. Bakt., 
II. Abt., 1909, 27. 

ZeUnow. Romanowski's F&rbung bei Bakterien. Zeitschr. f. Hyg., etc., 1899, 
XXX, 1, and Centralbl. f. Bakt., 1900, Abt. I, xxvii, 803. 



CHAPTER III. 

MICROSCOPIC METHODS. 

DRT#AND MOIST PREPARATIONS, STAINS, AND BUOROSOOPIO 

EXAMINATION OF BACTERIA. 

The direct microscopic examination of suspected substances for 
bacteria can be made either with or without staining. Unstained, 
the bacteria are examined living in a hanging drop or on transparent 
solid media, under daylight, or, better, artificial light, to note their num- 
ber, their motility, their size, form, and spore formation, their general 
arrangement and their reactions to specific serums; but for more exact 
study of their structure they can be so much better observed when 
stained in a dried film preparation on a glass slide or a cover-gla3s that 
this step is always advisable. 

Elimination of Foreign Bacteria from Preparations. — Since bac- 
teria are present in the air, in dust, in tap water, on our bodies, clothes, 
and on all surrounding objects, it follows that when we begin to examine 
substances for bacteria the first requisite is, that the materials we use, 
such as staining fluids, cover-glasses, etc., should be practically free 
from bacteria, both living and dead, otherwise we may not be able to 
tell whether those we detect belong originally in the substances examined 
or only in the materials we have used in the investigation. 

Film Preparation (spread, smear). — A cover-glass or slide prepara- 
tion is made as follows: A very small amount of the blood, pus, dis- 
charges from mucous membranes, cultures from fluid media, or other 
material to be examined is removed, usually by means of a sterile swab 
or platinum loop, and smeared undiluted in an even, thin film over a 
perfectly clean,* thin cover-glass or slide. From cultures on solid media, 
however, on account of the abundance of bacteria in the material, a little 

* To render new cover-slips clean and free from grease, the method recom- 
mended by Gage is useful: Place in following solution overnight. 

BichromAte of potaah (KiCrtO?) 200 grms. 

Water, tap or diatilled 800 c.c. 

Sulphuric acid 1200 c.c. 

The bichromate is dissolved in the water by heating in agate kettle; the sul- 
phurous acid is added very slowly and carefully on account of great heat devel- 
oped. After cooling, it is kept in glass vessel. It may be used more than once. 

Glasses are removed the next morning and cleansed m runniils tap water until 
the yellow color disappears. They are then placed in ammonia alcohol until used. 
When used wipe with soft, clean linen or cotton cloth. If old cover-slips are used, 
boil first in 5 per cent, sodium carbonate solution. 

Another procedure is, after washing with soap and water and rinsing in water. 
to soak the cover-glasses in alcohol, then wipe with soft linen, then place in a 
Petri dish, and heat in the dry sterilizer for one hour at 200° C. to burn off fatty 
substances. The heating mav be done by holding the cover-glass in the flame 
sufficiently to heat thoroughly without softening. A cover-glass is not clean 
when a drop of water spread over it does not remain evenly distributed, but 
gathers in droplets. 

27 



28 PATHOGENIC MICRO-ORGANISMS. 

of the growth is diluted by adding it to a tiny drop of filtered or distilled 
water, free from all suspended matter, which has been previously 
placed on the glass. The amount of dilution is learned after a few 
trials. It is best to add to the drop just enough of the culture to make 
a perceptible cloudiness. The mixture is then smeared thinly and 
uniformly over the glass. When blood or pus is to be studied it is well 
to put a small drop on a slide or cover-glass and then inmiediately to 
place on top of this another slide or cover-glass. The fluid will spread 
between the two, and when they are drawn apart a fairly thin, even 
smear will be left on each of them. If it is desired to preserve the 
blood cells intact the films are placed in a saturated solution of corrosive 
sublimate for two or three minutes and then washed in running water, 
or they may be exposed to the vapor of formalin, or be placed in methyl 
alcohol or absolute ethyl alcohol for a few seconds before staining. 

Milk films, after fixation, are cleared of fat by means of ether or 
alkaline solutions.* From whatever source derived the film is allowed 
to dry thoroughly at the usual air temperature, and then, in order to 
fix the film with its contained bacteria to the glass, the latter is grasped 
in any one of the several kinds of forceps commonly used, and is 
passed three times by a rather slow movement through the Bunsen 
or alcohol flame. Instead of this method the film may be fixed to the 
glass before becoming completely dried by placing it in any one of the 
already named fixatives for a few minutes. The smear thus prepared 
is usually stained either by the simple addition of a solution of an aniline 
dye, for from a few seconds to five minutes, or bj one of the more com- 
plicated special stains described later. When the stain is to be hastened 
or made more intense the dye is used warm. For ordinary staining, 
the bacteria are simply covered completely by the cold staining fluid, 
which is left the requisite length of time. 

The cover-glass or slide, with the charged side uppermost, may 
either rest on the table or be held by some modification of Comet's 
forceps. When the solution is to be warmed the cover-glass may be 
floated, smeared side down, upon the fluid contained in a porcelain 
dish resting on a wire mat, supported on a stand, or the solution may 
be poured on the glass which may then be held over the flame in the 
Cornet forceps. If a slide is used it is simply inserted in the fluid or 
covered by it. The fluid both in the dish and on the glass should be 
carefully warmed so as to steam without actually boiling. The glass 
should be kept completely covered with fluid. 

The bacteria having now been stained, the cover-glass or slide 
is grasped in the forceps and thoroughly but gently washed in clean 
water and then dried, first between layers of filter-paper and then in 
the air or high over a flame. A drop of balsam or water is then placed 
on a glass slide and the cover-glass put upon in with the bacterial side 
down. The cover-glass or slide preparation is now ready for micro- 
scopic examination after the addition of a drop of oil. 

Stains Used for Bacteria. — The protoplasm of mature bacteria 

* One-half to one per cent, sodium hydrate. 



MICROSCOPIC METHODS. 29 

reacts to stains much as nuclear chromatin, though sometimes more 
and sometimes less actively. 

Though bacteria may be stained with various dyes of very different 
chemical composition, such as heematoxyhn and certain plant dyes, 
the best stains are the basic aniline dyes, whrch are compounds 
derived from the coal-tar product aniline (C,H5NH2).* R. Koch was 
the first to recognize the affinity of bacteria for these dyes and to 
note their importance as a means of differentiating microorganisms 
from other corpuscular elements. 

Aniline Dyes. — The aniline dyes which are employed for staining pur- 
poses are divided into two groups according as the staining action 
depends on the basic or the acid portion of the molecule. The for- 
mer contain amido groups and are spoken of as nuclear stains, since 
they color the nuclear chromatin of both cells and bacteria. • The 
latter contain hydroxyl gi^oups and stain bacteria faintly; they are 
used chiefly for contrast coloring. The basic dyes are usually em- 
ployed as salts of hydrochloric acid, while the acid dyes occur as sodium 
or potassium salts. 

The following are the most commonly used basic aniline stains: 
Violet stains — methyl violet, gentian violet, crystal violet. 
Blue stains — methylene blue, thionin blue. 
Red stains — basic fuchsin, safranin. 
Brown stain — Bismarck brown. 
Green stain— methyl green. 

Of the above stains the violet and red stains are the most intense 
in action. It is correspondingly easy to overstain a specimen with them. 
Of the blue, methylene blue probably gives the best differentiation of 
structure and it is difficult to overstain with it. 

These dyes are all more or less crystalline powders, and while 
some are definite chemical compounds, others are mixtures. For 
this reason various brands are met with on the market and the exact 
duplication of stains is not always possible. Dyes should be ob- 
tained from reliable houses only; most bacteriologists obtain them 
from Griibler, of Leipzig. 

It is advisable to keep on hand not only the important dyes, but 
also stock solutions from which the staining solutions are made. The 
stock saturated alcoholic solutions are made by pouring into a bottle 
enough of the dye in substance to fill it to about one-quarter of its 
capacity. The bottle should then be filled with alcohol, tightly corked, 
well shaken, and allowed to stand twenty-four hours. If at the end of 
this time all the staining material has been dissolved, more should be 
added, the bottle being again shaken and allowed to stand for another 
twenty-four hours. This must be repeated until a permanent sediment 
of undissolved coloring matter is seen upon the bottom of the bottle. 
This bottle will then be labeled "saturated alcoholic solution," of what- 
ever dye has been employed. The alcoholic solutions are not themselves 

* For a good description of the composition and action of the various stains 
see A. B. Lee's ** Microtomist's Vade-Mecum," 6th edition, 1905. 



30 PATHOGENIC MICRO-ORGANISMS, 

employed for staining purposes. The solution for use is made by filling 
a small bottle three-fourths with distilled water, and then adding the 
concentrated alcoholic solution of the dye, little by little, until one can 
just see through the solution. It is sometimes desirable to use a more 
concentrated solution with dyes such as methylene blue. Care must 
be taken that the color does not become too dense; usually about one 
part to ten is sufficient. Small wooden cases come prepared for holding 
about one-half dozen bottles of the staining solutions. This number 
will answer for all practical purposes. 
Oeneral Observations on the Principles of Staining Bacteria. — 

The staining of bacteria is not to be considered simply as a mechan- 
ical saturation of the cell body with the dye, in which the latter is 
dissolved in the plasma. It is rather a chemical combination between 
the dye substance and the plasma. This union, however, is apparently 
an unstable one and easily broken up. Unna believes that the basic 
aniline dyes, from their chemical composition, are not really bases, but 
neutral salts — e. g,, fuchsin equals rosaniline chloride; they are called 
basic only because the staining components (as the rosaniline) are of a 
basic nature. The staining process is, therefore, not to be looked upon 
as if the dye substance separated into its component parts and only the 
staining ingredient attacked the cell body, because the tissues for which 
these "basic aniline dyes" have special affinity are themselves basic. 
On the contrary, the dyestuff unites as a whole with the plasma, forming, 
as it were, a double salt or unstable compound between the two. 

The dependence of the staining process upon the solvent condition 
of the dye is shown in the following observations : 

1. Entirely water-free, pure alcoholic dye solutions do not stain. 

2. Absolute alcohol does not decolorize bacteria, while diluted 
alcohol is an active decolorizing agent. The compound of dye sub- 
stance and plasma is therefore insoluble in pure alcohol. 

3. The more completely a dye is dissolved the weaker is its stain- 
ing power. For this reason pure alcoholic solutions are inactive; 
and the so-called weak dye solutions to which strong dye solvents 
have been added are limited in their action on certain bacteria in 
which the dye substance is closely united. This is the principle of 
Neisser's stain for diphtheria bacilli — viz., acetic acid methylene- 
blue solution. 

On the other hand, the addition of alkalies to the dye mixture ren- 
ders the solvent action less complete and the staining power more 
intense. According to Michaels, however, in Loeffler's methylene- 
blue solution the r6le of the alkali is purely of a chemical nature, 
by which it converts the methylene blue into methylene azure 
(azure II). 

The dependence of the staining process upon the nature of the 
bacteria is exhibited in the following facts: 

Certain bacteria stain easily, others with difficulty. To the latter 
belong, for example, the tubercle bacillus and lepra bacillus. Spores 
and flagella also stain with difficulty. The easily stained objects re- 



MICROSCOPIC METHODS. 31 

quire but a minimum of time to be immersed in a watery solution, 
while the others must be stained by special dyes with or without the aid 
of outside influences (heat, mordants, etc.). The difficultly stained ob- 
jects are at the same time not easily decolorized. The explanation 
of the resistance which these bacteria show to staining as well as to 
decolorizing agents is to be sought in two ways : either on the assump- 
tion that they possess a difficultly permeable or a resisting envelope, or 
that they have a special chemical constitution. The latter hypothesis 
holds good only, if at all, in regard to flagella and spores; while the 
assumption of the resisting envelope has reference more particulariy 
to the tubercle bacillus, and is probably correct. The presence of 
fatty and waxy bodies in the envelope of these microorganisms is 
capable of demonstration. Moreover, after extraction of these bodies 
by ether the tubercle bacillus loses its power of resisting acids, which 
peculiar resistance can also be artificially produced in other bacteria 
having normally no such resisting power. In many instances, doubt- 
less, both of these causes, viz., resistant envelope and chemically dif- 
ferent constitution, work together to produce the above-mentioned 
results. 

Individual differences in acid resistance among the difficultly 
stained bacteria have been observed in tubercle bacilli; according to 
Ziehl and Ehrlich, those having less individual r^istance are prob- 
ably the younger members. Individual differences in staining, in 
the easily stained bacteria, have also been noticed; for example, 
cholera vibrios and allied species are best stained with fuchsin, not 
so well with methylene blue, etc. 

The relation between staining and degeneration of bacteria is a 
complicated question. Decrease of staining power takes place dur- 
ing degeneration of the bacterial cell, but it is often difficult to deter- 
mine the exact moment when this loss of power occurs. Degener- 
ated forms of the cholera bacillus from the abdominal cavity of 
guinea-pigs thus soon lose their power of staining in methylene-blue 
solution, but stain well in diluted carbol fuchsin. Moreover, bac- 
teria killed by drying and moderate heating, as in the preparation 
of films, retain their power of staining. Kitasato found dead tuber- 
cle bacilli in sputum which took on normal staining. Bacteria killed 
by chloroform, formalin, etc., still retain their staining properties intact. 

Elective staining properties, whereby certain species of bacteria 
are exclusively or rapidly and intensely stained by certain dyes, have 
repeatedly been observed. Of the greatest practical importance in this 
respect is the Gram stain (see p. 33, and Chap. XVI), used for the 
differential diagnosis of many species of bacteria; although a distinct 
classification of bacteria into those which are stained and those which 
are not stained by Gram's solution has been shown to be impractic- 
able. There are some bacteria, however, which act uniformly toward 
Gram under all conditions; as, for example, the anthrax bacillus and 
the pyogenic cocci are always positive, the cholera and plague bacilli 
and gonococci are always negative to Gram. Other species again are 



32 PATHOGENIC MICRO-ORGANISMS. 

at one time stained and at another decolorized by Gram; thus 
pyocyaneus is stained only in young individuals. Previous heating or 
extraction with ether does not prevent the action of Gram's stain, 
but treatment with acids or alkalies renders it impossible. Bacteria 
so treated, however, after one hour's immersion in LoeflHer's mordant 
regain their property of staining with Gram. 

As to the nature of Gram's staining solution, it may be mentioned 
that only the pararosanilines (gentian violet, methyl violet, and 
Victoria blue) are suitable for the purpose, whereas the rosanilines 
(fuchsin and methylene blue) give negative results. The reason for 
this is that the iodine compounds with the pararosanilines are fast 
colors, while those with the rosanilines are unstable. These latter 
compounds when treated with alcohol break up into their constitu- 
ents, the iodine is washed out, and the dye substance remaining in 
the tissues stain them uniformly; that is, without differentiation. 
But iodine-pararosaniline compounds are not thus broken up and 
consequently stain those portions of the tissue more or less, accord- 
ing to the aflSnity which they have for the dye substance. The parts 
stained by Gram are thus distinguished from those stained violet, 
not only quantitatively, but qualitatively; it is not a gentian violet, 
but an iodine-pararosaniline staining which occurs. 

Use of Mordants and Decploimng Agents. — We have already 
noted that the protoplasm of unrelated bacteria may respond differently 
to the several dyes. There is, however, seldom any difficulty in 
selecting a dye which will stain sufficiently to make bacterial cells 
in pure cultures distinctly visible. When the bacteria are imbedded 
in tissue or mixed in a film with blood or pus, it is frequently 
difficult to prevent the stain from so acting on the tissue or pus ele- 
ments as to obscure the bacteria. Various methods are then em- 
ployed to stain the bacteria more intensely than the tissues or to 
decolorize the tissue more than the bacteria. Heating, the addition 
of alkali to the staining fluid and prolonging the action of the dyes 
increase the staining of the bacteria. We regulate these so as to 
give the best results. We also use mordants; that is, substances 
which fix the dye to the bacterial cell, such as aniline oil or solutions 
of carbolic acid and metallic salts. As decolorizing agents we use 
chiefly mineral acids, vegetable acids, diluted alcohols, and various oils. 

Formulae of the Most Commonly Used Stain Combinations. — 
Loeffler's Alkaline Methylene-blue Solution. — This consists of 
concentrated alcoholic solution of methylene blue, 30 c.c; caustic 
potash in a 0.01 per cent, solution, 100 c.c. The alkali not only 
makes the cell more permeable, but also increases the staining power 
by liberating the free base from the dye. 

Koch-Ehrlich aniline-water solution of fuchsin or gen- 
tian VIOLET is prepared as follows: To 98 c.c. of distilled water 
add 2 c.c. aniline oil, or, more roughly but with equally good results, 
pour a few cubic centimeters of saturated aniline oil into a test-tube, 
then add sufficient water nearly to fill it. In either case the mix- 



MICROSCOPIC METHODS. 33 

tures are thoroughly shaken and then filtered into a beaker through 
moistened filter-paper until the filtrate is perfectly clear. To 75 
c.c. of the filtrate (analine oil water) add 26 c.c. of the concentrated 
alcoholic solution of either fuchsin, methylene blue, or gentian violet, 
or add the alcoholic solution until the aniline water becomes opaque 
and a film begins to form on the surface. 

Carbouc-Fuchsin, or Ziehl-Nielsen Solution — Distilled water, 
100 c.c; carbolic acid (crystalline), 5 gm.; alcohol, 10 c.c; fuchsin, 1 
gm.; or it may be prepared by adding to a 5 per cent, watery solu- 
tion of carbolic acid the saturated alcoholic solution of fuchsin until 
a metallic lustre appears on the surface of the fluid. The carbolic 
acid, like the alkali, favors the penetration of the stain. 

The last two methods, combined with heating, are used to stain 
spores and certain resistant bacteria as the tubercle bacilli and other 
'* acid resisters," so that they retain their color when exposed to decolor- 
izing agents. 

Carbolic-methylene blue, first used by Kuhne, consists of 1.5 gm. 
of methylene blue, 10 gm. of absolute alcohol, and 100 c.c of a 5 
j>er cent, solution of carbolic acid. Carbolic-ihionin consists of 10 
parts of a saturated alcoholic solution of thionin and 100 parts of a 
1 per cent, solution of carbolic acid. 

Gram's Stain. — Another differential method of staining which is 
employed is that known as Gram's method. In this method the 
objects to be stained are floated on or covered with the aniline or 
carbolic gentian-violet solution described above. After remaining 
in this for a few minutes they are rinsed in water and then immersed 
in an iodine solution (Lugol's), composed of iodine, 1 gm.; potas- 
sium iodide, 2 gm.; distilled water 300 c.c In this they remain 
for from one to three minutes and are again rinsed in water. They 
are then placed in strong alcohol until most of the dye has been 
washed out. If the cover-glass as a whole still shows a violet color, it 
is again treated with the iodine solution, followed by alcohol, and 
this is continued until no trace of violet cplor is visible to the naked 
eye. It may then be washed in water and examined, or before ex- 
amination it may be counter-stained for a few minutes by a weak 
solution of a contrasting dye, such as eosin, fuchsin, carmine, or Bis- 
marck brown. This method is useful in demonstrating the capsule 
which is seen to surround some bacteria — particularly the pneumo- 
eoecus — and also in differentiating between varieties of bacteria, for 
some do and others do not retain their stain when put in the iodine 
solution for a suitable time (see Chap. XVI, for further remarks upon 
Gram's stain). 

Staining of Capsules. — Many methods of demonstrating the cap- 
sule have been devised. Two only will be given here. 

Welch's glacial acetic acid method is as follows: 1. Cover the prepa- 
ration with glacial acetic acid for a few seconds. 2. Drain off and 
replace with aniline gentian-violet solution; this is to be repeatedly 
added until all the acid is replaced. 3. Wash in 1 to 2 per cent. 
3 



34 



PATHOGENIC MICRO-ORGANISMS. 



Fig 18 




solution of sodium chloride and mount in the same. Do not use 
water at any stage. The capsule stains a pale violet. 

Hiss' Copper Sulphate Method (Fig. 18). — The organisms are 
grown, if possible, on ascitic fluid or serum media. If not, the organ- 
isms should be spread on the cover-glass mixed with a drop of serum, 
or, better, with a drop of one of the diluted serum media. Dry in 
the air and fix by lieat. 

The capsules are stained as follows : A 5 per cent, or 10 per cent, 
aqueous solution of gentian violet or fuchsin (5 c.c. saturated alco- 
holic solution gentian violet to 95 
c.c. distilled water) is used. This 
is placed on the dried and fixed 
cover-glass preparation and gently 
heated for a few seconds until steam 
arises. The dye is washed off with 
a 20 per cent, solution of copper 
sulphate (crystals). The prepara- 
tion is then placed between filter- 
paper and thoroughly dried. 

Staining Spores and Acid-fast 
Bacteria.^ — We have alreadv noted 
that during certain stages in the 
growth of a number of bacteria 
spores are formed which refuse to 
take up color when the bacteria are 
stained in the ordinary manner. 
Special methods have been devised for causing the color to 
penetrate through the resistant spore membrane. In the simplest 
method a coverslip after having been prepared in the usual way is 
covered with Ziehl's carbolic fuchsin solution and held over the 
Bunsen flame until the fluid steams. This is continued for one or two 
minutes. It is then washed and dipped in a decolorizing acid solu- 
tion, such as a 2 per cent, alcoholic solution of nitric acid, or a 1 
per cent, solution of sulphuric acid in water, until all visible color has 
disappeared, then it is washed and dipped for one-half minute in a 
saturated watery solution of methylene blue. The bodies of the 
bacilli are blue and the spores red. This same method is used for 
staining acid-fast bacilli. Sometimes the spores refuse to take the 
stain in this manner. We then can adopt Moeller's methody which is 
designed still further to favor the penetration of the coloring matter 
through the spore membrane. The prepared cover-slip is held for 
two minutes in chloroform, then washed off in water, and placed from 
one-half to three minutes in a 5 per cent, solution of chromic acid, 
again washed off in water, and now stained by carbolic fuchsin, 
which is steamed for several minutes. The staining fluid is then 
washed off and the preparation decolorized in a 3 per cent, solution of 
hydrochloric acid or a 5 per cent, solution of sulphuric acid. The 

* Special staining methods for the individual organisms are given in Part II. 



Capsule atain by Hiss' method. Rhinoecle- 
roma baciUi 



lufl. X 1000. (Thro.) 



MICROSCOPIC METHODS. 35 

preparation is finally stained for a minute in methylene-blue solution. 
The spores will be red and the body of the cells blue. The different 
spores vary greatly in the readiness with which they take up the dyes, 
and we have, therefore, to experiment with each variety as to the length 
of time it should be exposed to the maceration of the chromic acid. 
Even under the best conditions it is almost impossible to stain some 
spores. 

Staining Flagella. — ^For the demonstration of flagella, which are 
possessed by all motile bacteria, we are indebted first to LoeflHer. The 
staining of flagella satisfactorily is one of the most diflScult of bacterio- 
logical procedures. Special stains devised by him, by Van Ermengem, 
by Pitfield, and others are employed. In all methods young (twelve-to 
eighteen-hour) cultures of agar should be chosen. Enough of the cul- 
ture to produce slight cloudiness is placed in a few cubic centimeters 
of filtered tap water in a test-tube. This may be used immediately, or 
allowed stand in the thermostat at blood heat for from one to two hours 
to permit shght development. A tiny drop of this rather thin emulsion 
is allowed to spread with as little manipulation as possible over the 
cover-glass so that it may dry quickly. This latter point seems to 
be the important one since slow drying allows the bacteria to shed 
their flagella. We have gotten very good results by placing on the 
cover-glass with considerable force the tiny drop held in the plat- 
inum loop, in order to spatter extremely tiny drops which may dry 
in a minimum of time. 

Bunge's modification of LoeflHer's method is carried out as follows: 
Cover-glasses which have been most carefully cleaned are covered by 
a very thin smear. After drying in the air and passing three times 
through the flame the smear is treated with a mordant solution, which 
is prepared as follows: To 3 parts of saturated watery solution of 
tannin add 1 part of a 25 per cent, solution of ferric chloride. This 
mordant should be allowed to stand for several weeks before using. 
After preparing the cover-slip with all precautions necessary to clean- 
liness, the filtered mordant is allowed to act cold for five minutes, after 
which it is warmed and then in one minute washed off. After drying, 
the smear is stained with the carbol-fuchsin solution or carbol-gentian 
violet, and then washed, dried, and mounted. 

Frequently the flagella appear well stained, but often the process 
has to be repeated a number of times. Overheating of the film prevents 
the staining of the flagella. The cell membrane may also show by 
this method. 

Van Ermengem's method gives good results. It is as follows: The 
films are placed for one hour at room temperature, or are heated for 
five minutes over a water-bath at lOO^'C. in the following solution: 

Solution A. 

Osmic acid, 2 per cent, solution 1 part. 

Tannin, 10 to 25 per cent, solution 2 parts. 

Wash successively with water, absolute alcohol, and water, then place 
in the following solution for a few seconds: 



36 PATHOGENIC MICRO-ORGANISMS, 

Solution B. 
0.5 per cent, solution of AgNO, in distilled water. 
Without washing transfer them to a third solution: 

Solution C. 

Gallic acid 5 grms. 

Tannin 3 grms. 

Fused potassium acetate 10 grms. 

Distilled water 350 c.c. 

Aft^r keeping in this for a few seconds, place again in solution B until 
film begins to turn black. Then wash and examine. 

Examination of Bacteria in Tissues. — Occasionally it is of impor- 
tance to examine the bacteria as they occur in the tissues themselves. 
The tissues should be obtained soon after death, so as to prevent as 
much as possible post-mortem changes, with consequent increase or 
decrease in the number of bacteria. Selected pieces of tissues can 
be frozen by ether or carbon dioxide and sections cut, but the best 
results are obtained when the material is embedded in paraffin or 
in celloidin. From the properly selected spots small portions, not 
larger than one-quarter of an inch by one-eighth inch, are removed 
and placed in absolute alcohol for from four to eight hours, and 
longer if thicker. For the larger pieces it is better to change the alcohol 
after eight hours. The pieces of tissue should be kept from falling 
to the bottom as the higher layers of alcohol remain nearer absolute. 
If along with the bacteria one wishes to study the finer structure of 
the tissue, it is better to employ another fixative, formalin or corrosive 
sublimate. Corrosive sublimate (saturated solution in 0.75 per cent, 
sodium chloride solution) is an excellent fixing agent. Dissolve the 
sublimate in the salt solution by heat, allow it to cool; the separartion of 
crystals will show that saturation is complete. For pieces of tissue 
one-eighth inch in thickness four hours' immersion is sufficient, for 
larger, twenty-four hours may be necessary. They should then be 
placed in pieces of gauze and left in running water for from twelve to 
twenty-four hours, according to the size of the pieces, to wash out the 
excess of sublimate. They are then placed successively for twenty- 
four hours each in the following strengths of methylated spirit (free 
from naphtha) : 30 per cent., 60 per cent., and 90 per cent. Finally they 
are placed in absolute alcohol for twenty-four hours and are then 
ready to be embedded in paraffin (see Sec. III). The paraffin sections 
of tissue having been prepared and cut, they are ready for staining. 
If all of the sublimate has not been remove;^ by the water the sections 
may be immersed in iodine-alcohol for ten minutes. For fixing in 
formalin the tissue is put in 4 to 10 per cent, formalin solution for 
three to twenty-four hours, and then in the alcohols. 

Loeffler's Staining Method. — The section is placed in LoeflBer's 
alkaline methylene-blue solution for 5 to 30 minutes, then placed for a 
few seconds in 1 per cent, acetic acid. It is then placed in absolute 
alcohol, xylol, and Canada balsam. The number of seconds during 
which the preparation remains in the acetic acid must be tested by trials. 



MICROSCOPIC METHODS. 37 

The bacteria should be dark blue, the nuclei blue, and the cell bodies 
light blue. 

Thionin solution, carbol-fuchsin solution, and gentian violet can 
be used instead of LoeflHer's methylene blue. Gram's method, with 
3 per cent, hydrochloric acid in alcohol as a tissue decolorizer for ten 
seconds, is also valuable. 

Preservation of Specimens. — Dry stained preparations of bacteria 
keep indefinitely, but if mounted in Canada balsam, cedar oil, or 
dammar lac they tend gradually to fade, although many preparations 
may be preserved for many months or years. Dry unstained spreads 
should be kept in the ice-box until stained. 

THE MICROSCOPE AND THE MICROSCOPIC EXAMINATION OF 

BACTERIA. 

Different Parts of the Microscope (Figs. 19 and 20). — A complete 
instrument usually has four oculars, or eye-pieces {A)^ which are num- 
bered from 1 to 4, according to the amount of magnification which 
they yield. Nos. 2 and 4 are most useful for bacteriologic work. The 
objective — the lens at the distal end of the barrel (B) — serves to give 
the main magnification of the object. For stained bacteria, the 
j^ achromatic oil-immersion lens is regularly employed; for pho- 
tographic purposes the apochromatic lenses are needed, although 
even here they are not indispensable. A ^^ lens may at times be useful, 
but hardly necessary; a No. 4 ocular and a y^ lens give a magnifica- 
tion of about 1000 diameters (Fig. 21). For unstanied bacteria we 
employ either the yV immersion or \ dry lens, according to the purpose 
for which we study the bacteria; for the examination of colonies where, 
as a rule, we do not wish to see individual bacteria, but only the general 
appearance of whole groups, we use lenses of much lower magnifi- 
cation (Fig. 22). 

The stage C — the platform upon which the object rests — should be 
large enough to support the Petri plates if culture work is to be done. 
The distance between the optical axis of the instrument and the pillar 
must be great enough to permit one to examine rather more than half 
the surface of the Petri dish without revolving it. The iris diaphragm 
D, which is now regularly used in bacteriologic work, opens and closes 
similar to the iris of the eye, and so controls the amount of light. 
Its opening is diminished or increased by moving a small arm, which 
is underneath the stage, in one or another direction. The reflector 
or mirror (E) placed beneath the stage serves to direct the light to the 
object to be examined. It has two surfaces — one concave and one 
plane. The concave surface must not be employed when the substage 
condenser is being used, otherwise the rays of light reaching the stage 
from the condenser will not be correctly focused. The concave sur- 
face may be used when unstained objects, such as colonies, hanging 
drops, are examined. At the same time the Abbe condenser should 
be lowered and the iris blender regulated. The coarse adjustment 



38 PATHOGENIC MICRO-ORGANISMS. 

F is the rack-and-pinion arrangement by which the barrel of the 
microscope can be quickly raised or lowered. It is used to bring the 
bacteria roughly into focus. If the bearings become loose tighten 
the little screws at the back of the pinion box. Keep the teeth cleao. 
If the bearings need oiling use an acid-free lubricant, such as paraffin 
oil. The fine adjustment G serves to raise and lower the barrel very 
slowly and evenly, and is used for the exact study of the bacteria when 



r lenses are used. It is necessarily of limited range and 
I its mechanism. If, when looking into the eye-piece, no 
focus is noticed by turning the micrometer head, or if the 
r head ceases to turn, the adjustment has reached its hmit. 
barrel of the microscope by means of the coarse adjustment, 
the micrometer back to bring the fine adjustment midway 
range. When the fine adjustment head stops do not force 
e microscopic study of bacteria it is essential that we magnify 



MICROSCOPIC METHODS. 39 

the bacteria as much as possible and still have their definition clear 
and sharp. For this purpose the microscope should be provided 
with an oil-immersion system and a substage condensing apparatus. 
In using the oil-immersion lens a drop of oil (oil of cedar) of the same 
index of refraction as the glass is placed upon the face of the lens, so 
as to connect it with the cover-glass when the bacteria are in focus. 
There is thus no loss of sight through deflection, as is the case in the 
dry system. If the lenses become dirty they should be wiped gently 
with Japanese lens paper or a clean, soft, old-linen handkerchief. 




If necessary breathe on the lens before wiping, and if this does not 
succeed use a little xylol or chloroform. These substances are not to be 
used unless necessary. An immersion objective should always be 
cleaned immediately after using. The objective should always be 
kept covered so as to prevent dust dropping in, 

ZJS^ht. — The best light is obtained from white clouds or a blue sky 
with a northern exposure. Avoid direct sunlight. If necessary use 
white shades to modify the sunlight. Artificial light has one advan- 
tage over daylight in that it is constant in quality and quantity. 
The Welsbach burner and a whitened incandescent bulb give a good 
light, A blue glass between the artificial light and the lens is often 
of value. An eye-shade may be helpful. 

Snbstage Oondensing Apparatus H is a. system of lenses situate<l 
beneath the central opening of the stage. It serves to condense the 
light passing through the reflector to the object in such a way that 
it is focused upon the object, thus furnishing the greatest amount of 
luminosity. Between the conden,ser and the reflector is placed the iris 
diaphragm. 

Foctlsillg. — Focus the body tube down by means of the coarse 
abjustment until the objective approaches very near to the cover- 



40 PATHOGENIC MICRO-ORGANISMS. 

glass, being careful not to touch it. Then with the eye at the eye- 
piece focus up carefully with the coarse adjustment until the speci- 
men comes plainly into view. Be careful not to pass by this focal 
point without noticing it. This is likely to occur if the light be too 
intense and the specimen thin and transparent. If the sliding tube 
coarse adjustment is used, focus carefully by giving the tube a spiral 
movement. 

When the object is brought fairly well into focus by means of the 
coarse adjustment, use the fine adjustment to focus on the particular 
spot desired, for if this spot is in the centre of the field of the low 
power it should be somewhere in the field of the higher power. It is 
too much to ask of the maker that the lenses be made absolutely 
parfocal and centred. The delicacy of the centring can be appre- 
ciated when the magnification and the extremely small portion ex- 
amined are considered. When the objectives are not thus fitted to 
the nose-piece, refocusing and again hunting up the object are neces- 
sary. In so doing we repeat the caution always to focus up before 
turning the nose-piece. When no revolving nose-piece is used the 
change of objectives means the unscrewing of one and the screwing 
of the other into its place, and refocusing. 

The beginner should always use the low-power objectives and ocu- 
lars first. The low-power objectives have longer working distances 
and are not so apt to be injured. They always show a larger por- 
tion of the specimen and thus give one a better idea of the general 
contour. After obtaining this general idea the higher powers can 
be used to bring out greater detail in any particular part. Gener- 
ally speaking, it is best to use a high-power objective and low-power 
eye-piece in preference to a low-power objective and high-power eye- 
piece. In the latter case any imperfections in the objective are mag- 
nified unduly by the eye-piece, giving, as a rule, poor definition. 

Tube Length and Oover-glass. — All objectives are corrected to a 
certain tube length (160 mm. by most makers — Leitz, 170 mm.) 
and all objectives in fixed mounts of over 0.70 N. A. are corrected 
to a definite thickness of cover-glass as well (Zeiss, 0.15 mm., 0.20 
mm.; Leitz, 0.17 mm.; Bausch & Lomb and Spencer, 0.18 mm.). 
These objectives give their best results only when used with the 
cover-glass and tube length for which they are corrected. As indi- 
cated in Fig. 53 the tube length extends from the eye lens of the 
eye-piece to the end of the tube into which the objective or nose- 
piece is screwed. If a nose-piece is used the draw tube must be 
correspondingly shortened. If the cover-glass is thinner than that 
for which the objective is corrected, the tube must be lengthened to 
obtain best results; if thicker, shortened. 

The more expensive objectives are provided with adjustable 
mounts by which the distances between the lens systems may be 
changed to compensate for difference of thickness of cover. They 
are successfully used only in the hands of an expert. One of them 
out of adjustment is worse than an ordinary objective. 



MICROSCOPIC METHODS. 41 

Examination of Bacteria in the Hanging Drop. — As we stated at 
the beginning of this chapter, it is often valuable to observe bacteria 
alive, so as to study them under natural conditions. We can thus note 
the method and rate of their multiplication, the presence or absence 
of spore formation, and their motility in fluids and their agglutination 
with specific serums. For this examination special slides and methods 
are desirable. The slide used is one in which there is ground out 
on one surface a hollow having a diameter of about half an inch (Fig. 
23). According to the purpose for which the hanging drop is to be 
studied, sterilization of the slide and cover-glass may or may not be 
necessary. The technique of preparing and studying the hanging 
drop is as follows: The surface of the glass around the hollow in the 
slide is smeared with a little vaselin or other inert substance. This 
has for its purpose both the sticking of the cover-glass to the slide 

Fio. 23 
Hollow slide with cover-glass. 

and the prevention of evaporation in the drop placed in the little 
chamber, which is to be formed between the cover-glass when placed 
over the hollow, and the slide. 

For the purpose of studying the bacteria we place, if they are in 
fluids, simply a large platinum loopful upon the centre of the cover- 
glass and, to avoid drying, immediately invert it by means of a slender 
pair of forceps over the hollow in the slide, being verj' careful to have 
the drop over the very centre of the cover-glass. The cover-glass 
is then pressed on the slide so as to spread the vaselin and make a 
perfect seal. If the bacteria, on the contrary, are growing on solid 
media, or are obtained from thick pus or tissues from organs, they 
are mixed with a suitable amount of bouillon or sterile physiological 
salt solution either before or after being placed upon the cover-glass. 
If we wish to observe the bacteria under natural conditions we must 
keep the tiny drop of fluid at the proper temperature for the best growth 
of the bacteria. If, however, we simply wish to observe their form and 
arrangement this is not necessary. 

In the study of living bacteria we often wish to observe their grouping 
and motion rather than their individual characters, and so use less 
magnification than for stained bacteria. In studying unstained 
bacteria and tissues we shut off as large a portion of the light with our 
diaphragm as is compatible with distinct vision, and thus favor con- 
trasts which appear as lights and shadows, due to the differences in 
light transmission of the different materials under examination. It is 
necessary to remember that they are seen with difficulty, and that we 
are very apt, unless extremely careful in focusing, to allow the lens 
to go too far, and so come upon the cover-glass, break it, destroy our 
preparation, and, if examining parasitic bacteria, infect the lens. 



42 PATHOGENIC MICRO-ORGANISMS. 

This may be avoided by first finding the hanging drop with a low- 
power lens and thus exactly centre it. The lens of higher magnification 
is now very gradually lowered, while at the same time gently moving 
the slide back and forth to the slightest extent possible with the left 
hand. If any resistance is felt the lens should be raised, for it has 
gone beyond the jxiint of focus and is touching the cover-glass. 

Hanging Mass or Hanging Block Cnltores.— In order to study the 
morphology and manner of multiplication of bacteria to better advan- 
tage than in the hanging drop, we have used hanging masses of agar, 
made by placing a large platinum loop full of m^ted agar on a sterile 
cover-glass and allowing it to harden, protected from dust. The bac- 
teria are placed on the free surface of this mass which is then in- 
verted over a hollow slide and studied as in a hanging drop. 

Hill devised the following procedure: Melted nutrient agar is 
poured into a Petri dish to a depth of about one-eighth to one-quarter 
of an inch. When cool a block is cut out about one-quarter of an inch 
square. The block is placed, under surface down, on a slide and 
protected from dust. A very dilute suspension of the growth to be 
examined is then made in sterile bouillon and spread over the upper 
surface of the block. The slide and block are then put in the incu- 
bator for ten minutes to dry slightly. A clean cover-slip is now 
placed on the agar block in such a way as to avoid large air bubbles. 
The slide is then removed. With the aid of a platinum loop a drop or 
two of melted agar is run along each side of the block to fill any angles 
between it and the cover-glass. After drying In the incubator for five 
minutes it is placed over a hollow slide and sealed with paraffin. 

We consider the hanging mass method better than that of the hang- 
ing block in many instances, because in the former method no press- 
d on the bacteria, and more oxygen is allowed them. 
Lgglutinative Properties of Semm. — By agglutination is 
aggregation into clumps of uniformly disposed bacteria 
)y sedimentation, the formation of a deposit composed of 
1 when the fluid is allowed to stand; sedimentation is thus 
I'e evidence of agglutination. 

I serum of animals is found to acquire the clumping power 
■very variety of motile bacteria, and for many non-motile 
infection with each variety. The substances causing the 
e called agglviinins. (For a discussion of agglutination 
ipter.) 

itinins were discovered by Gruber and Durham. Their 
icteria can be observed either macroscopically or micro- 
For example, if a serum from an animal which has passed 
yphoid infection is added to a twenty-four-hour culture 
aciUi, and the mixture placed in a thermostat, the following 
1 will be noticed: The bacteria, which previously clouded 
uniformly, clump together into little masses, settle to the 
test-tube, and gradually fall to the bottom until the fluid 
tirely clear. In a control test, on the contrary, to which 



MICROSCOPIC METHODS. 43 

no active serum is added the fluid remains uniformly cloudy. The 
reaction is completed in from one to twelve hours. If the reaction is 
observed in a hanging drop, a gradual formation of clumps is seen. 
Frequently one sees bacteria which have recently joined a group make 
violent motions as though they were attempting to tear themselves 
away; then they gradually lose their motility completely. Even the 
larger groups of bacteria may exhibit movement as a whole. After not 
more than one or two hours the reaction is completed; in place of the 
bacteria moving quickly across the field, one sees one or several groups 
of absolutely immobile bacilli. Now and then in a number of prepa- 
rations one sees a few separate bacteria still moving about among 
the groups. If the reaction is feeble, either because the immune 
serum has been highly diluted or because it contains very little agglu- 
tinin, the groups are small and one finds comparatively many isolated 
and perhaps also moving bacteria. It is essential each time to make a 
control test of the same bacterial culture without the addition of 
serum. Under some circumstances the reaction proceeds with ex- 
traordinary rapidity, so that the bacilli are clumped almost imme- 

Fio. 24 Fio'. 25 




MicitMcopio field, showing the top of a Microscopic field, showing a cross-section 

bangmg drop in a normal typhoid culture. of the drop in Fig. 24. 

diately. By the time the microscopic slide has been prepared and 
brought into view, nothing is to be seen of any moving or isolated 
bacteria, and only by means of the control test is it possible to tell 
whether the culture possessed normal motility. 

In order to help the student thoroughly to understand what com- 
prises a reaction, Wilson prepared a set of drawings, which are here 
reproduced. The culture to be tested should be of about twenty 
hours' growth, either in bouillon or on agar. If on the latter a sus- 
pension is made in broth or normal salt solution. A loopful of the 
fluid containing the bacteria is placed on the cover-glass, and to it an 
equal quantity of the desired serum dilution is added. 



44 



PATHOGENIC MICRO-ORGANISMS. 



In making the hanging drop to be examined it is necessary to have 
it of such a depth that it will show at least three focal planes, other- 
wise the examination will be incomplete and unsatisfactory. The 
moist chamber must be well sealed by vaselin so as to prevent drying, 
and kept at a temperature of at least 20° and not over 35° C. 

Fig. 24 shows a microscopic field of the top of a hanging drop of a 
normal bouillon culture of typhoid bacilli. The culture is twenty 
hours old and the organisms are freely motile. This represents the 
control drop used for comparison with the drop of the same culture to 
which has been added a little of the blood of a person suspected to have 
typhoid. Note these points in Fig. 24; the organisms are evenly 
distributed throughout the field, except at the edge of the drop, where 
they are gathered in great numbers; they show great activity here, 
seemingly trying to crowd to the very edge. This attraction is probably 
due to the action exerted on the organisms by the oxygen in the air, 
which naturally exerts positive chemotaxis on all aerobic organisms. 

Fig. 25 shows a cross-section of the drop represented in Fig. 24, and 
it will be noticed that the bacilli are evenly distributed throughout the 



Fio. 26 



Fio. 27 







Microecopio field, showing the top of a 
drop with the typhoid reaction. 



Microscopic field, showing a cross-section of 
the drop in Fio. 26. 



drop, except at one place in the focal plane a, and again in the focal 
plane c. 

It sometimes happens that there is a substance adhering to a sup- 
posedly clean cover-glass which attracts the bacilli to that point, where 
they appear as fairly well-defined clumps, more or less like the true 
clumps due to the agglutinating substance in typhoid blood. The 
increase in organisms at the bottom of the drop in the focal plane c 
is easily accounted for by the fact that gravity naturally carries the 
dead and non-motile organisms to the bottom, these frequently assum- 
ing the character of clumps. 

If a field can be found in any focal plane of the hanging drop free 



MICROSCOPIC METHODS, 



45 



from clumps, one can be quite sure that any clumping present is not 
due to any agglutinating substance which necessarily will affect organ- 
isms in every focal plane. 

Fig. 26 shows the microscopic appearance of the io'p of a drop 
where the reaction is present. Notice first that the organisms have 
been drawn together in groups and that the individuals of each group 
appear to be loosely held together. Viewed under the microscope 
these clumps are practically quiescent, there being very little move- 
ment either of the individual organisms or of the clump as a whole. 
The edge of the drop is practically free from organisms, showing that 
the air no longer exerts any influence on them. 

Fig. 27 shows a cross-section of the hanging drop shown in Fig. 26. 
The clumps are evenly distributed throughout the drop, with perhaps 
some increase in the numbers and compactness of the clumps at the 
bottom. 



Fig. 28. 



Fio. 29. 




Microscopic field, showing the top of a drop of 
culture with reaction not due to typhoid. 



Microscopic field, showing a cross- 
section of Fig. 28. 



Fig. 28 shows the microscopic appearance of the top of a hanging 
drop of a bouillon culture to which has been added some blood of a 
patient suffering from a febrile condition not caused by typhoid 
infection, but which exerts a slight non-specific influence on the 
typhoid organisms. It will be seen that the reaction is incomplete 
and that there are many organisms at the edge of the drop. The air 
exerts the same influence on the bacilli that it did before the addition 
of the blood. Note the character of the clumps, generally small and 
compact at the centre, with the bacilli at the edge of the clump, usually 
attached by one end only. 

Very frequently these clumps have the appearance of being built up 
around a piece of detritus present in the clump. All the organisms 
comprising the clump seem to have retained part, at least, of their 
motility, those on the edges being particularly motile, so far as their 
free ends are concerned. 



46 PATHOGENIC MICRO-ORGANISMS. 

When motility is very much inhibited these clumps have a peculiar 
trembling movement, which is like the molecular movement described 
by Brown. 

Fig. 29 shows a cross-section of the drop represented in Fig, 28. 
Note the same character of the clumps in every focal plane: the large 
number of motile bacilli and the number attracted at the edge of the 
drop by the air. 

Dark Oroand nimnination and the Examination of Ultramiero- 
SCOpic Particles. — ^The apparatus constructed by Siedentopf and 
Zsigmondy' makes visible, and in solutions otherwise apparently 
homogeneous, very minute particles, which heretofore could not be 
seen even with the highest magnifications. Particles l/i/i (a milli- 
micron = one millionth of a millimeter) are thus rendered visible. 



VimlenC diptheriu builli. Cultures two days old. UmUiaed. X 2100. (After Siabeit.) 

This increased power in microscopic analysis is made possible by 
intense (electric arc lamp) focal lateral illumination of the objects 
examined, making them appear as minute luminous points. The 
greater the difference between the refractive index of the particles 
colloidally dissolved or otherwise held in suspension and the fluid which 
surrounds them, the brighter will be the appearance of the particles, 
and, therefore, the more readily visible. 

The jmicroscopic field, as will be seen by the photograra herewith 
(Fig. 30), is dark; the objects which refract the light show as brightly 
illuminated, sharply defined pictures, in vhich the black margin cor- 
responds to the contour of the object. The illuminated portion is sur- 

• Annalen der Physik, 4tc Folge, Bond 10. 



MICROSCOPIC METHODS, 47 

rounded by a fine dark zone, this in turn by alternate bright and dark 
zones, in which the illumination rapidly decreases. 

Reichert, of Vienna, has recently simplified this apparatus by 
devising a new condenser/ The light which illuminates the object 
has a greater refraction than the cone of light entering the objective 
which produces the image. Its advantages over the first method are: 
(1) It utilizes the source of light better; (2) any dry objective can be 
used without alterations; (3) small particles are seen without the dis- 
turbing refraction rings. With this apparatus such living organisms 
as the Sfirocheta pallida, and the fiagella on certain bacteria, which 
can scarcely be seen by ordinary microscopes on account of their low 
refractive indices, may be demonstrated with great clearness. 

The use of microphotograpky with uUra-violet light (according to 
A. Kohler*) makes visible particles that cannot be seen by ordinary 
light, because of the inability of the violet rays to pass through certain 
substances, e. g,, chromatin. The few discoveries claimed by these 
means for diseases of unknown origin have so far lacked suflScient 
corroboration to constitute them proved. 

Burri's Indio'ink method^ of demonstrating bacteria. In 1907, 
1908 and 1909 Burri recommended the following method for isolat- 
ing and studying single bacterial cells. A solution of India ink 
(flussige Perltusche) in water 1 : 10 [better 1-4] is sterilized in test- 
tubes in the autoclave for fifteen minutes. A small drop of this ink 
is mixed carefully with a drop of the fluid to be examined. If cul- 
tures from isolated cells are desired the fluid should first be diluted 
so that a drop contains presumably a single organism; then drops of 
the mixture are placed in rows upon nutrient agar plates. If the 
bacteria are to be examined immediately a drop of the mixture (ink 
plus undiluted bacterial fluid) is allowed to dry upon a glass slide 
and then examined under an oil-immersion lens. The bacteria 
appear a brilliant white upon a dark field, particles of the ink sur- 
rounding the organisms like a capsule. This method is especially 
applicable for the demonstration of such organisms as the Tr. pal- 
lidum which have poor staining qualities and a low index of refraction. 

• 

* Joum. R. Micr. Soc., 1907, p. 364, gives full description and instructions for 
use. 

« A. Kohler. Ztschr. f. wiss. Mikroskopie, 1904, 21, 129. 

' Burri, Robt. Das Tuscheverfahren als einfaches Mittel zur Ldsung einiger 
schwieriger Aufgaben der Bakterioskopie, 1909. Jena, O. Fischer. 



CHAPTER IV. 
EFFECTS OF SURROUNDING FORCES UPON BACTERIA. 

FOOD, OXTGEN, TEMPERATURE, LIGHT, ETC. 

1. Food. — Naturally, the eflFect of food upon bacteria is marked. 
Though the majority of bacteria grow easily on certain artificial foods 
(culture media), some we have not yet been able to cultivate outside 
of the body of their host. Those bacteria which depend entirely upon a 
Uving host for their existence are known as strict 'parasites; those which 
live only upon dead organic (a few on inorganic) substances are called 
strict saprophytes; those which can lead a saprophytic existence, but 
which usually thrive only within the body of a living animal, are called 
faxndtative parasites. The strict saprophytes, which represent the larger 
majority of all bacteria, are not only harmless to living organisms, but 
perform many exceedingly important functions in nature, such as the 
destruction of dead organic matter and its preparation for plant food 
through decomposition, putrefaction, and fermentation, while one group 
(see below, the nitrifying bacteria) are constructive in their activities. 
The parasites, on the contrary, are harmful invaders of the body 
tissues, exciting by their growth and products many forms of disease. 
The substances essential for the majority of those forms which can be 
grown artificially are organic material as a source of carbon and nitro- 
gen, an abundance of water and certain salts (either calcium or mag- 
nesium and sodium or potassium salts are usually required, also sulphur 
and phosphorus salts. Iron is demanded by a few varieties). The de- 
mands of bacteria for food of a certain composition vary considerably. 
The greater number of important bacteria and all the pathogenic species 
thrive best in media containing abundant albuminoid substances and of 
a slightly alkaline reaction to litmus. Some species of water bacteria, 
on the other hand, require so little organic material that they will grow 
in water that has been twice distilled. A certain species will grow 
abundantly in water containing ammonium carbonate in solution and 
no other source of carbon and nitrogen. Then there is a whole group 
of soil bacteria, the so-called nitrifying organisms which develop in the 
presence of very simple mineral salts (ammonium salts and nitrites). 
These show the power of some bacteria to produce cell substance 
from the simplest materials — a power formerly supposed to belong 
only to the higher plants which obtain their nourishment from the air 
through their chlorophyll and the assistance of sunlight. The bac- 
teria, however, of any importance in disease are not so easily satis- 
fied, though there are many species which are able to develop without 

48 



EFFECTS OF SURROUNDING FORCES UPON BACTERIA. 49 

the presence of albumin and in comparatively simple culture media, 
such as the culture liquid proposed by Uschinsky, or the simpler one 
of Voges and Fraenkel, which consists of water, 1000; sodium chloride, 
5; neutral sodium phosphate, 2; ammonium acetate, 6; and asparagin, 
4. In these media many bacteria grow well. 

When we consider in detail the source of the more important chemi- 
cal ingredients of bacteria we find that their nitrogen is most readily 
obtained from diffusible albuminoid material and less easily from 
ammonium compounds. Their carbon they derive from albumin, 
peptone, and sugar, as well as from other allied carbohydrates: gly- 
cerin, fats, and other organic substances. It is an interesting fact that 
even compounds which in considerable concentration are extremely 
poisonous, can, when in sufficient dilution, provide the necessary 
carbon and even act as stimulants to growth; in this way carbolic 
acid in very dilute solutions may be used by some bacteria. 

The value of substances as a source of nutrition is often influenced 
by the presence of other materials, as, for instance, the value of as- 
paragin is increased by the presence of sugars. Further, materials 
from which nitrogen and carbon cannot be directly obtained still 
become assimilable after being subjected to the influence of bac- 
terial ferments. The profound and diverse changes produced by the 
different ferments make it almost impossible to establish, except in 
the most general way, the nutritive value of any mixture for a large 
number of bacteria through a simple knowledge of its chemical 
composition. 

The special culture media, such as bouillon, blood serum, etc., used 
for the development of bacteria, will be dealt with in a later chapter. 

While it is true that very wide differences in relative composition 
and total concentration of food media may have slight effect upon the 
development of a given bacterium, slight changes in composition and 
reaction of the media often have a great effect upon morphology, 
rate of growth, motility, and specific products of growth. 

Reaction of Media. — ^The reaction of the media is of very great im- 
portance. Most bacteria grow best in those media that are slightly 
alkaline or neutral to litmus. Only a few varieties require an acid 
medium, and none of these belong to the parasitic bacteria. An 
amount of acid or alkali insufficient to prevent the development of 
bacteria may still suffice to rob them of some of their most important 
functions, such as the production of poison. The different effect upon 
closely allied varieties of bacteria of a slight excess of acid or alkali 
is sometimes made use of in separating those which may be closely 
allied in many other respects. 

Influence of One Species upon the Orowth of Another. — When one 
species of bacteria is grown in a food medium, that medium usually 
becomes less suitable for the growth of its kind and of other bacteria. 
This is due partly to the impoverishment of the food stuffs, but more 
to the production of chemical substances or enzymes. When different 
species are grown together, the antagonistic action of one upon the 
4 



50 PATHOGENIC MICRO-ORGANISMS. 

other may be shown from the beginning. Some species, however, 
have a cooperative or symbiotic action with other species. 

In nature, bacteria usually occur in mixed cultures (e. g,, water, 
milk, intestinal contents of all animab), and here we may see antago- 
nistic action in the prevalence of one species over others (e. g., the lactic 
acid formers in the intestines), or cooperative action in the equal and 
luxuriant growth of two or more species (e. g., pneumococcus and 
influenza bacillus in the lungs). 

Experimentally, the existence of antagonisms can be demonstrated 
by inoculating gelatin streak cultures of various bacteria. It is found 
that many species will grow not at all or only sparingly when in close 
proximity to some other species. This antagonism, however, is often 
only one-sided in character. Again, when gelatin or agar plates are 
made from two different species of bacteria it may be observed that 
only one of the two grows. A third method of making this experi- 
ment is simultaneously to inoculate the same liquid medium with 
two species, and then to examine them later, both microscopically and 
by making plate cultures; not infrequently one species may take prece- 
dence of the other, which after a time it may entirely overcome. 

Finally, it may be shown experimentally that bacteria may oppose 
one another as antagonists in the animal body. For instance, Emme- 
rich has shown, that animals infected with anthrax may often be cured 
by a secondary infection with the streptococcus. 

The symbiotic or cooperative action of bacteria may be demonstrated 
experimentally in the following examples: 

a. Pneumococci when grown together with a bacillus obtained from 
the throat, produces very large, succulent colonies. The influenza 
bacillus which will not grow alone upon ordinary nutrient agar ^lU 
grow well there in the presence of certain other bacteria. Some anae- 
robic species grow even with the admission of air if only some aerobic 
species are present (tetanus bacilli with diphtheria bacilli). 

6. Certain chemical effects, as, for instance, the decomposition of 
nitrates to gaseous nitrogen, cannot be produced by many bacteria 
alone, but only when two are associated, 

2. Behavior toward Oxygen and other Oases.— The majority of 
bacteria absolutely require free oxygen for their growth, but a consider- 
able minority fail to grow unless it is excluded. This latter fact, 
noted first by Pasteur, led him to divide bacteria into aerobic and 
anaerobic forms. Between these two groups we have those that can 
grow either with or without the presence of oxygen, called respectively 
facultative aerobic and facultative anaerobic bacteria. 

a. Aerobic Bacteria. — Growth only in the presence of free oxygen: 
the slightest restriction of air inhibits development. Spore formation, 
especially, requires the free admission of air. 

5. Anaerobic Bacteria. — Growth and spore formation only in the 
total exclusion of free oxgyen. Among this class of bacteria are the 
bacillus of malignant oedema, the tetanus bacillus, the bacillus of 
symptomatic anthrax, and many soil bacteria. Exposed to the action 



* • •• • 

• • ; . •• • 

V " • • • 

^ ••: : .. 



EFFECTS OF SURROUNDING FORCES UPON BACTERIA. 51 

of oxygen, the vegetative forms of these bacteria are readily destroyed; 
the spores, on the contrary, are very resistant. Anaerobic bacteria 
being deprived of free oxygen — the chief source of energy supplied to 
the aerobic species, by which they oxidize the nutritive substances in 
the culture media — are dependent for their oxygen upon decomposable 
substances, such as grape-sugar. 

e. Facultative A^obic and Facultative Anaerobic Bacteria. — The 
greater number of aerobic bacteria, including most of the pathogenic 
species, are capable of withstanding, without being seriously affected, 
some restriction in the amount of oxygen admitted, and many, indeed, 
grow equally luxuriantly with the partial exclusion of oxygen. Life in 
the animal body, for example, as in the intestines, necessitates existence 
with diminished supply of oxygen. If in any given variety of bacteria, 
the amount of oxygen present is unfavorable, there will be more or 
less restriction in some of the life processes of this variety, such as 
pigment and toxin production, spore formation, etc. Pigment forma- 
tion almost always ceases with the exclusion of oxygen, but poisonous 
products of decomposition may be more abundantly produced. 

It is important to note that, according to recent investigations, it 
has been shown that the aerobic development of the anaerobes may 
be facilitated by the presence of Uving or dead aerobes. 

It has also been observed not infrequently that certain species which 
on their isolation at first show more or less anaerobic development — 
that is, a preference to grow in the depth of an agar stick culture, for 
instance — after a while seem to become strict aerobes, growing only 
on the surface of the medium. This observation proves that the 
simple fact of an organism showing aerobic or anaerobic growth is 
not a sufficient basis for its separation into a distinct species. 

Other Gases. — While all facultative bacteria as well as strict anaer- 
obes grow well in nitrogen and hydrogen, they behave very differently 
toward carbonic acid gas. A large number of these species do not grow 
at all, being completely inhibited in their development until oxygen is 
again admitted — for example, B, anthracis and B. svbtilis and other 
allied species. It has been found in some species, as glanders and 
cholera, that the majority of the organisms are quickly killed by CO,, 
while a few, such as staphylococci, offer a great resistance, rendering 
impossible complete sterilization by means of this gas. Another group, 
again — ^viz., streptococcus and staphylococcus — exhibits a scanty 
growth. A mixture of one-fourth air to three-fourths carbonic acid gas 
seems to have no injurious effect on bacteria which cannot grow in an at- 
mosphere of pure COj. Under pressure COj is more effective (p. 56). 

Sulphuretted hydrogen in large quantity is a strong bacterial poison, 
and even in small amounts kills some bacteria. 

3. Effect of Temperature upon Bacteria.— Some form of bacterial 
life is possible within the limits of 0° and 70° C. The maximum and 
minimum temperature for each individual species ordinarily lies from 
10° to 30° C. apart, and the optimum covers about 5°. Usually the 
temperature of the soil in which the bacteria are deposited is the con- 



52 PATHOGENIC MICRO-ORGANISMS. 

trolling factor in deciding whether growth will or will not take place. 
Thus, nearly all parasitic bacteria require a temperature near that of 
the body for their development, while many saprophytic bacteria can 
grow only at much lower temperatures. Bacteria when exposed to 
lower temperature than suflBces for their growth, while having their 
activities decreased, are not otherwise injured unless actually frozen 
for a certain time; while exposure to higher temperatures than allows 
of growth more or less quickly destroys the life of the bacteria. Bac- 
teria have been classified according to the temperatures at which they 
develop, as follows: 

Psychrophilic Bacteria. — Minimum at 0° C, optimum at 15*^ to 20® 
C, .maximum at about 30° C. To this class belong many of the water 
bacteria, such as the phosphorescent bacteria in sea-water. 

Mesophilic Bacteria. — Minimum at 5° to 25° C, optimum about 37° C, 
maximum at about 43° C. To this class belong all pathogenic bac- 
teria, most parasitic and many saprophytic forms. 

Thermophilic Bacteria. — Minimum at 25° to 45° C, optimum at 50° 
to 55° C, maximum at 60° to 70° C. This class includes a number 
of soil bacteria which are almost exclusively spore-bearing bacilli. 
They are also found widely distributed in feces. 

By carefully elevating or reducing the temperature the limits 
within which a variety of bacteria will grow can be altered. Thus, 
the anthrax bacillus was gradually made to accommodate itself to a 
temperature of 42° C, and pigeons, which are comparatively im- 
mune to anthraxl, partly on account of their high body temperature 
(42° C), when inoculated with this anthrax succumbed to the infec- 
tion. Another culture accustomed to a temperature of 12° C. killed 
frogs kept at 12° C. We have cultivated a very virulent diphtheria 
bacillus so that it will grow at 43° C. and produce strong toxin. 

Effect of Low Temperature. — The rapidity of bacterial growth 
is retarded by temperatures lower than those required for the opti- 
mum of each species. It is the usual custom in laboratories to pre- 
serve bacteria which die readily (such as streptococci) by keeping 
them in the refrigerator at about 5° to 10° C, after cultivation for 
two days at 30° C, as a means for retaining their vitality without 
repeated transplantation. Temperatures even far under 0° C. are 
only slowly injurious to bacteria, different species being affected 
with varying rapidity. This has been demonstrated by numerous 
experiments in which they have been exposed for weeks in a refriger- 
ating mixture at — 18° C. If a culture of typhoid bacilli is frozen, about 
50 to 70 per cent, of the organisms are killed at the time. At the end 
of one week not more than 10 per cent, survive, and at four weeks not 
over 1 per cent. After six months none survive. More resistant 
bacteria live longer and spores may survive in ice for years. Bacteria 
have even been subjected to a temperature of — 175° C. by immersing 
them in liquid air kept in an open tube for two hours, and 15 to 80 per 
cent, were found still to grow when placed in favorable conditions. 
We found about 10 per cent, of typhoid bacilli alive after thirty minutes' 



EFFECTS OF SURROUNDING FORCES UPON BACTERIA. 53 

exposure to this low temperature. Staphylococci were more resistant. 
Spores were scarcely affected at all. 

Effect of High Temperatures. — Temperatures from 5° to 10*^ C. 
over the optimum affect bacteria injuriously in several respects. 
Varieties are produced of diminished activity of growth, the viru- 
lence and the property of causing fermentation are decreased, and 
the power of spore formation is gradually lost. These effects may 
predominate either in one or the other direction. 

If the maximum temperature is exceeded, the organism dies; the 
thermal death point for the psychrophilic species being about 37° 
C, for the mesophilic species about 45*^ to 55*^ C, and for the ther- 
mophilic species about 75*^ C. There are no non-spore-bearing 
bacteria which when moist are able to withstand a temperature of 
100*^ C. even for a few minutes. A long exposure to temperatures 
between 60° and 80° C. has the same result as a shorter one at the 
higher temperatures. Ten to thirty minutes' exposure to moist heat 
will at 60° C. kill the cholera spirillum, the streptococcus, the typhoid 
bacillus, and the gonococcus, and at 70° C. the staphylococcus, the 
latter being among the most resistant of the pathogenic organisms 
which have no spores. A much shorter exposure will kill a large per- 
centage of any mass of these bacteria. 

Effect of Dry Heat. — When microorganisms in a desiccated con- 
dition are exposed to the action of heated dry air the temperature 
required for their destruction is much above that required when they 
are in a moist condition or when they are exposed to the action of 
hot water or steam. A large number of pathogenic and non- 
pathogenic species are able occasionally to resist a temperature 
of over 100° C. dry heat for from ten minutes to one hour. In any 
large number of bacteria a few are always more resistant than the 
majority. A temperature of 120° to 130° C. dry heat maintained 
for one and a half hours will destroy all bacteria, in the absence of 
spores. 

ResiBtance of Spores to Heat. — Spores possess a great power of resist- 
ance to both moist and dry heat. Dry heat is comparatively well- 
bome, many spores resisting a temperature of over 130° C. for as long 
as three hours. Exposed to 150° C. for one hour, practically all spores 
are killed. Moist heat at a temperature of 100° C, either boiling water 
or free-flowing steam, destroys the spores of most varieties of bacteria 
within fifteen minutes; certain pathogenic and non-pathogenic species, 
however, resist this temperature for hours. The spores of a bacillus 
from the soil required five and a half to six hours' exposure to stream- 
ing steam for their destruction. They were destroyed, however, by 
exposure for twenty-five minutes in steam at 113° to 116° C. and in 
two minutes at 127° C. The spores from tetanus bacilli may require 
longer than fifteen minutes' exposure to kill them. 

The resistance of spores to moist heat is tested by suspending threads, 
upon which the spores have been dried, in boiling water or steam. 
The threads are removed from minute to minute and laid upon agar 



54 PATHOGENIC MICRO-ORGANISMS. 

or in broth, which is then placed at a suitable temperature for growth, 
should any spores be living. 

Practieal Points on Heat Disinfection. — In the practical application of 
steam for disinfecting purposes it must be remembered that while 
moist steam under pressure is more effective than streaming steam, it 
is scarcely necessary to give it the preference, in view of the fact that 
most known pathogenic bacteria produce no spores and the spores of the 
few that do develop them are quickly destroyed by the temperature 
of boiling water, and also that "superheated" steam is less effective 
than moist steam. When confined steam in pipes is "superheated" 
after its generation it has about the same germicidal power as hot, dry 
air at the same temperature. Esmarch found that anthrax spores 
were killed in streaming steam in four minutes, but were not killed in 
the same time by superheated steam at a temperature of 114° C. 
It should also be remembered that dry heat has but little penetrating 
power, and that even steam requires time to pass through heavy goods. 
Koch and WolflFhiigel found that registering thermometers placed in 
the interior of folded blankets and packages of various kinds did not 
show a temperature capable of killing bacteria after three hours' expo- 
sure in a dry hot-air oven at 133*^ C. and over. We have put a piece of 
ice in the middle of several mattresses and recovered it after exposing 
the goods to an atmosphere of live steam for ten minutes. 

Fractional Sterilization. — Certain nutrient media, such as blood- 
serum and the transudates of the body cavities, as well a& certain 
fluid foodstuffs, need at times to be sterilized, and yet cannot be sub- 
jected to temperatures high enough to kill spores without suffering 
injury. The property of spores, when placed under suitable con- 
ditions, to germinate into the non-spore-bearing form, is here taken 
advantage of by heating the fluids up to the highest non-injurious 
point for a certain time on each of several consecutive days, and keep- 
ing the fluid at about 20° C. during the intervals. By this means 
we kill, upon each exposure, all bacteria in vegetative form, and allow 
during the intervals, for the development of any still remaining in the 
spore stage, or which have reproduced spores, to change again into the 
vegetative form. Experience has shown that, with but few exceptions, 
in the case of blood serum and body transudates, an exposure for six 
consecutive days at 55° to 70*^ C. for one hour will completely sterilize 
the fluids so exposed. 

With the usual culture media a temper.ature of 100*^ C. for twenty 
minutes does little or no harm, while one of 120° C. is sometimes 
deleterious. With heating to 100° C. an exposure on three consecutive 
days, and to 115° C. on one or two days suffices. 

Pasteurization. — It is sometimes undesirable to expose food, such 
as milk, to a temperature that will destroy spores, because of the 
deleterious effects of such high temperatures upon food values, and 
yet a partial destruction of the contained bacteria is necessary. In 
these cases we heat the foodstuffs for from twenty to forty minutes 
at 60° C. or from two to five minutes at 70° C. This degree of heat 



p 



EFFECTS OF SURROUNDING FORCES UPON BACTERIA. 55 

will kill the bacteria in the vegetative form, but allow the spores to 
remain alive. These exposures kill about 98 to 99 per cent, of the 
bacteria in milk. The exposure to this degree of heat alters the 
chemical composition of the milk but little. 

4. Influence of Light. — A large number — perhaps the majority — of 
bacteria are inhibited in growth by the action of bright daylight, 
all are by that of direct sunlight, and when the action of the latter 
is prolonged they lose their power of developing when later placed in 
the dark. 

In order to test the susceptibility of bacteria to light, it is best, 
according to H. Buchner, to suspend a large number of bacteria in 
nutrient gelatin or agar and pour the media while still fluid in Petri 
dishes, upon which has been pasted a strip of black paper on the side 
upon which the light is to act. The action of heat may be excluded 
by allowing the ray of light first to pass through a layer of water or 
alum of several centimetres' thickness. After the plates have been 
exposed to the light for one-half, one, one and a half, two hours, 
etc., they are taken into a dark room and allowed to stand at 20 
or 35° C. a suflScient length of time to allow of growth, and then 
examined to see whether there are colonies anywhere except on 
the spot covered by the paper; when the colonies exposed to the 
light have been completely destroyed there is lying in a clear sterile 
field a sharply defined region of the shape of the paper strip crowded 
with colonies. 

Dieudonn^, in experiments upon the Bacillus prodigiosus, found 
that direct sunlight in March, July, and August killed these bacilli in 
one and a half hours; in November in two and a half hours. Diffuse 
daylight in March and July restrained development after three and a 
half hours' exposure (in November four and a half hours) and com- 
pletely destroyed their vitality in from five to six hours. The electric 
arc light inhibited growth in five hours and destroyed vitality in eight 
hours. Incandescent light inhibited growth in from seven to eight 
hours and killed in eleven hours. Similar results have been obtained 
with B. colt, B. typhosus y and JS. anthracis. According to Koch, the 
tubercle bacillus is killed by the action of direct sunlight in a time 
varying from a few minutes to several hours, depending upon the 
thickness of the layer exposed and the season of the year. Diffuse 
daylight also had the same effect, although a considerably longer time 
of exposure was required— when placed close to a window, from five 
to seven days. B, diphiherice protected by clear, non-colored glass is 
not materially affected by diffuse daylight or by direct sunlight. Un- 
protected they are quickly killed by the latter and sjowly by the 
former. 

Only the ultraviolet, violet, and blue rays of the spectrum seem to 
possess bactericidal action; green light has very much less; red and 
yellow light none at all. The action of light is apparently assisted by 
the admission of air; anaerobic species, like the tetanus bacillus, and 
facultative anaerobic species, such as the colon bacillus, are able to 



56 PATHOGENIC MICRO-ORGANISMS, 

withstand quite well the action of sunlight in the absence of oxygen, 
the B. coli intense direct sunlight for four hours. 

According to Richardson and Dieudonn^, the mechanism of the 
action of light may be at last partially explained by the fact that in 
agar plates exposed to light for a short time (even after ten minutes' 
exposure to direct sunlight) hydrogen peroxide (H2O2) is formed. 
This is demonstrated by exposing an agar plate half covered with 
black paper, upon which a weak solution of iodide of starch is poured, 
and over this again a dilute solution of sulphate of iron; the side 
exposed to the light turns blue-black. In gases conta ling no oxygen, 
hydrogen peroxide is not produced, and the light has no injurious 
effect. Access of oxygen also explains the effect which light produces 
on culture media which have been exposed to the action of sunlight, 
as standing in the sun for a time, when afterward used for inoculation. 
The bacteria subsequently introduced into such media grow badly — 
far worse than in fresh culture media which are kept in the shade. 

Influence of Radium. — Radioactive fluids have a slight inhibiting 
effect on bacterial growth, but nothing decided enough to be used for 
therapeutic purposes has been evolved up to the present time. 

Influence of X-Rays. — These rays have a slight inhibiting effect on 
bacteria when they are directly exposed to them. 

6. Influence of Electricity on Bacteria. — ^The majority of the obser- 
vations heretofore made on this subject would seem to indicate that 
there is no direct action of the galvanic current on bacteria; but the 
effect of heat and the electrolytic changes in the culture liquid resulting 
from the electrolysis may destroy them. 

6. Influence of Agitation. — Meltzer has shown that the vitality of 
bacteria is destroyed by protracted and violent shaking, which causes 
a disintegration of the cells. Appel found that moderate agitation 
of the bacteria caused no injury, even when long continued. 

7. Influence of Pressure. — Bacteria in fluids which are subjected to 
great pressure are for a time inhibited in their growth. When oxygen 
or nitrogen are used the same moderate inhibition occurs. 

Influence of Carbonic Acid Under Pressure. — D'Arsonval and Char- 
rin submitted a culture of Bacillus 'pyocyaneus to a pressure of 
fifty atmospheres under carbonic acid. At the end of four hours 
cultures could still be obtained, but the bacillus had lost its power 
of pigment production. A few colonies were developed after six 
hours' exposure to this pressure, but after twenty-four hours no de- 
velopment occurred. Other bacteria subjected to pressure have ex- 
hibited more resistance. We have subjected broth and milk contain- 
ing typhoid,, dysentery, diphtheria, and colon bacilli to the gas under 
a pressure of seventy-five and one hundred and fifty pounds. Within 
twenty-four hours 99 per cent, of those in the broth and 98 per cent, of 
those in the milk were destroyed. Within one week the broth was 
sterile and within four weeks the milk was sterile. Tubercle bacilli 
and staphylococci were much more resistant, but little effect being 
noticed in twenty-four hours. The results were the same whether 



EFFECTS OF SURROUNDING FORCES UPON BACTERIA. 57 

the cultures were kept at 10° or 25°. Bottled waters charged with 
carbonic acid are usually sterile. 

8. Life in Absence of Moisture. — For growth, bacteria require 
much moisture. Want of water affects them in different ways. 
Upon dried culture media development soon ceases; but in media 
dried gradually at the room temperature (nutrient agar, gelatin, 
potato) they live often for a long time, even when there are no 
spores to account for their longevity A shrunken residue of such 
cultures in bouillon has often been found, after a year or more, to 
yield living bacteria. The question as to how long the non-spore-bear- 
ing forms are capable of retaining their vitality when dried on a cover- 
glass or silk threads has been variously answered. We know now 
that there are many factors which influence the retention of vitality; 
spores, of course, being more resistant than vegetive forms. The 
following table of the results obtained by Sirena and Alessi with some 
non-spore-bearing forms, gives some idea of the extent and effect of 
such influences. In the experiments silk threads were saturated with 
bouillon cultures or aqueous suspensions of the bacteria, and some 
then enclosed in tubes containing sulphuric acid or calcium chloride, 
while others were left exposed to various outside influences: 



Desiccation 



With sul- With calcium t„ :„«„u„*«^ In dry room t„ ,««:o* 
phuric .cid.. chloride. ^°t3'?.7^^' mAfde J^T^Li 



Cholera spirilla 1 day 1 day 1 day 1 day 12 day« 

B. of fowl cholera 2 days 1 day 1 day 5 days 59 dasrs 

B. typhosus 41 days 1 day 18 days 64 days ^ 68 days 

B. maOei 35 days 44 days 31 days 

Diploooc. pneumoniae. . . 114 da3rs 31 days 131 days 164 days 192 days 



The results of all investigators, however, would seem to indicate 
that the greatest possible care must be exercised in desiccation experi- 
ments to come to any positive conclusions; but recently most aston- 
ishing results have been obtained with regard to many species usually 
supposed to be particularly sensitive to desiccation, showing that 
under certain conditions they may retain their vitality in a dry state 
for a very long time. Thus, Koch found that cholera spirilla lived 
only a few hours when dry; Kitasato determined their life duration 
at fourteen days at most; while various French observers have found 
that they may, under favorable conditions, live 150 to 200 days. The 
varying results sometimes reported by different observers in such 
experiments may be explained by the fact that the conditions under 
which they were made were different, depending upon the desiccator 
used, the medium upon which the cultures were grown, and the use of 
silk threads or cover-glasses. In all these experiments, of course, it 
should be previously determined that in spore-bearing species there are 
no spores present. Even when a dried culture lives for a long time the 
majority of the organisms die in a few hours after drying. We have 
found 1,500,000 colon bacilli to be reduced to 100,000 after three 



58 PATHOGENIC MICRO-ORQANtSMS. 

hours' drying. When protected by a covering of mucus, as in expecto- 
ration, they live much longer than when unprotected. 

Dnntdon of Life in Pure Water. — When bacteria which require 
much organic food for their development {and these include most of 
the pathogenic species) are placed in distilled water they soon die — 
that is, within a few days; even in sterilized well water or surface 
water their life duration does not usually exceed eight to fourteen days, 
and they rarely multiply. Instances, however, of much more extended 
life under certain conditions are recorded. 

9. Tactic Effect of Chemicals.— CAemo/fm*.— The deleterious effect 
of chemicals, especially those used as germicides, will be considered in 
a separate chapter. 

Some chemical substances exert a peculiar attraction for bacteria, 
known as -posUive ekemotaxis, while others repel them — negath'c 
chemotaxis. Moreover, all varieties are not affected alike, for the same 
substances may exert on some bacteria an attraction and on others a 
repulsion. Oxygen, for example, attracts aerobic and repels anaerobic 
bacteria, and for each variety there is a definite proportion of oxygen, 
which most strongly attracts. The chemotactic properties of sub- 
stances are tested by pushing the open end of a fine capillary tube, 
filled with the substance to be tested, into the edge of a drop of culture 
fluid containing bacteria and examining the hanging drop under the 
microscope. W'e are able thus to watch the action of the bacteria and 
note whether they crowd about the tube opening or are repelled from 
it. Among substances showing positive chemotaxis for nearly all 
bacteria are peptone and urea, while among those showing negative 
chemotaxis are alcohol and many of the metallic salts. Such experi- 
ments are of course rough. The diffusion of the substances from the 
tube into the surrounding medium must play an extremely active 
r6le in the final result. 



CHAPTER V. 

THE MATERIALS AND METHODS USED IN THE CULTIVATION 

OF BACTERIA.. 

The methods employed for the artificial cultivation of bacteria are 
of fundamental importance in bacteriology. The study of the char- 
acteristics of any bacterium requires that it be examined growing apart 
from all others in pure cultures. In order to separate one species from 
others and to study its morphologic, biochemic, and cultural char- 
acteristics we have to prepare a number of sterile solid and liquid 
media and employ them in various technical ways. In the first place, 
however, we have to take the greatest precautions to insure that the 
materials that we make use of for the growth of bacteria, the flasks 
and tubes that hold these materials, and the instruments with which 
we transfer the bacteria are sterile. 

Cleansing and Sterilization of Apparatus. — In bacteriologic work 
sterilization is practically always done by means of dry and moist heat, 
for no antiseptic substances can be allowed to remain in any of the 
media used for the growth of bacteria or on any of the apparatus which 
would come in contact with them, as such substances would inhibit 
the growth of the bacteria which we desire to study. 

The platinum wires and loops (Fig. 54) used in transferring bacteria 
are sterilized by holding them for a moment until red-hot in a gas or 
alcohol flame. They should not be used until time enough has elapsed 
for them to cool suflSciently not to injure the bacteria touched by them. 
Knives, instruments, etc., are, after thorough cleansing, placed in 
boiling 1 per cent, washing soda solution for three to five minutes. 
Hypodermic needles are sterilized by boiling in soda solution, or, when 
this is impossible, they are first frequently rinsed with boiling or with 
very hot water, and then filled with a 5 per cent, carbolic acid solution 
for at least thirty minutes and then rinsed again with sterile water. 
New tubes and flasks sometimes require to be washed in a solution 
of dilute hydrochloric acid, so as to remove any free alkali which may 
be present. They are finally thoroughly rinsed in pure water. Old 
tubes, flasks, and other glassware are boiled for about thirty minutes 
in a 5 per cent, solution of washing soda in soapsuds, and then thor- 
oughly rinsed off with water until perfectly clean. If necessary, any 
dirt clinging to the insides of the flasks and tubes can be removed by 
bristle brushes or suitable swabs. After the tubes and flasks have been 
thoroughly cleaned they are plugged loosely with ordinary cotton- 
batting, or, if that is not at hand, the more expensive absorbent cotton. 
The tubes and flasks with their cotton plugs, and all other glassware 

59 



60 PATHOGENIC MICRO-ORGANISMS. - 

are sterilized by dry heat at 150° C. for one hour in the dry-heat 
sterilizer (Fig. 31). 

Freparatioii of Golture Media. — Before we can get a suitable 
growth of any special variety of bacteria, we must have the substances 
necessary for growth present in the proper proportion and concen- 
tration. Certain species of bacteria require special foodstuffs, so that 
for each kind the proper food must be found through experimentation. 
The most commonly used media have b^ their basis the watery extract 
of meat and peptone. The addition to this by Koch of gelatin gave 
us a transparent solid medium which had, however, the objection of 
melting below the temperature required for 
''"'■ ^' the growth of many pathogenic bacteria. 

Another substance, of vegetable origin 
(agar), was found, which melted just below 
the boiling point of water. This has been 
substituted for gelatin whenever we desire 
to grow bacteria at temperatures above 
20° C. or desire other characteristics of the 
agar media. 

Preparation of Mett Intosion and Simple 
Botullon.— One pound (500 grams) of finely 
chopped, fresh, lean meat is macerated in 
Dry-beat ■teriiiier. 1000 c.c. of watcr and put in an ice-chest 

for from eighteen to twenty-four hours; 
or it may be warmed at a temperature not exceeding 60° C. for 
one hour. Any fat present is skimmed off. The last traces can 
be removed by stroking the surface with filter-paper. The infusion 
is now strained through a fine cheese-cloth into a flask, and the remain- 
ing meat placed in a cloth and squeezed by hand or in a press. The 
resulting fluid contains the soluble albumin, the soluble carbohydrates, 
the soluble salts, extractives, and coloring matter of the meat. This 
meat extract is then exposed to live steam, either without pressure in 
the Arnold steam sterilizer (Fig. 32) for thirty minutes, or, if the 
changes produced by a temperature of 110° to 115°C. are not objection- 
able, in the autoclave at a pressure of one atmosphere for fifteen 
minutes, or boiled over a free flame for ten minutes. During this 
process all the albumins are coagulated. While still hot the fluid is 
filtered through filter-paper or through absorbent cotton, and the 
reaction Is tested and sufficient normal hydrochloric acid solution or 
sodium hydroxide added to give it the desired reaction, which is for 
most bacteria slightly alkaline to litmus (1 per cent, acid to phenol- 
pthalein, the standard indicator).' If in the boiling there has been 
any evaporation, suflicient water is added to bring the fluid up to its 
original bulk. If the fluid is clear it is put into flasks and tubes and 
sterilized; if not clear, the white of one or two eggs beaten up in water 
(50 c.c. to an egg) is added to the fluid after cooling it down to about 
55° C. After thoroughly mixing with the eggs, the bouillon is boiled 
' The method of titration ia giveo later od p. 67. 



METHODS USED IN CULTIVATION OF BACTERIA. 



61 



Fia. 32 




STERILIZING CHAMBER 



briskly for a few minutes, its reaction adjusted, and then again filtered 
and distributed in flasks and put in the Arnold sterilizer for one hour on 
each of three consecutive days, or in the autoclave for twenty minutes 
for sterilization/ Instead of meat 2 to 4 grams of Liebig's or some 
other meat extract may be added to each litre of water. It is best to 
dissolve the extract in a small amount of cold water and filter through 
a cold wet filter-paper to remove the 
excess of fat which occurs frequently 
in certain meat extracts. For some 
purposes the extract is as good as the 
fresh meat, but for others it is inferior. 
This simple bouillon contains very 
little albuminous matter, and consists 
chiefly of the soluble salts of the 
muscle, certain extractives, and any 
slight traces of soluble proteid not 
coagulated by heat. It is not, there- 
fore, a suitable medium for most 
bacteria. 

We use this or the infusion as a basis 
for the following more useful media: 

Nutrient ftonillon Media. — These con- 
sist of meat infusion plus certain 
nutrient substances. 

(a) Peptone or Nutrient Bouillon, — 
This has the following composition: 
meat infusion, 1000 c.c; sodium 
chloride, 5 grams; peptone (Witte), 
10 grams. Warm moderately and 

stir until the ingredients are dissolved, then boil for thirty minutes 
in the Arnold sterilizer or the autoclave and treat as in making simple 
bouillon. For the careful study of bacteria the exact reaction of the 
media should be carefully determined. For this purpose standard 
solutions are used with phenolphthalein or litmus as an indicator. 
This subject will be taken up in detail later in this chapter. For 
water bacteria sodium chloride is omitted and the reaction is made 
■f 1 per cent. 

(6) Sugar-free Nutrient Bouillon. — A quantity of a culture of bacillus 
coli or of bacillus lactis aerogenes is added to the meat extract and 
incubated at 37*^ for twenty-four hours. The acidity is neutralized, 
peptone and salt added, and treated as described under bouillon. 

(c) Sugar Nutrient Bouillon, — ^To the sterile peptone broth from 
which, before its completion, the fermentable sugars have been removed 
1 to 2 per cent, of glucose, lactose, saccharose, or other sugar is added. 
No more boiling than necessary to sterilize should be used after the 
addition of the sugars, since they become altered by heat. Temper- 

* After heating the reaction may become more acid by the releasing of free H 
iona from the phosphates present. 




Arnold steam steriliser. 



62 PATHOGENIC MICRO-ORGANISMS, 

atures higher than 100° C. should never be employed. These media 
are used to determine the efiFect of bacteria upon the different sugars. 

(d) Glycerin-peptone Nutrient Bouillon. — After filtration, 3 to 5 
per cent, of glycerin is added to the peptone bouillon and the whole 
again sterilized. This medium is used especially for the growth of the 
tubercle baciUi. 

(e) Mannite-peptone Bouillon. — ^This is prepared by adding 1 per 
cent, mannite to the peptone bouillon. It is used especially in dif- 
ferentiating between the varieties of dysentery bacilli, some ferment- 
ing mannite and others not. In careful work the bouillon must be 
rendered sugar free. 

Bouillon for Production of Diphtheria Toxin. — This is now prepared 
as follows, in the Research Laboratory of the Health Department: 

The clean muscle of young veal, preferably " Bob veal," is chopped 
up and tap water added in the usual manner. This is allowed to 
ferment over night at room temperature, about 24° C. It is then di- 
gested for two hours at 55*^ C. The infusion is now boiled for thirty 
minutes. The boiled fluid is strained from the meat and receives 2 
per cent, peptone and ^ per cent. salt. The broth is then titrated 
at room temperature using phenolpthaline for an indicator. The 
first faint pink color is used to indicate the end of the reaction. 
Sufficient normal sodium hydrate is added to bring the acidity down 
to 1.2 per cent, normal acid. The broth is boiled again for twenty 
minutes and filtered clear. 

Gelatin Media. — These are simply the various bouillon and peptone 
media to which gelatin is added as follows: To the nutrient bouillon 
already prepared as described add 10 per cent, of sheet gelatin and 
neutralize. Add the whites of two eggs for each litre, and boil for a 
few minutes. Filter, place in tubes or flasks, and sterilize. After 
sterilization the gelatin should be placed at once in a cool place. This 
procedure prevents a further lowering of the original melting point. 
Instead of adding gelatin to bouillon already prepared, it may be 
added to the meat infusion at the same time the peptone and salt were 
added in preparing nutrient bouillon as just described. Different 
preparations of gelatin differ greatly as to their melting point. Boiling 
lowers the melting point, so that heat should not be applied any 
longer than necessary. The melting point of different samples of 
nutrient gelatin varies between 20° to 27° C. The "gold-label " gelatin 
is employed. 

Agar Media. — These are the various bouillon and peptone media 
to which 1 to 2 per cent, of agar-agar are added. When sugars are 
needed, in order to lessen the effect of heat on them, simple nutrient 
agar is first prepared and then the sugar added. Nutrient agar is 
prepared by adding to stock bouillon 1 to 2 per cent., as desired, of 
thread agar, melting it by placing over a free flame or in the auto- 
clave or steam sterilizer. When the agar is brought into solution 
over a free flame there may be considerable loss of fluid by evapora- 
tion. This should be compensated for by adding additional water 



■. 



METHODS USED IN CULTIVATION OF BACTERIA, 63 

before boiling. Agar may be added directly to the meat infusion 
along with the peptone and salt. Indeed, this is an advantage, as 
agar-agar is very diflScult to bring into solution, and is not injured in 
the least by prolonged boiling. The agar may be added to water 
alone in double the amount finally desired. To this is added an 
equal quantity of nutrient broth, which is also double its usual 
strength. Nutrient agar begins to thicken at a fairly high tempera- 
ture, and should be filtered as hot as possible. When small amounts 
are made it is well to place the filter and receiving flask in the steril- 
izer while filtering. 

Glycerin agar is simply nutrient agar plus 3 to 6 per cent, of gly- 
cerin. It is added to the hot nutrient agar just previous to putting it 
in the flasks. 

The following special media are also used in the cultivation of 
bacteria: 

Peptone Solution (Dunham's). — This is a simple 1 to 2 per cent, 
solution of peptone in tap or distilled water to which 0.5 per cent, 
of sodium chloride is added. The peptone and sodium chloride are 
dissolved by heating. The fluid is filtered, placed in tubes, and ster- 
ilized. A reaction slightly alkaline to litmus is suitable for most pur- 
poses. It can be altered or standardized if desired. 

Sugar-peptone Soltdiofiy etc, — The various sugars and mannite, inulin, 
glycerin, etc.,* are added to the peptone solution just as previously 
described for bouillon. 

Milk.— This fluid is a good culture medium for most pathogenic 
bacteria. It should be obtained as fresh as possible, so that but little 
bacterial change has occurred. It is first put in the ice-chest for twelve 
hours to allow the cream to rise. The milk is then siphoned ofiF from 
below the cream into a flask and its reaction tested. After correction 
it is put in tubes or flasks and sterilized. If acid to phenolphthalein, 
normal sodium hydrate should be added to make it — 1 per cent. 

litmus Media. — When it is desirable to determine whether bacteria 
produce in their growth acid or alkali from one or more of the con- 
stituents of the media, litmus is frequently added. To prepare the 
litmus solution take Merck's purified litmus, powder finely, and make 
a 5 per cent, solution in distilled water. Steam this in Arnold's 
sterilizer for two hours, shaking frequently. Filter and then boil for 
thirty minutes on two successive days. The litmus solution is added 
to the neutral media in sufficient quantity to give the desired depth 
of color. The less heating that is done after mixing the better the 
results. 

Petruflky'B litmus Whey (as modified by Durham). — ^Fresh milk is 
slightly warmed and clotted by means of essence of rennet. The 
whey is strained ofiF and the clot is hung up to drain in a piece of muslin. 
The whey, which is somewhat turbid, is then cautiously neutralized with 
4 per cent, citric acid solution, neutral litmus being used as an indicator. 
When it gives a good neutral violet color with the litmus it is heated at 
100° C. for one hour; thereby nearly the whole proteid is coagulated. 



64 PATHOGENIC MICRO-ORGANISMS, 

It is thus filtered clear, and neutral litmus is added to a convenient 
color for observation. 

Neutral Red. — ^This dye is added to the peptone and bouillon-sugar 
media to the amount of 1 to 5 per cent, of a concentrated solution. 
Its reduction by the growth of bacteria is a valuable point in differentia- 
tion in certain cases. 

Nitrate Bouillon. — Dissolve 10 grams of peptone in 1 litre of spring 
or tap water and add 0.02 gram of potassium nitrate (which is free of 
nitrites). This is placed in test-tubes and sterilized. 

Potatoes. — ^Potatoes are used for some special purposes. The pota- 
toes may, after thorough scrubbing and removal of **eyes," be soaked 
in bichloride of mercury (1 : 1000) for twenty minutes, placed in running 
water twenty-four hours to prevent darkening, and then sterilized on 
three consecutive days for one-half hour in the steam sterilizer. To use 
they are cut in thick slices and put in deep Petri dishes. When desired 
the potatoes are first cut into proper sizes for tubes, and then soaked for 
twelve hours in one per cent, sodium carbonate solution to remove the 
acidity. 

Bile. — ^Fresh bile of cattle is sterilized and used without additions or 
to it is added 1 per cent, of peptone or again 10 per cent, of peptone and 
10 per cent, of glycerin. The bile inhibits the coagulation of blood and 
also the development of many varieties of bacteria. The bacilli of 
the colon-typhoid growth are, however, Uttle affected. This medium 
is used especially for obtaining the typhoid bacillus from the blood 
and from water, and the colon bacillus from polluted water. 

Blood Media. — (a) Fresh Shod Media, — ^These are made by streak- 
ing sterile defibrinated or fresh human, rabbit, or other blood over 
nutrient agar contained in tubes or dishes. Sometimes fresh blood is 
added to fluid nutrient agar at 40° C. or to bouillon and a mixture thus 
obtained. Media made with fresh blood contains not only the haemo- 
globin, but also intact red blood cells. Blood media are used for the 
growth of the influenza bacillus, for pneumococci and other bacteria, 
and for the observation of the production of hemolysis by the growth 
of certain bacteria. 

(6) Heated Blood Media, — ^The clot containing the red cells, after 
the separation of the serum, is broken up and added to the bouillon 
and heated to 80° to 90° C. This makes a muddy fluid which is 
fitted only for the development of bacteria where no exact observation 
of their growth characteristics is required. 

Blood -serum Media. Ascitic or Pleuritic Fluid. — Blood serum may 
be sterilized by fractional sterilization and remain fluid, or it may be 
rendered solid by the degree of heat used in sterilizing. The blood may 
be obtained from an ox, horse, sheep, dog, or rabbit and collected into 
jars, flasks, or tubes, where it is allowed to stand until it clots. When 
the serum is to be used in a fluid state the blood should be drawn in an 
aseptic manner into a flask from a vein by means of a sterile cannula 
and rubber tube. When the serum is to be solidified, less care is 
necessary. It is here sufficient to catch the blood from the cut artery 
or vein into sterile jars or tubes. To facilitate clotting it is well to 



METHODS USED IN CULTIVATION OF BACTERIA. 65 

have in the jar or tube something upon which the clot may contract, 
such as nickel-plated wire or broken glass. 

Loeffier'g Blood Serum. — Three parts of calf's or sheep's blood serum 
is mixed with one part of neutral peptone bouillon containing 1 per cent, 
of glucose. The serum mixture is run into tubes, which are plugged 
and then placed in a slanting position in the serum coagulator. 

Serum may be solidified and still remain translucent at a temperature 
of 76° C, but when heated to a higher degree a more definite coagu- 
lation takes place, and the medium becomes opaque. Care must be 
taken in coagulating blood serum at the higher temperature to run the 
temperature up slowly, and not to heat above 95° C. until the serum 
has firmly coagulated ; for, unless 
these precautions are taken, ^°- ^ 

ebullition is hkely to occur, 
which will lead to the formation 
of bubbles and an unevenness of 
the surface upon which growth 
is to be obtained and studied. 
Serum may be solidified at the 
temperature mentioned in an in- 
cubator, water-oven, or even in 
an Arnold stenlizer with the top 

covered by a cloth instead of the Biood^Mmm concuiator. 

usual lid, and when coagulated 

firmly (90° C.) the tubes and their contents may, on the following day, 
be sterilized in streaming steam at 100° C. without danger of the sub- 
sequent formation of bubbles. Koch's serum coagulator (Fig. 33) 
is, however, the most convenient apparatus. A modification of this 
which we made is very useful. The water holder is 10 inches high 
and into it are built three boxes having the proper slant, and open- 
ing in front. Each compartment has a cover. The serum-holding 
tubes are inserted in the boxes. In this way the warm water is above 
as well as below, so that the heating is uniform. Some bacteriologists 
prepare the tubes of solidified serum in the autoclave, gradually in- 
creasing the temperature to 110° C. This is a very rapid and conven- 
ient method. It has seemed to us, however, that the high temperature 
injured the medium somewhat. 

Alkaline Blood Senim. — To each 100 c.c. of blood serum add 1 to 
l.Sc.c. of a 10 percent, solution of sodium hydrate. Treat asLoeffler's 
serum. This will give a solid, clear medium consisting chiefly of 
alkali albuminate. 

Serum-bouillon Media (Marmorek's Media): 

1. Human serum, 2 parts; nutrient bouillon, 1 part. 

2. Ascitic or pleuritic fluid, I part; nutrient bouillon, 2 parts. 

3. Horse serum, 1 to 2 parts; nutrient bouillon, I to 2 parts. 
These media were first used extensively by Marmorek in cultivating 

streptococci. The ascitic fluid bouillon has been found by Williams 
to he of great use in enriching cultures of diphtheria bacilli. It is 



66 PATHOGENIC MICRO-ORGANISMS, 

also one of the best media for the growth of pneumococci, streptococci, 
and many other pathogenic bacteria. 

Seruwrwater Media (Hiss* Serum Media). — When diluted with 2 
to 10 parts of water, many sera can be steamed without coagulating. 

1. Ox serum, 1 part; distilled water, 2 parts; normal sodium hydrate, 
. 1 per cent. 

2. The same, with inulin 1 per cent, substituted for the sodium 
hydrate. 

• For the sterilization of undiluted fluid serum and of ascitic and 
pleuritic fluids, it is requisite that they be exposed to a temperature of 
from 62^ to 66° C. for one hour on each of six consecutive davs. The 
best apparatus for obtaining and maintaining this temperature (about 
65° C.) is a small and well-regulated incubator or chamber surrounded 
by a water space, into which the tubes and flasks containing serum 
are to be put each day, and in which they are to be left for the pre- 
scribed time after having been warmed to the desired temperature. 

Serum may be preserved by placing it in flasks which, after the 
addition of 5 per cent, of chloroform, are sealed. When it is to be 
used it is poured into sterilized culture (test) tubes and sterilized by 
exactly the same methods as are employed in sterilizing fresh serum. 
The chloroform, being volatile, tends to disappear at ordinary tem- 
peratures, but is quickly and surely driven off at the temperatures 
used in sterilizing. 

Serum may be efficiently sterilized, when great care is used, by 
passing it through a well-tested Pasteur filter, under pressure. WTben 
so treated the fluid is very clear and light colored. The first few cubic 
centimetres are deficient in blood proteids because of adhesion to 
the filter. 

Important media used for special varieties of bacteria will be noted 
in the chapters devoted to these bacteria. 

Reaction of Culture Media. — ^The reaction of media is a matter of 
the greatest importance, since slight variations will often aid or in- 
hibit the growth of bacteria and also produce marked differences 
in the microscopic and macroscopic characters of a growth. 

Formerly it was customary to use litmus as the indicator in neu- 
tralizing media, adding normal soda solution or hydrochloric acid 
solution until the red litmus turned blue, or the blue litmus just a 
tinge less blue. This was considered the neutral point. This 
method is still a satisfactory one for those who are only going to cul- 
tivate the common pathogenic bacteria for diagnostic purposes or for 
the routine development of toxin. Most parasitic bacteria which 
grow at all on artificial culture media develop best in them when 
they have a neutral or slightly alkaline reaction to litmus. If a cer- 
tain alkalinitv is desired a definite number of cubic centimetres of 
normal soda solution can be added for each litre of neutral media; 
if an acidity is desired, normal hydrochloric acid solution is added. 

Many bacteriologists consider that litmus is not delicate enough 
to be entirely satisfactory, especially when experiments are to be 



METHODS USED IN CULTIVATION OF BACTERIA. 67 

reported or exactly repeated. This objection is made chiefly by those 
investigating water bacteria who are watching cultural and bio- 
chemic characteristics in simple peptone-beef media. For these pur- 
poses phenolphthalein has been generally selected. It is of great 
importance to remember that different indicators not only differ in 
delicacy, but that they react differently to different substances. A 
medium which is slightly alkaline to litmus is usually slightly acid to 
phenolphthalein, showing that there are present in such media sub- 
stances possessing an acid character which litmus does not detect. 
These substances are weak organic acids and organic compounds, theo- 
retically amphoteric, but in which an acid character predominates. 
Thus, a litre of bouillon becomes, on the addition of 1 per cent, of 
peptone, more alkaline to litmus, but decidedly more acid to phenol- 
phthalein; 100 c.c. of water with 1 per cent, of peptone is acid to phenol- 
phthalein to such an extent that about 3.5 c.c. of decinormal NaOH 
is required to neutralize it. To litmus it is alkaline and requires 3 . 4 
c.c. of decinormal HCl. Two per oent. of peptone doubles the dif- 
ference. The same figures hold approximately true for peptone broth. 
We should find by growing the bacteria just what reaction we want for 
any variety, and then test the fluid with phenolphthalein or litmus as 
the indicator. With precisely similar ingredients we can then exactly 
reproduce at any time in the future the same reaction, but with dif- 
ferent materials one would again have to study the reaction. 

Titration of Culture Medki. — We must have accurately standard- 
ized normal and decinormal solutions of sodium hydrate and hydro- 
chloric acid; also a 0.5 per cent, solution of phenolphthalein in 30 
per cent, alcohol and a neutral 1 per cent, solution of Merck's litmus. 
Care should be taken to prevent the absorption of carbon .dioxide 
by the soda solution, by arranging that all air which comes in contact 
with the latter, either in the stock bottle or in the burette, shall first 
pass through a strong solution of sodium or borium hydrate. The 
arrangement of the apparatus is described in any work on chemical 
analysis. The medium is brought to the desired volume with water 
and boiled four minutes to expel the carbon dioxide. Media are com- 
monly warm or hot when measured, hence it must be remembered 
that true volumes cannot be thus obtained; for instance, a litre meas- 
ured at, say, 80° C. would be only 973 c.c. if measured at 20° C, 
the temperature at which litre flasks are calibrated. Since many 
media cannot be cooled to 20° C. because of solidification, as in the 
case of agar or gelatin, it is a better plan when accuracy is' important 
to determine measures of volume by weight. For this, place a clean, 
dry saucepan, in which the medium is to be prepared, upon one side 
of a trip scale, and counterbalance its weight exactly. The weight 
of a Utre of bouillon, gelatin, or agar having been determined once 
for all, the necessary weights added Jto the weight of the pan will 
give the amount which the pan and its contents must balance when 
the volume is exactly one litre. A portion of the medium brought 
to the exact volume is then taken and cooled to room temperature 



68 PATHOGENIC MICRO-ORGANISMS. 

(20° C), or to a point a few degrees above solidification, and 10 
c.c. withdrawn, placed in a small beaker, 50 c.c. of distilled water 
and 1 c.c. of the phenolphthalein solution added. If the medium is 
acid the j^ NaOH solution is then run in cautiously until a pale 
but decided pink color is obtained. The number of cubic centimetres 
of the solution used, multiplied by ten, will ^ve the number of cubic 
centimetres of normal sodium hydrate per litre necessary to effect 
complete neutralization. The question as to what is the best reac- 
tion of media for general work is not an easy one to settle, and one 
on which bacteriologists differ. What is the proper reaction for one 
variety of bacteria is often far from the best for some other variety. 
Reactions are now commonly expressed by plus or minus signs, the 
former representing an acid and the latter an alkaline condition, the 
number following the sign representing the percentage of normal acid 
or alkali present in the medium. Thus, +1.5 would indicate that 
the medium contained 1 .5 parts per 100 or 1 .5 per cent, of free nor- 




LoDg-decked fluk. Pai 

mal acid, while —1 5 would indicate that the medium contained an 
equivalent quantity of free alkali. The committee of the American 
Public Health Associatmn in 1898 adopted for nutrient bouillon or agar 
a reaction of + 1 5 as the best for general work in water examinations. 
In 1905 this was changed to +1.0percent. A medium whose reaction 
is +0.5 per cent, acid to phenolphthalein is still better adapted for 
many bacteria. It cannot be too strongly impressed upon the reader 
that whatever the reaction, its measure should be stated in all descrip- 
tions of cultural characters. The Htmus solution is added in the 
same way As that of phenolphthalein. 

Storag^e of Media. — The nutrient media are stored in glass flasks 
(Figs. 34 and 35). From these, as needed, glass tubes are filled. When 
small amounts of media are taken frequently from flasks, Pasteur's 
flasks (Fig- 36) are of great convenience. They consist of a flask with 
a ground-glass neck, over which fits a cap. This cap may or may not 
terminate, as desired, in a narrow tube, which is plugged with cotton. 
The cap keeps the edges of the flask free from bacteria and prevents 
the cotton from sticking. A tumbler or a simple cap of paper over the 



METHODS USED IN CULTIVATION OF BACTERIA, 69 

neck answers much the same purpose. Stock media, unless protected 
from drying by sealing, should be kept in a cool moist place until 
needed. 

Preparation and Filling of Tubes. — ^The cheaper grades of test-tubes 
should be avoided. They are thin and break easily, and also fre- 
quently frost on heating, from the separation of silicic acid. The tubes 
of the better class can be used after rinsing with hot water; they should 
have no lip. Cheap tubes are very alkaline and must first be soaked 
in dilute hydrochloric acid. The sterilization of glassware has al- 
ready been spoken of (p. 59). 

The sterile tubes and flasks are filled with the media, when small 
quantities are used, by means of a sterile glass funnel. The main pre- 
caution to be observed is not to let the media soil the neck of the 
tubes and flasks, as this would cause the fibres of the cotton plugs to 
adhere to the sides of the tubes when the media dried, and make it 
difficult to remove the plugs wholly when we wished to inoculate the 
contents of the tubes. 

The tubes and flasks, plugged with sterile cotton and containing 
media, are sterilized by fractional sterilization at 100° C. for one-half 
hour on three consecutive days; or they may be sterilized by steam 
under pressure (in autoclave for fifteen minutes) on two consecutive 
days. A portion of the tubes containing nutrient agar are laid in a 
slanted position before cooling, after the final sterilization, so that a 
larger surface may be obtained. 

THE CULTIVATION OF BACTERIA. 

Bacteria can seldom be identified by their microscopic and staining 
characteristics alone. By these methods only their individual forms, 
arrangement, and motility or lack of motility can be studied. To go 
beyond this we have to grow the microorganism in pure culture on 
the various culture media and perhaps also in animals. It is neces- 
sary, as well, to have the proper conditions as to temperature, mois- 
ture, access of oxygen, etc. 

When we make cultures from any material, we are very apt to find 
that instead of one variety of bacteria only there are a number present. 
If such material is placed in fluid media contained in test-tubes, we 
find that the different varieties all grow together and become hopelessly 
mixed. When, on the other hand, the bacteria are scattered over or 
through solid media they develop about the spot where they happen 
to light, forming small colonies each composed of a single variety of 
organism. If different varieties, however, are placed too near together, 
they overgrow one another; it is thus advisable to have a greater surface 
of nutrient material than is given on the slanted surface of nutrient 
agar or blood-serum contained in test-tubes. This need is met by 
pouring the media while warm on flat, cool, glass plates or into shallow 
dishes. From the isolated colonies thus formed new growths may be 
obtained of a single variety, and thus we have a pure culture (see p. 75). 



70 PATHOGENIC MICRO-ORGANISMS, 

Technique of Making Plate Onltures. — In making plate cultures 
two methods are carried out. In the first the material with its contained 
bacteria is scattered throughout the fluid before it hardens; in the 
second it is streaked over the surface of the medium after that has so- 
lidified. Nutrient agar and nutrient gelatin, the two substances used 
for plate cultures, differ in two essential points, which cause some differ- 
ence in their uses. Nutrient 1 per cent, agar melts, near the boiling 
point and begins to thicken at about 36^ C. It is not liquefied by 
bacterial ferments. Nutrient 10 per cent, gelatin melts, according to 
the variety used, at the low temperature of about 23° to 27° C, and 
solidifies at a point slightly below that. It is liquefied by many 
bacterial ferments. When we wish to inoculate fluid nutrient agar 
for plate cultures we have to take great care that in cooling it to a 
point which will not injure the bacteria, about 41° C, we do not allow 
it to cool too much and thus solidify and prevent our pouring it into 
the plates. The correct way to proceed when a number of tubes are 
to be inoculated, to place them while still hot in a basin of water which 
has been heated to about 45° C. Then when the temperature of the 
agar in the tubes as shown by a thermometer placed in one of them, 
has fallen to 42° C, the water, milk, feces, bacterial culture, or other 
substance to be tested is added to the other tubes or placed in the dishes 
in whatever quantity is thought to be proper up to 1 c.c. A greater 
quantity of fluid would dilute and cool the nutrient agar too much. 
After inoculation, the contents of the tubes are thoroughly shaken and 
poured out quickly into round, flat-bottomed, glass Petri dishes (Pig. 
37), the covers of which are raised on one side for the required time 
only. Instead of placing the fluid containing the bacteria in the tube it 
is often placed directly in the Petri dish. In this case no bacteria are 
Fio 37 ^^'* *" *^^ medium sticking to the tube from which 

it is poured, and hence organisms lost. The melted 

nutrient gelatin or agar is then poured in the dish, 

and by gently tipping the fluids are mixed. It is 

^^^^ somewhat more diflScult to scatter the bacteria 

^P^^"^^ evenly when they are mixed with the media in 

plates rather than in tubes so that there is little 
to choose in point of accuracy between the two methods. The 
bacteria are now scattered throughout the fluid, and as it quickly 
solidifies they are fixed wherever they happen to be, and thus, as each 
individual multiplies, clusters are formed about it at the spot where it 
was fixed at the moment of solidification. The number of colonies 
of bacteria thus indicate to us roughly the number of living bacteria 
in the quantity of fluid added to the liquid gelatin (Fig. 38) or agar. 
Groups or chains of bacteria which in spite of shaking remain at- 
tached produce single colonies. Bacteria which do not grow on the 
media or at the temperatures employed produce of course no colonies. 
Nutrient gelatin is used exactly as agar, except that as the average 
product does not congeal until cooled below 22° C. we have no fear 
of its cooling too rapidly. 




METHODS USED Iff CULTIVATION OF BACTERIA. 71 

In order not only to count the number of colonies and to obtain 
a characteristic growth, but also to prevent the inhibition of the 
growth of some and the fusing of others, it is desirable not to inoculate 
the nutrient agar or gelatin to be poured in one plate_with too large a 
number of bacteria. We therefore use the following dilution methods 
in making culture plates of suspected material. 

DUation Methods. — As it is impossible to know the number of bac- 
teria in any suspected fluid, it is usual to make a set of from two to four 
different plates, to each of which a different amount of material is added, 
so that some one of the series may have the required number of colonies. 
The dilutions are made in bouillon or sterile distilled water. In the 



Pbotocrmph of m Urge aumber of colonial developiu in a lfty«r of gelAtJn roDl4un«d Id a Petri 
duh, 8«&s ooloniea are only pinpoiDtio uie; some selaiie as the eDiT of ■ pencil. The coloaia 
brn appear in tbair ulual iiu. 

first tube we place an amount which we believe will surely contain 
sufficient and probably too many bacteria. To the second tube we 
add 10 per cent, of the amount added to the first, and to the third 10 
percent.of the second, and to the fourth lOpercent. of the third. Thus, 
if the first contained 60.000 bacteria the second would have 6000 (Fig. 
38), the third 600, and the fourth 60 (Fig. 39). If, however, the first 
contained but sixty, the second would have about 6, and the remaining 
two would probably contain none at all. When there are many colon- 
ies present the dishes are covered by a glass plate (Fig. 40), ruled in 
larger and smaller squares, Wolffhugel's apparatus. With the eye 
or when necessary aided by a hand tens the colonies in a certain num- 
ber of squares are counted and then the number for the whole contents 
estimate. It is verj- important to remember that when more than 
200 or 300 bacteria start to develop in the agar or gelatin contained in 
a plate some develop colonies which fu.se together, while others are 
inhibited before they develop visible colonies. Thus if si.xty thousand 



72 PATHOGENIC MICRO-ORGANISMS. 

separated bacteria were placed in the agar of one dish tbey would 
probably not produce over ten thousand colonies, while one-tenth as 
much would produce about three thousand and one one-hundredth as 
much would produce about five hundred. Unless this effect of over- 
crowding is taken into account gross inaccuracies will occur in esti- 
mating the number of bacteria present in the material from which the 
plates were made. If possible, dilutions should be made so that plates 
will contain between forty and four hundred colonies. It is often 
advisable to examine the material to be tested in hanging drop and 
stained spreads under the microscope in order to determine roughly 
the number of bacteria present and so decide what dilutions to make. 
When the material to be tested is crowded with bacteria it is often 



Well-diitributed coloDita in agmr in WolShfisel'a Bppanitiu (or counling coloDiss. 

portion oF Petri dish. 

best to make an emulsion of a portion of it, and use this rather than 
the original substance for making the dilutions to be used. Meas- 
ured quantities of the diluted material can be transferred most accu- 
rately through a sterilized, long, glass pipette graduated in one one- 
hundredth cubic centimetres, or, more roughly, by a platinum loop of 
known size. 

Streaked Surface Plate Cnltures.— About 8 c.c. of agar-agar are 
poured into a Petri dish and allowed to harden. The substance to 
be tested bacteriologically, or a dilution of it, is then drawn lightly 
across the surface of the medium in a series of parallel streaks by 
means of a platinum loop. Each successive streak is made with the 
same needle or loop without replenishing the material to be tested. 
Each streak will therefore leave le.ss deposit of bacteria and fewer 
colonies will develop. While in the former method (poured plate) 
most of the bacteria developed under the surface, here all develop 
upon it. This is an advantage, as many forms of bacteria develop 
more characteristically on the surface than in the midst of the media, 
and it is easier to remove them free from other bacteria with the 
platinum needle. Instead of streaking the material by means of the 
platinum wire over the agar, a loopful may be deposited on the agar 
and then smeared over its surface by a sterile swab or a glass rod bent 



METHODS USED IN CULTIVATION OF BACTERIA. 73 

SO that the last two inches strokes the plate horizontally. The old 
method of using glass plates upon a coohng stage (Koch's method) 
has now been practically given up for the more convenient one of 
Petri dishes. In warm weather the dishes may be cooled before using, 
so as to harden quickly the agar or gelatin that is poured into them. 
An old method, which is still sometimes used to find the number 



of hving bacteria, is, instead of pouring out the media which has 
been inoculated, to congeal it on the sides of the test-tubes. This 
is best done by laying the tube flat on its side on a cake of ice and 
rotating it. Tubes come especially formed for this by having a slight 
neck, which prevents the media running up to the plugged end of the 
tube. This method (Esmarch's) is used only when the Petri dishes 
are'not obtainable or cannot easily be transported. 



Flo^ 44. — Od« Large irTp«uLar colony of colon and (wo flmaller colonics of typhoid badlli in soft 
crLatia. (Pi(i, 42-44 fram photographs by Dunham. I 

Study of Oolonies in Plate Cultures in Nutrient Agar. — The plates 
should be studied after twelve to forty-eight hours' growth at blood 
temperature and after two to five days at 70° F. {21" C). The special 
time allowed varies according to the rapidity of the growth of the varie- 
ties developing; thus, bacteria, such as the streptococci and influenza 



74 PATHOGENIC MICRO-ORGANISMS. 

bacilli, reach the charactenstic development of thdr colonies in from 
ten to sixteen hours, while others continue to spread for several days. 
If we wait too long where numerous varieties of bacteria are growing 
the colonies of heavier growth may cover up the finer and more delicate 



marldDg- (Fisi. 4S-5S 



Moiat mined coloni« with no vi 

'Deep colonifls^ UBually atber Uiht brown, ^ 



Fia. 47.— The colony 1 




in color, opaque, willi UtUe 



lighter borden. The maisin la co 
ia Hwreely imiular in putly I 




form, compoiieil of 
etwork of threnda 



Ihin border fhn 



ones. As a rule, the younger colonies are more characteristic, except 
where the development of pigment is sought. 

The colonies are first examined with the naked eye (Fig. 39), then 
with magnification of about 60 diameters (Figs. 4! to 52), and then, 



METHODS USED LV CULTIVATION OF BACTERIA. 75 

if necessary, at from 400 to 500 diameters (Fig. 53), We note every- 
thing we can about them, such as their size, surface elevation, form, 
internal structure, edges, and optical characters; if grown in gelatin, 
whether they have or have not caused liquefaction. The accompany- 
ing schematic representations from Lehman and Neumann (Figs. 
45 to 52) illustrate some of these points. 

At the higher magnification we begin to detect the individual bac- 
teria (Fig. 53). After studying the colonies we remove a few of 
the bacteria from one or more of them by touching each with the 



>loai« of diphdwris bacilli upon 
1* ^ (nn ^£*m*tAii. 



X MO diuncten. 



tip of a sterile platinum needle (Fig. 54), and thus transfer them to 
a cover-glass for microscopic examination, or to new media where they 
may develop in pure cultures and show their growth characteristics. 

In using nutrient gelatin one must always remember not to allow 
it to stay where the temperature is over 22° C, for if that happens 
the media, as a rule, will melt; nor must the liquefying colonies be 
allowed to grow for too long a time, or the entire media will become 
fluid. 

Pure OnltoreB. — If bacteria from a colony formed from a single or- 
ganism are transferred without contamination to new media, and these 
grow, we have what is known as a pure culture of that variety. When 
these are transferred to the solid media we call the growth which takes 
place from smearing the bacteria over the surface a surface or smear 



76 PATHOGENIC MICRO-ORGANISMS. 

culture, and that formed in the depth of the media by plunging the 
needle carrying the bacteria into it a stab culture (Figs. 55 and 56). 

Wliile transferring bacteria from one tube to another we slant the 
tubes so that no dust may fall within and contaminate with other 
p,^ jj bacteria the special variety we wish 

to transplant. The greatest care must 
be taken that the sterilized platinum 
needle used to transfer the bacteria 
is not infected by touching any non- 
sterile matter. The upper rim of 
culture tubea should be passed through 
the flame so as to destroy any bacteria 
resting there. Even with our utmost 
care bacteria will from time to time 
pass from the air or edges of our 
tubes into the culture media, and thus 
the possibility of contamination must 
always be kept in mind. When this 
sub cuUq™ of thiH cholera .piriiia id occufs upoo solid media we, as a rule, 
?o!liVderaKS^i"«5SSX';Jin"nt«*e'SdS' ^^^lly detect it, for we notice at some 
point the growth of bacteria of differ- 
ent colony characteristics; but in fluid media, on account of the 
complete mingling of the bacteria, we are not so apt to notice the 
additional growth. 

The Study of Pure Cultures in Tubed Media. — A few points 
of the many which should be observed are the following: 
Gelatin stab cultures. 

A. Non-liquefying. 
Line of puncture. 

Filiform, uniform growth, without special characters. 
Beaded, consisting of loosely placed, disjointed colonies. 
Arborescent, branched, or tree-like. 
Some of these points are illustrated in Fig. 56, sketched by 
Chester. 

B. Liquefying. 

Crateriform, a saucer-shaped liquefaction of the gelatin. 
Saccate, shape of an elongated sac, tubular (Rg. 55). 
Statiform, liquefaction extending to the walls of the tube. 

Nutrient agar tube cultures give fewer points for observation, but 
should be studied in the same way. The agar in the tubes is usually 
slanted and the culture growth is not only in the stab, but along the 
streaked surface. The characteristics of each should be noticed. 

Opposite page 94 is appended the chart devised by a committee of the 
Society of American Bacteriologists comprising a set of rules and of 
descriptive terms to be used in giving a complete description of a bac- 
terium. The chief advantage of using such a chart in whole or in part 
is that the observations of different workers may be cleariy compared. 



METHODS USED IN CULTIVATION OF BACTERIA. 



77 



Apparatus for Obtaining a Suitable Temperature for the Growth 
of Bacteria. — Incubators. — In order to have a constant and proper 
temperature for the growth of bacteria, forms of apparatus called 
incubators have been devised. These consist, in their simplest form, 
of an inner air chamber surrounded by a double copper wall contain- 
ing water (Fig. 57). The apparatus externally is lined with asbestos, 
to prevent radiation. It is supplied with doors and with openings for 
thermometers and a thermoregulator. The thermoregulators are of 



B 




TcQ 



2 



\J 




Fio. 5e 

T 
1 



/g''?^ 




\y 






c::^ 



^^^L 




^lowinc charactere of gelatin stab cultures: A . Characters of surface elevation : 1 , flat; 2, raised ; 
3. convex- 4, pulvinate; 5, capitate; 6, umbilicate; 7, umbonate. B. Characters of growth in 
depth: 1. filiform; 2, beaded; 3, tuberculate-ecinulate; 4, arborescent; 5, villous. (From Chester.) 



various kinds; those in most use depend upon the expansion or con- 
traction of the fluid in the bulb A (Fig. 58), which rests within the 
water-jacket, to lessen or increase the space between the surface of the 
mercury B and the inner tube D, thus allowing of the passage of a 
greater or less quantity of gas to the burner through the tube D. 
Other forms are used in very large incubators or in incubator rooms. 
These usually depend upon the contraction or expansion of metal, 
or the use of the electric current to control the flow of the gas. 

The temperature in the air chamber is kept above that of the sur- 
rounding air by means of a gas flame regulated as above described, or, 
when that cannot be obtained, a lamp. 

When temperatures lower than that of the surrounding air are 
wanted, heat is reduced by passing a stream of cool water through the 
water chamber, which is itself regulated. When very accurate 
investigations are to be made a gas-pressure regulator is added to the 



78 PATHOGENIC MICRO-ORGANISMS. 

thermoregulator. Incubators may be also both warmed and regulated 
by electricity. 

In emergencies, a culture may be developed at the blood temperature 
by placing it in a closed jar or bottle which is placed in a larger vessel 
filled with water heated at 38° C. By adding a little hot water from 
time to time to the outer vessel the temperature can readily be kept 
between 34" and 38° C, which is sufficiently uniform for bacteria such 
as the diphtheria bacilli to grow. A Thermos bottle will answer the 
purpose. As a temporary expedient during the night, when haste is 
necessary, it is possible, when the culture medium is solid and within a 




strong glass lube or metal case, to make use of the body heat by putting 
it under the clothing next to the body or sleeping uf>on it. Naturally, 
this should only be done when other means fail. 

MeUiods for Obtaining Anaerobic Conditions (or Bacteria.— 
Pasteur excluded the oxygen by pouring a layer of oil on the culture 
fluid. A simple device is that of Koch, who placed a thin strip of 
sterile mica upon the agar or gelatin while still fluid in the Petri dish, 
after inoculation. After the solidification of the media the portion 
under the mica is excluded from the air and anaerobic growth can 
develop. 

A second simple method (Liborius) is to fill the tubes with media 
fuller than usual and to inoculate the bacteria deep down to near 
the bottom of the tubes while the media are still semisolid. An 
anaerobic growth will take place in the lower part of the tube. In 
a similar way the clo.sed arm of the fermentation tube will suffice for 
anaerobic growth, if the opening connecting it with the open bulb is 



METHODS USED IN CULTIVATION OP BACTERIA. 79 

quite small and the medium has been freshly heated to expel any 
dissolved oxygen. Wright devised the following procedure: A 
short glass tube with constricted ends is used. 
Each end has a piece of rubber .tubing attached. ^"'" *" 

One of these is connected with a glass tube, 
which projects through the cotton plug of the 
test-tube. The test-tube contains bouillon. The 
whole is sterilized and then the test-tube inocu- 
lated. The bouillon is then drawn up into the 
constricted tube, which is sealed by simply push- 
ing down the tube so that both rubber ends are 
sealed by being bent 
'"■ ™ on themselves. When 

spores are present, a 
simple method sug- 
gested, I believe, by 
McFarland, can be 
successfully em- 
ployed. Vessels 
plugged with stoppers 
perforated by gla^s 
tubes drawn to a point 
are filled to such a 
height that when the 

NoYy i«r (or anaiiobie culMina. fluid is heated tO 80° 

C. it will just fillthem. 
They are inoculated when the bouillon is at 
about 60° C, heated to 80° C, and then sealed 
by closing the tube's point by means of a flame. 
After inoculating and heating, instead of sealing 
the glass tube a sterile rubber cork can be 
inserted. 

If much fermentation is expected, the cork 
should be clamped or tied to the bottle, so that 
it will not blow out. One advantage of this 
method is that any contaminating organisms 
which have no spores will be killed. 

When sealed the bottles should be cooled and 
then placed in the incubator. 

A very convenient modification of Pasteur's 
method for the growth of bacteria in fluid media Buchoer'Bsnikembiciube, 
is to cover the fluid with albolene or paraffin. ^iu.SXl^"^ £"Io 
In boiling, all the oxygen is driven out. We S^Jb™*,' ^!h^'','i^°iuhii 
prepare all our tetanus toxins in this way: Litre "^ charged •■ith piewo of 

■: \ „,, , , 1 - T 1 .»» cauBlic poioflh covervd with 

flasks are nlled to near the neck with bouillon, pyrogailic acid. 
This is covered with a one-half inch layp r of albo- 
lene mixed with sufficient paraffin to yield a nearly solid substance at 
37° C The bouillon after boiling is quickly cooled by setting the 



80 PATHOGENIC MICRO-ORGANISMS. 

flask containing it in a shallow layer of cool water, so as to lower the 
temperature of the lower portion of the bouillon to 40° C. or under, 
while leaving the paraflSn on the surface still fluid. While in this con- 
dition it is inoculated with a spore-bearing tetanus culture. Bits of 
tissue suspected to contain tetanus bacilli may be dropped into smaller 
flasks filled and prepared in the same way. 

Displacement of Air. — In the more complicated methods the plates 
or tubes are placed in jars of a type devised by Novy (Fig. 59), in which 
the oxgyen is displaced by a stream of hydrogen developed by the 
Kipp apparatus, through the action of pure granulated zinc and a 
25 per cent, solution of pure sulphuric acid. When all the oxygen 
has been displaced the jars are sealed by rotating the stopper. 

Absorption of Oxygen. — In another .method the oxygen is ex- 
tracted by a mixture of pyrogallic acid and caustic potash. To each 
100 c.c. of air space in the jar 1 gram of pyrogallic acid and 10 c.c. of 6 
per cent, solution of potassium hydroxide are added and the jars 
immediately sealed. A very simple modification has been described 
by Wilson. In a large test-tube a small piece of solid caustic potash 
is placed and over this powdered pyrogallic acid is poured. This is 
stored until wanted. A smaller culture tube with the desired me- 
dium is inoculated. Water is now added to the large test-tube, which 
works its way slowly through the pyrogallic acid. The small tube is 
quickly inserted and the whole sealed by water or a rubber cork (Fig. 
60). Solid culture media in test-tubes can be inverted over the acid 
soda mixture, which is then covered by a layer of albolene to prevent 
the absorption of oxygen from the air. The displacement method is 
often used along with that of absorption. 

AssociAtED WITH Aerobic Bacteria. — Anacrobic bacteria mixed 
with aerobic bacteria will frequently grow in the apparent presence 
of oxygen, the aerobic bacteria robbing the media of it. Thus, tetanus 
and diptheria grow together in an open flask of bouillon. 

Method for Adapting Bacteria to Animal Fluids.— The placing of 
cultures in collodion sacs in the abdomens of animals has been used 
extensively by the Pasteur school for exalting the virulence of bacteria 
or trying to adapt them to species of animals differing from the one 
from which they were isolated. 

The underlying idea is to grow the organisms in the peritoneal 
cavity of an animal under such conditions that the waste products 
of the germs will be removed, an abundant supply of nutrient mate- 
rial furnished, and the germs themselves protected from the action 
of the phagocytes. The hermetically sealed collodion sacs answer 
this purpose. The collodion used is the U. S. Pharmacopoeia solu- 
tion, which by exposure to the air has been concentrated one-third. 

The sealed inoculated sacs are to be inserted into the peritoneal 
cavity with every possible precaution for asepsis. The sacs are left in 
place for days or months, as th^ experiment requires. 



CHAPTER VI. 
PRODUCTS OF BACTERIAL GROWTH. 

LIGHT, HEAT, GHEBIIGAL GOMPOUKDS, ETC. 

Bacteria not only are acted upon by their surroundings, as has 
already been shown, but they themselves act, often markedly, upon 
these surroundings. We have spoken, under the effect of food upon 
bacteria (p. 48) of the great changes which may be produced in 
bacterial growths by slight changes in the food medium. So, many 
of the products, as noted below, are influenced to a greater or a less 
extent by environment. 

Production of Light. — Bacteria which have the property of emitting 
light are quite widely distributed in nature, particulariy in media 
rich in salt, as in sea-water, salt fish, etc. Many of these, chiefly 
bacilli and spirilla, have been accurately studied. The emission of 
light is a property of the living protoplasm of the bacteria, and is not 
usually due to the oxidation of any photogenic substance given off by 
them; at least only in two instances has such substance been claimed 
to have been isolated. Every agent which is injurious to the existence 
of the bacteria affects this property. Living bacteria are always 
found in phosphorescent cultures; a filtered culture free from germs is 
invariably non-phosphorescent; but while these organisms cannot emit 
light except during life, they can live without emitting light. They are 
best grown under free access of oxygen in a culture medium prepared 
by boiling fish in sea- water (or water containing 3 per cent, sea-salt), 
to which 1 per cent, peptone, 1 per cent, glycerin, and 0.5 per cent, 
asparagin are added. Even in this medium the power of emitting 
light is soon lost unless the organism is constantly transplanted to fresh 
media. 

Thennic Effects. — The production of heat by bacteria does not 
attract attention in our usual cultures because of its sHght amount, 
and even fermenting culture liquids with abundance of bacteria cause 
no sensation of warmth when touched by the hand. Careful tests, 
however, show that heat is produced. The increase of temperature in 
organic substances when stored in a moist condition, as tobacco, hay, 
manure, etc., is due, partly at least, to the action of bacteria. 

Ghemical Effects. — The changes which substances undergo as they 
are split up by microorganisms depend, first, on the chemical nature 
of the bodies involved and the conditions under which they exist, 
and, secondly, on the varieties of bacteria present. A complete 
description of these chemical changes is at present impossible. Chem- 
ists can as yet only enumerate some of the final substances evolved, 
6 81 • 



82 PATHOGENIC MICRO-ORGANISMS, 

and describe, in a few cases, the manner in which they were produced. 
Bacteria are able to construct their body substance out of various kinds 
of nutrient materials, as well as to produce fermentation products or 
poisons; they are able to do these things either analytically or synthetic- 
ally with almost equal ease. Anabolic and katabolic power exists, 
according to Hueppe, among bacteria to an extent known as yet 
among no other living things. 

In the chemical building up of their body substance we can dis- 
tinguish, as Hueppe concisely puts it, several groups of phenomena: 
Polymerization, a sort of doubling up of a simple compound; synthesis, 
a union of different kinds of simple compounds into one or more com- 
plex substances; formation of anhydride, by which new substances 
arise from a compound through the loss of water; and reduction or 
loss of oxygen, which is brought about especially by the entrance of 
hydrogen into the molecule. The breaking down of organic bodies of 
complicated molecular structure into simpler combinations takes 
place, on the other hand, through the loosening of the bonds of poly- 
merization, through hydration or entrance of water into the molecule, 
and through oxidation. 

The chemical effects which take place from the action of bacteria 
are greatly influenced by the presence or absence of free oxygen. 
The access of pure atmospheric oxygen makes the life processes of 
most bacteria more easy, but is not indispensable when available 
substances are present which can be broken up with suflScient ease. 
The standard of availability is very different for diflFerent bacteria. 

In the presence of oxygen some of the decomposition products that 
are formed by the attack of the anaerobic bacteria are further decom- 
posed and oxidized by the aerobes; they are thereby rendered, as a 
rule, inert and consequently harmless as well as odorless in most 
cases. Some bacteria have adapted themselves to the exclusive use 
of combined oxygen, using those compounds from which oxygen can 
be obtained, and others — the obligatory aerobes — are able to live 
only in the presence of free oxygen. The facts of anaerobiosis are 
of great importance to technical biology and to pathology. Many 
parasitic bacteria are found to produce far more poison in the ab- 
sence of air than in its presence. The following four types of chemi- 
cal activity can be separated: 1. Production of substances which help 
in some way the life of the cell. These substances may be secreted 
and retained within the cell, or liberated from it; e.g., ferments or 
enzymes; true toxins (?). 2. Production of substances liberated by 
the bacteria as waste products. 3. . Production of substances by the 
breaking down of the food media; e.g., putrefactive products, due 
largely to enzyme action. 4. The production of substances which 
help form the protoplasm of the bacterial cell itself. 

Fermentation. — ^The term fermentation is differently used by dif- 
ferent authors. Some call every kind of decomposition due to micro- 
organisms or their products a fermentation, speaking thus of the putre- 
factive fermentation of albuminous substances; others limit the term 



PRODUCTS OF BACTERIAL GROWTH. 83 

to the process when accompanied by the visible production of gas; 
others, again, take fermentation to mean only the decomposition of 
carbohydrates, with or without gas-production. 

Fermentation may be defined as a chemical decomposition of an 
organic compound, induced by the life processes of living organisms 
(organized ferments), or by chemical substances thrown off from the 
organisms (unorganized or chemical ferments or enzymes)* In the 
first case the action is due to the life processes necessary for the 
growth of the organisms producing the ferment, as in the formation 
of acetic acid from alcohol by the action of the vinegar plant; in the 
second case the enzyme, either within or outside of the organism and 
having no direct connection with the growth of the organism, causes 
a structural change without losing its identity, as in digestion. E. 
Buchner {Berichte d, Devisch, chem. Gesellsch., xxx., 117-124 and 
1110-1113) has shown that, even in those cases of fermentation in 
which formerly it was believed the organized cell itself was necessarily 
concerned, the cell protoplasm squeezed from crushed cells and sepa- 
rated by filtration is able to cause the same changes as the organ- 
ized celb. This brings fermentation by unorganized and organized 
ferments very closely together, the one being a substance thrown off 
from the cell, the other a substance ordinarily retained within the 
cell. The elaboration of both ceases with the death of the bacteria 
producing them. Fermentation, therefore, requires the living agent or 
its enzyme. It furthermore demands the proper nutriment, tempera- 
ture, and moisture and the absence of deleterious substances. 

Fermentation yields products that are poisonous to the ferment; 
hence fermentation ceases when the nutriment is exhausted or the 
fermentation is in excess. Often, however, the process will begin 
again after diluting the fermented medium, showing that the con- 
cerUration of the harmful products plays an important part in the 
inhibitory action. 

Specific names are applied to various well-known fermentations 
according to the product — e. g.y acetic, yielding acetic acid; alcoholic 
or vinous, yielding alcohol; ammoniacal, yielding ammonia; amylic, 
yielding amylic alcohol; benzoic , yielding benzoic acid; butyric , yield- 
ing butyric acid; lactic, yielding lactic acid; and viscous, yielding a 
gummy mass. 

Gharacteiifltics of Ferments or Enxymes. — Ferments are non-dialyz- 
able. They withstand moderate dry heat, but are usually destroyed 
in watery solutions on exposure of 10 to 30 minutes to a temperature 
of 60 to 70° C. They are injured by acids, especially the inorganic 
ones, but are resistant to all alkalies. They, even when present in 
the most minute quantities, have the power of splitting up or decom- 
posing complex organic compounds into simpler, more easily soluble 
and diffusible molecules. The changes thus made may greatly aid in 
rendering the food stuff suitable for bacterial growth. A simple ex- 
ample of bacterial fermentation of carbohydrates produced by an 
enzyme is that of grape-sugar: 



PATHOGENIC MICRO-ORGANISMS. 



Far Less common is oxidizing fermentation, such as occurs, for 
example, in the production of acetic acid from alcohol. Here the 
energy is acquired not from the decomposition, but by the oxidation 
of the alcohol. 

The ProteolTtie Ferm«titB. — The proteolytic ferments which are 
somewhat analogous to trypsin — being capable of changing albumin- 
ous bodies into soluble and diffusible substances — are very widely 
distributed. The liquefaction of gelatin, which is chemically allied 
to albumin, is due to the presence of a proteolytic ferment or trypsin. 
The production of proteolytic ferments by different cultures of the 
same variety of bacteria varies considerably — far more than is gen- 
erally supposed. Even among the freely liquefying bacteria, such 
as the cholera spirillum and the staphylococcus, poorly liquefying 
strains have been repeatedly found. These observations have 
taught us that gelatin cultures must be observed for at least one month 
before deciding that no hquefaction will occur. Most conditions 
which are unfavorable to the growth of bacteria seem to interfere also 
with their liquefying power. 

Bitter-tasting products of decomposition may be formed by cer- 
tiun liquefying bacteria in media containing proteid, &s, for example, 
in milk. 

DiMtatic FeimantB.— Diastatic ferments convert starch into sugar. 
This action is demonstrated by mixing starch paste with suitable 
cultures to the resulting mixture of which thymol has been added, 
and keeping the digestion for six to eight hours in the incubating 
oven; then, on the addition of Fehling's solution and heating, the 
reaction for sugar appears— ^the reddish-yellow precipitate due to the 
reduction of the copper. 

Livertingf Ferments. — Inverting ferments (that is, those which 
convert polysaccharides into monosaccharides) are of very frequent 
occurrence. Bacterial invertin withstands a temperature of 100° 
C. for more than an hour, and is produced in culture media free from 
proteid. The presence or absence of such a ferment is often an impor- 
tant means of differentiating between closely related varieties of organ- 
~~ lore details as to the action of ferments on sugars see chap- 
ol on -typhoid groups, 

B Ferments. — Rennin-like ferments (substances ha^■ing 
f coagulating milk with neutral reaction, independent of 
und not infrequently among bacteria. The B. prodigiosus, 
in from one to two days coagulates to a solid mass milk 
een sterilized at 55° to 60° C. 



PRODUCTS OF BACTERIAL GROWTH, 85 

Alkaline Products and the Fermentation of Urea. — Aerobic bac- 
teria always produce alkaline products from albuminous substances. 
Many species also produce acids from sugars, which explains the fact 
that neutral or slightly alkaline broth often becomes acid at first from 
the fermentation of the sugar contained in the meat used for making 
the media. When the sugar is used up the reaction often becomes 
alkaline, as the production of alkalies continues. The substances 
producing the alkalinity in cultures are chiefly ammonia, amine, and 
the ammonium bases. 

The conversion of urea into carbonate of ammonia affords an ex- 
ample of the production of alkaline substances by bacteria: 

COCNH,)^ + 2H,0 - COjCNH,), 

Urea. 2 Water. Ammonium carbonate. 

The power of decomposing urea is not widespread among bacteria. 

Pigment Production. — Pigments have no known importance in 
connection with disease, but are of interest and have value in identi- 
fying bacteria. Their chemical composition is not generally known. 

Red and Yellow Pigments. — Of the twenty-seven red and yellow 
chromogenic bacteria studied by Schneider, almost all produce pig- 
ments soluble in alcohol and insoluble in water. The large majority 
of these pigments possess in common the property of being colored 
blue-green by sulphuric acid and red or orange by a solution of pot- 
ash. Though varying considerably in their chemical composition 
and in their spectra, they may be classified, for the most part, among 
that large group of pigments common to both the animal and vegetable 
kingdoms known as lipochromes, and to which belong the pigments 
of fat, yolk of eggs, the carotin of carrots, turnips, etc. 

^olet Pigments. — Certain bacteria produce violet pigments, also in- 
soluble in water and soluble in alcohol, but insoluble in ether, ben- 
zol, and chloroform. These are colored yellow when treated in a 
dry state with sulphuric acid, and emerald-green with potash solution. 

Bine Pigments. — Blue pigments, such as the blue pyocyanin pro- 
duced by B. pyocyaneiis; the fluorescent pigment common to many 
so-called fluorescent bacteria is different (bacteriofluorescence). In 
cultures the pigment is at first blue; later, as the cultures become 
alkaline, it is green. 

Numerous investigations have been made to determine the cause 
of the variation in the chromogenic function of bacteria. All condi- 
tions which are unfavorable to the growth of the bacteria decrease the 
production of pigment, as cultivation in unsuitable media or at too 
low or too high a temperature, etc. The B, prodigiosus seldom makes 
pigment at 37° C, and when transplanted at this temperature, even 
into favorable media, the power of pigment production is gradually 
lost. B. pyocyaneus does not produce pigment under anaerobic 
conditions. 

Ordinarily colorless species of bacteria sometimes produce pigments. 
Occasionally colored and uncolored colonies of the same species of bac- 



86 PATHOGENIC MICRO-ORGANISMS, 

teria may be seen to occur side by side in one plate culture, as, for 
example, in the case of staphylococcus pyogenes. 

Ptomains. — Nencki, and later Brieger, Vaughan, and others, suc- 
ceeded in isolating organic bases of a definite chemical composition 
out of putrefying fluids — meat, fish, old cheese, and milk — as well 
as from pure bacterial cultures. Some of these were found to exert 
a poisonous effect, while others were harmless. The poisons may be 
present in the decomposing cadaver (hence the name ptomain, from 
wrStfjiaf putrefaction), and, in consequence, have to be taken into 
consideration in questions of legal medicine. They may be formed 
also in the living human body, and, if not made harmless by oxida- 
tion, may come to act therein as self-poisons or leucomains. They 
possess the characteristics of alkaloid bodies and are different from 
the specific poisonous toxins. 

Many ptomains are known already and among them are some whose 
exact chemical constitution is established. Especially interesting is 
the substance cadaverin, which was separated by Brieger from portions 
of decomposing dead bodies and from cholera cultures, by reason of 
the fact that Ladenburg prepared it synthetically and showed it to 
be pentamethylenediamin [(NH2)2(CH2)5]- The cholin group is 
particularly interesting. Cholin itself (CsHjsNOj) arises from the 
hydrolytic breaking-up of lecithin, the fat-like substance found in 
considerable amounts in the brain and other nervous tissue. By the 
oxidation of cholin there can be produced the highly toxic muscarin, 
found by Schmiedeberg in a poisonous toadstool and isolated by Brieger 
in certain decomposing substances: 

C,H,,NO, + o - C,H,,Np, 

Cholin Muscarin 

The ptomain tyrotoxicon was obtained from cheese, milk, and ice- 
cream by Vaughan. 

Pyocyanin (Cj^Hj^NjO), which produces the color of blue or blue- 
green pus, is a ptomainic pigment. Similar bodies of a basic nature 
may be found in the intestinal contents as the products of bacterial 
decomposition. Some of these are poisons and can be absorbed into 
the body. Since the name ptomain was given to the poisonous products 
of bacterial growth before these products were chemically understood 
it is by many wrongly applied to all poisons found in food. Such 
poisoning may be due to true toxins or even living bacteria. 

The isolation of these substances can here be only briefly referred 
to. According to Brieger's method, which is the one now generally 
employed, the cultures having a slight acid reaction (HCl) are boiled 
down, filtered, and the filtrate concentrated to a syrupy consistency, 
dissolved in 96 per cent, alcohol, purified and precipitated by means 
of an alcoholic solution of bichloride of mercury. 

The Bacterial Toxins. — Any poisonous substance formed in the 
growth of bacteria or other microorganism may be called a toxin. 



PRODUCTS OF BACTERIAL GROWTH. 87 

The different bacterial toxins vary greatly in their characteristics. As 
little is known concerning their chemical nature, we are not able to 
classify them. There are certain known differences among them 
which are important and which may be made use of for purposes of 
study to divide the bacteria into two groups: 

1. Those varieties of bacteria that excrete in ordinary culture media 
water-soluble very specific toxic products, extracellular toxins. Type: 
diphtheria, tetanus. 

2. Those varieties which possess apparently only endotoxins, that 
is, true toxins which are more or less closely bound to the living cell, 
and which are only in a small degree separable in unchanged condi- 
tion outside of the body. On death of the cell they partly become 
free, partly remain united, or become secondary poisonous modifica- 
tions, no longer of the nature of toxins. Type: cholera, typhoid, 
pneumococcus. 

Among the intracellular poisons some are heat resistant. To these 
the name proteins is frequently given. 

Kztracalliilar Toxins. — Among the properties of the extracellular 
toxins are the following: They are, so far as known, uncrystallizable, 
and thus differ from ptomains; they are soluble in water and they 
are slowly dialyzable, through thin membranes, but not through thick 
membranes such as are used in refining antitoxic globulins; they are 
precipitated along with proteids by Concentrated alcohol, 65 per cent, 
or over, and also by ammonia sulphate; if they are proteids they are 
either albumoses or alUed to the albumoses; they are relatively unstable, 
having their toxicity diminished or destroyed by heat as well as by 
chemical manipulation (the degree of heat, etc., which is destructive 
varies much in different cases). Their potency is often altered in the 
precipitations practised to obtain them in a pure or concentrated 
condition, but among the precipitants ammonium sulphate has but 
moderate harmful effect. A remarkable characteristic of the group 
is that they are highly specific in their properties and have the power 
in the infected body to excite the production of antitoxins. The 
diphtheria and tetanus bacilli are the best known extracellular toxin 
prcxlucers. 

Predpitotion of Extracellular Toxins. — Ammonium sulphate erys- 
tab are added to the fluid containing the toxin until it is saturated. 
A large excess of ammonium sulphate crystals is then added 
and the whole kept at about 37° C. for twelve to eighteen hours. 
The toxin is precipitated and rises to the surface. This is skimmed 
off and dried in a vacuum or in an exsiccator containing strong 
sulphuric acid. The dried powder is placed in vacuum tubes and 
stored in the dark. Under these conditions the toxins deteriorate 
very slowly. During the process there may be a considerable loss 
of toxin, even when every care is taken. Tetanus toxin is espe- 
ciallv liable to deterioration. With the toxin other substances are 
precipitated. The diphtheria toxin is best precipitated from the 
bouillon by adding alcohol sufficient to produce a 65 per cent, solution. 



88 PATHOGENIC MICRO-ORGANISMS. 

Tlie precipitate is removed from the alcohol by filtration with the least 
possible delay. 

Intracellnlar Tozins. — Regarding the intracellular toxins which are 
more intimately associated with tlic bacterial cell and are produced 
by all bacteria we know much less, but it is probable that their chemical 
nature is somewhat similar, though they differ in their resistance to 
heat, — e. g., some of the toxins elaborated by tubercle bacilli with- 
stand boiling, while others do not. In the case of all toxins the 
fatal dose for an animal varies with the body weight, age, and 
general conditions. 

Terment Oharactciistics of Toxins. — The comparison of the action of 
bacteria in the tissues in the production of these toxins to what takes 
place in the gastric digestion has raised the question of the possibility 
of the elaboration by these bacteria of ferments, by which the process 
may be started. It would not be prudent to dogmatize as to whether 
the toxins do or do not belong to such an ill-defined group of substances 
as the ferments. It may be pointed out, however, that the essential 
concept of ferments is that of a body which can originate change without 
itself being appreciably changed, and no evidence has been adduced 
that toxins fulfil this condition. Another property of ferments is that, 
so long as the products of fermentation are removed, the action of a 
given amount of ferment is indefinite. In the case of toxins no evidence 
of such an occurrence has been found. A certain amount of a toxin is 
always associated with a given amount of disease effect. 

Similar Vogetablo and Animal Poisons.^-Substances similar to the 
bacterial endotoxin ferments and soluble toxins are formed by many 
varieties of cells other than bacteria. The ricin and abrin poisons 
obtained from the seeds of the Ricinus commtmis and the Abrus pre- 
calorius have a number of properties similar to those of the diphtheria 
and tetanus poisons. The active poisons contained in ricin and abrin 
have not yet been isolated, but the impure substances are extremely 
poisonous. When injected Into suitable animals anti-poisons are pro- 
duced and accumulate in the serum. These neutralize the poisons 
wherever they come in contact with them. 

They resemble the toxins in a general way in the manner in 
which they react to heat and chemicals. They are precipitated 
by alcohol. Through animal membranes they are less dialyzable 
than albumoses. Substances having these characteristics are called 
toxalbumins. 

Poisonous snakes secrete poisons which have many of the char- 
^nt^^.,t:^= nt the bacterial albumoses. The venom contains some 
milar to peptone and others similar to globulin. The 
general nervous symptoms and paralysis of the respiratory 
the latter cause intense local reaction with hemorrhages 
)int of injection. The injection of venins into animals is 
he production of antivenins which neutrahze the venins, 
-um -containing abundant suitable antevenins is injected 
ed person it has considerable therapeutic value. 



PRODUCTS OF BACTERIAL GROWTH. 89 

Ehilich's Theories as to the Nature of Extracellular Toxins. — 

From a large number of most carefully conducted experiments with 
the toxin and antitoxin of diphtheria, Ehrlich has formulated a theory 
concerning the former. This theory has undergone several modifi- 
cations since it was first proposed, and it is difficult to give an exact 
statement of its present status. Generally speaking, however, in con- 
densed form its essential points are as follows: 

Toxins and antitoxins neutralize one another after the manner of 
chemical reagents. The chief reasons for this belief lie in the ob- 
served facts: (a) that neutralization takes place more rapidly in con- 
centrated than in dilute solutions, and (b) that warmth hastens and 
cold retards neutralization. From these observations Ehrlich con- 
cludes that toxins and antitoxins act as chemical reagents do in the 
formation of double salts. A molecule of the poison requires an exact 
and constant quantity of the antitoxin in order to produce a neutral 
or harmless substance. This implies that a specific atomic group 
in the toxin molecule combines with a certain atomic group in the anti- 
toxin molecule. 

The toxins, however, are not simple bodies, but easily split into 
other substances which differ from one another in the avidity with 
which they combine with antitoxin. 

These derivatives Ehrlich calls prototoxins, deuterotoxins, and 
tritotoxins. 

All forms of toxins are supposed to consist of two modifications, 
which combine in an equally energetic manner with antitoxin or with 
suitable substance in the cells, but differ in their resistance to heat and 
other destructive agents. 

The less resistant form passes readily into a substance called tox- 
oid which has the same affinity for the antitoxin as the original toxin, 
but is not poisonous. The facts observed, Ehrlich thinks, are best 
explained on the supposition • that the toxic molecule contains two 
independent groups of atoms, one of which may be designated as the 
haptophorous and the other as the toxophorous group. It is by the 
action of the former that toxin unites with antitoxin or cell molecule 
and allows the latter to exert its poisonous effect. 

The toxophorous group is unstable, but after its destruction the 
molecule still unites with the antitoxin or the sensitive molecule 
through its retained haptophorous group. 

Bordet has shown that toxin unites in different multiples with anti- 
toxin, so that the toxin molecule may have its affinity slightly, partly, 
or wholly satisfied by antitoxin. Slightly satisfied, it is still feebly 
toxic; combined with a larger amount of antitoxin, it is not toxic; but 
still may, when absorbed into the system, lead to the production of 
antitoxin. Fully saturated,, it has no poisonous properties and no 
ability to stimulate the production of antitoxin. 

The most important of the extracellular toxins are those produced 
by the diphtheria and tetanus bacilli. These are very powerful; 
0.0000001 gram of the dried filtrate of a tetanus culture will frequently 



90 PATHOGENIC MICRO-ORGANISMS. 

kill a white mouse, while 100 times of that amount of dried diphtheria 
filtrate has killed a guinea-pig. 

The same bacterium may produce several entirely distinct toxins, 
thus, according to Madsen and Ehrlich, the specific tetanus poison 
consists of two toxins, tetanospasmin and tetanolysin. To the first 
of these the tetanic convulsions are due, while the second has a hemo- 
lytic action. 

Altogether different from the poison effects are the immunization 
processes produced by the cell substances of bacteria, whether they 
be obtained from bacterial bodies or from chemical preparations. 
These processes have .little or nothing to do with the toxic action of 
the cell proteids, but rather depend upon the introduction of suitable 
receptors, that is, substance capable of union with the molecules of the 
cell.s which give rise to the antibodies. 

The pyogenic action of their proteids is common to all bacteria, 
this depending principally upon their being extraneous albuminous 
substances. Pyogenic effects may be produced in like manner by 
extraneous albumins of non-bacterial origin. That every extraneous 
albuminous substance is harmful to the organism which seeks to 
resist its action is shown by those specific precipitating ferments, the 
precipitins, which are produced in the organisms after the introduction 
of every extraneous albumin. 

Redaction Processes. ^The following processes depend wholly or 
in part upon the reducing action of nascent hydrogen. 

1. Sulphuretted Hydrogen (HjS). All bacteria, according to Petri 
and Maassen, possess the power of forming sulphuretted hydrogen, 
particularly in liquid culture media containing much peptone (5 to 10 
per cent,); only a few bacteria form H,S in bouillon in the absence 
of peptone, white about 50 per cent, in media containing 1 per cent, 
peptone pos.sess the property of converting sulphur into sulphuretted 
hydrogen, for which purpose is required the presence of nascent 
hydrogen. The presence of HjS is determined by placing a piece 
of paper moistened with lead acetate inside the neck of the flask con- 
taining the culture, closing the mouth with a cotton-wool stopper, and 
over this again an india-rubber cap (black rubber free from sulphur). 
The paper is colored at first brownish and later black; repeated ob- 
servation is necessary, as the color sometimes disappears toward the 
end of the reaction. Apparently negative results should not be rashly 
accepted as conclusive. 

2. The reduction of blue litmus pigments, methylene blue, and 

ubstances. The superficial layer of cultures in 
shows often no reduction, only the deeper layers 
agitation with access of air the colors may be 
at the same time, if acid has been formed, the 
Tied red. 
of nitrates to nitrites, ammonia, and free 
of these properties seems to pertain to a great 



PRODUCTS OF BACTERIAL GROWTH. 91 

The test for nitrites is made as foUows: Two bouillon tubes containing 
nitrates are inoculated, and, along with two uninoculated tubes, are allowed 
to remain in the incubator for several days; then to the cultures and con- 
trol test is added a small quantity of colorless iodide of starch solution (thin 
starch paste containing 0.5 per cent, potassium iodide) and a few drops of 
pure sulphuric acid. The control tubes remain colorless or become gradually 
slightly blue, while if nitrites are present a dark blue or brown-red coloration 
is produced. A test may be made also by sulfonilic acid and a naphthylamin 
hydrochloride, which give a brown-red coloration proportional to the amount 
of nitrite present. 

The demonstration of ammonia is made by the addition of Nessler's reagent 
to culture media free from sugar. In bouillon, if ammonia be present, 
Nessler's reagent is almost immediately reduced to black mercurous oxide. 
A strip of paper saturated with the reagent can also be suspended over the 
bouillon tube, or this can be distilled at a low temperature with the addition 
of magnesium oxide and the distillate treated with Nessler's reagent. A yel- 
low to red coloration indicates the presence of ammonia. Controls are neces- 
sary. Place 1 c.c. of bouillon and 49 c.c. NH, free H,0 in Nessler jar with 
controls. Add reagent to each, allow to stand fifteen minutes and read color 
which is compared with standards. 

Aromatic Products of Decomposition. — Many bacteria produce 
aromatic substances as the result of their growth. The best known of 
these are indol, skatol, phenol, and tyrosin. Systematic investigations 
have only been made with regard to the occurrence of indol and phenol. 

Test for Indol. — To a bouillon culture, which should, if possible, 
be not under eight days old and free from sugar, is added half its 
volume of 10 per cent, sulphuric acid. If in heating to about 80° 
C. a pink or bluish-pink coloration is immediately produced it indicates 
the presence of both indol and nitrites, the above-described nitroso- 
indol reaction requiring the presence of both of these substances for 
its successful operation. This is the so-called "cholera-red reaction," 
but it may be applied to many other spirilla besides cholera and to 
certain bacilli also. As a rule, however, the addition of sulphuric 
acid alone is not sufficient, and a little nitrite must be added; this may 
be done later, the culture being first warmed without nitrite, when, 
if there is no reaction or a doubtful one, 1 to 2 c.c. of 0.005 per cent, 
solution of sodium nitrite is added until the maximum reaction is 
obtained. The addition of strong solutions of nitrite colors the acid 
liquid brownish-yellow and ruins the test. Out of sixty species 
examined by Lehmann, twenty-three gave the indol reaction. 

Decomposition of Fats. — ^Pure melted butter is not a suitable culture 
medium for bacteria. The rancidity of butter is brought about (1) 
as the result of a purely chemical decomposition of the butter by the 
oxygen of the air under the influence of sunlight, and (2) through the 
formation of lactic acid from the milk-sugar left in the butter. Fats 
are, however, attacked by bacteria when mixed with gelatin and used 
as culture media,. with the consequent production of acid. 

Pntrefaction. — By putrefaction is understood in common parlance 
every kind of decomposition due to bacteria which results in the 
production of malodorous substances. Scientifically considered, putre- 
faction depends upon the decomposition of albuminous substances. 



92 PATHOGENIC MICRO-ORGANISMS. 

which are frequently first peptonized and then further decomposed. 
Typical putrefaction occurs only when oxygen is absent or scanty; 
the free passage of air through a culture of putrefactive bacteria — an 
event which does not take place in natural putrefaction — very much 
modifies the process: first, biologically, as the anaerobic bacteria are 
inhibited, and then by the action of the oxygen on the products or 
by-products of the aerobic and facultative anaerobic bacteria. 

As putrefactive products we have peptone, ammonia, and amines, 
leucin, tyrosin, and other amido substances; oxyfatty acids, indol, 
skatol, phenol, ptomains, toxins, and, finally, sulphuretted hydrogen, 
mercaptans, carbonic acid, hydrogen, and, possibly, marsh-gas. 

Nitnfying Bacteria. — According to recent observations, nitrification 
is produced by a special group of bacteria, cultivated in the laboratory 
with diflSculty, which do not grow on our usual culture media. From 
the investigations of Winogradsky it would appear that there are 
two common microorganisms present in the soil, one of which converts 
ammonia into nitrites and the other converts nitrites into nitrates. 

Oonversion of Nitrous and Nitric Acids into Free Ktrogen.— 

This process is performed by a number of bacteria. 

The practical importance of these organisms is that by their action 
large quantities of nitrates in the soil, and especially in manure, may 
become lost as plant food by being converted into nitrogen. 

By the aid of certain root bacteria, which gain entrance to the roots of 
legumes and there produce nodular formations, the leguminous plants 
are enabled to assimilate nitrogen from the atmosphere. It is not 
known exactly how this assimilation of nitrogen occurs, but it is assumed 
that the zoogloea-like bacteria, called bacteroids, constantly observed in 
the nodules, either alone or in a special degree, possess the property of 
assimilating and combining nitrogen. It seems, moreover, to have 
been recently established that, independently of the assistance of the 
legumes, certain nodule bacteria exist free in the soil, which accu- 
mulate nitrogen by absorbing it from the air. These various nitrifying, 
denitrifying, and nitrogen-fixing bacteria are described in detail in the 
special chapter upon bacteria in nature. 

Formation of Acids from Oarbohydrates.— Free acids are formed 
by many bacteria in culture media containing some form of sugar or 
other fermentable carbohydrates, such as the alcohol mannite; (he 
production of acid in ordinary bouillon takes place on account of 
the presence of meat-sugar, which is usually derived in small quan- 
tities from the meat.^ According to Theobald Smith, all anaerobic 
or facultative anaerobic bacteria form acids from sugar; the strict 
aerobic species do not, or do so very slowly that the acid is concealed 
by the almost simultaneous production of alkali. The formation of 
acid occurs sometimes with and sometimes without the production of 
gas. Excessive acid production may cause the death of the bacteria 
from the increase in acidity of the culture media. 

* According to Theobald Smith, 75 per cent, of the beef ordinarily bought in 
the markets contains appreciable quantities of sugar (up to 0.3 per cent.). 



PRODUCTS OF BACTERIAL GROWTH. 93 

If after the sugar is consumed, not enough acid has been formed 
to kill the bacteria, the acid is neutralized gradually and in the end 
the reaction becomes less acid or even alkaline. 

Among the acids produced the most important is lactic acid; also 
traces of formic acid, acetic acid, propionic acid, and butyric acid, 
and not infrequently some ethyl-alcohol and aldehyde or acetone are 
formed. Occasionally no lactic acid is present, and only the other 
acids are formed. 

Various bacteria, as yet incompletely studied, possess the prop- 
erty of producing butyric acid and butyl-alcohol from carbohydrates. 

Some bacteria also seem to have the power of decomposing cellulose. 

Formation of Oas from Oarbohydrates and Other Fermentable 
Substances of the Fatty Series. — ^The only gas produced in visible 
quantity in sugar-free culture media is nitrogen. If sugar is vigor- 
ously decomposed by bacteria, as long as pure lactic acid or acetic 
acid is produced there may be no development of gas, as, for instance, 
with the B. typhosus on grape-sugar; but frequently there is much 
gas developed, especially in the absence of air. About one-third of 
the acid-producing species also develop gas abundantly, this consisting 
chiefly of CO2, which is always mixed with H. Marsh-gas is seldom 
formed by bacteria, with the exception of those decomposing cellulose. 

In order to test the production of gas, a culture medium composed of 
solid or semi-solid nutrient agar, containing about 1 per cent, glucose, 
lactose, or other carbohydrate, may be used. At the end of eight to 
twelve hours in the incubator (or twenty-four hours' room ^^^ ^^ 
temperature) the agar will be seen to be full of gas- 
bubbles or broken up into holes and fissures. 

For the determination of the quantity and kind of gas 
produced by a given microorganism the fermentation tube 
recommended by Theobald Smith is the best. This is a 
bent tube, constricted greatly at its lowest portion, sup- 
ported upon a glass base, as shown in Fig. 61 . Fermenta- 
tion tubes should have the following essential points: 
The neck should be narrow, to prevent as far as possible 
the diffusion of gas; this is particularly necessary to pre- 
vent the entrance of oxygen which would of course 
destroy the anaerobiosis. The bulb should be large 
enough to hold all the fluid in the vertical arm together *"tSbe. 
with the amount normally in the bulb itself. The tube is 
filled with a culture medium consisting of peptone bouillon (without 
air bubbles) to which 1 per cent, of glucose, lactose, or other sugar 
has been added, and sterilized in the steam sterilizer. It is then 
inoculated with a loopful of a culture of the organism in question, 
and observations taken: 

1. If there is a turbidity produced in the open bulb it indicates the 
presence of an aerobic species; if this clouding occurs only in the closed 
arm, while the open bulb remains clear, it is an anaerobic species. 

2. The quantity of gas produced daily should be marked on the 




94 PATHOGENIC MICRO-ORGANISMS. 

upright arm; if the tube is graduated a note of it is taken and the 
percentage calculated on the fourth to the sixth day after gas produc- 
tion has ceased. 

3. A rough analysis of the gas produced may be made as follows: 
Having signified by a mark on the tube the quantity of gas produced, 
the open bulb is completely filled with a 10 per cent, solution of soda, 
the mouth tightly closed with the thumb, and the mixture throughly 
shaken. After a minute or two all the gas is allowed to rise to the top 
of the closed arm by inclining and turning the tube, and then, removing 
the thumb, the volume of gas left- after the union of the NaOH with the 
COj is noted. The remainder is nitrogen, hydrogen, and marsh-gas. 
If it is desired to test for the presence of hydrogen, the thumb is again 
placed over the open end and the gas collected under it. As the thumb 
is moved a lighted match is brought in contact with the gas. If hydro- 
gen is present a slight explosion occurs. 

Formation of Acids from Alcohol and Other Organic Adds.— 

It has long been known that the Bacterium aceti and allied bacteria 
convert dilute solutions of ethyl-alcohol into acetic acid by oxidization : 

CH, + O, - CH, + H,0. 
CHjOH COOH. 

The higher alcohols — ^glycerin, dulcit, mannite, etc. — are also con- 
verted into acids. 

Finally, numerous results have been obtained from the^' conversion 
of the fatty acids and their salts into other fatty acids by bacteria. 
As a rule, the lime-salts of lactic, malic, tartaric, and citric acids 
have been employed, these being converted into various acids by the 
action of bacteria, as, for example, butyric, propionic, valerianic, 
and acetic acids; also succinic acid, ethyl-alcohol, and, more rarely, 
formic acid have been produced. The gases formed were chiefly CO, 
and H. 

Thus Pasteur found that anaerobic bacteria convert lactate of lime 
into butyric acid. 

Important Oharacteristics which should be Noted in the Complete 
Study of a Bacterium. — The accompanying descriptive chart which 
gives the points decided upon by the Society of American Bacteriolo- 
gists (1907) as necessary for the complete identification of an organism 
is inserted in order to insure unity of methods and thus make com- 
parative studies easier. Some pathogenic bacteria require special 
media for their growth; moreover, they do not need testing with all 
of the tests mentioned in this chart in order to identify them. With 
some varieties the cultural characteristics are of the greatest impor- . 
tance, while with others pathogenic or toxic effects occupy the chief I 
place. J 



I 



Dim of bokiloa 



DBTAIL6D F6ATURB8. 



uneaie. daraU, curptd. 



VD^UkI 



I OneoLttioa (■nHE^iic) - - - 

{ Chum (No. ol elunfinU) 

-, S/toTi cfviint, teng chtnnt 
jOrienCaUon of chuoi. varaliiU 

Klium uApd t«mp . . . . - 

>«e d.y. 

Form, eUipheoI, tKon rodtt tptndled, datiaU, drum' 

Idioito of Sii«- ■ 



.-.«•. J ,„...u» (No. of elcmenls) 

iKDciua-block I OrieutatioD at chuiu 

Loc&tioD of £iid«pon«. antral, polar. 



eqMOXi/Tial, obliqi 



'. InVaiutiOB Formi. on in dky* kt ° C 

I. StalniDC RaactioDt. 

1 : 10 wktciy furhnn, gnitiiui- violet, oftrboMuobaii 

laffler't kluline aiethy)«n*-blue. 

Special Staiiu 

Unun Glyeofen 

Fat Acid-^t 



n. CULTURAL FEATURB8 (•) 
1. Ani Strok*, 

Growth. invUihte, ucanty, maderott. abtindanl. 
Form of groirlh. JUtfon ■ " ■ ■ ■ ' ■ 



inufnlr, bnded, iprnut- 



, h.ftal, rffu^e, n 

Luatre. fflitttnin^j. dull, crftactout. 
Topocnidiy. (imwfA, 
Optical r"- — 




I, Gelatin Stab. 

Line *^ puacuire. filiform, ba 

Liquefaotioik cratrfiform, n 

compleMia ...a. 

Medium if lurfKffiT. broxtmad. . 
I, Nutrient Brolb. 

Surf HCB growth, ring, pel 

CLoiidinff tligM, moderate, tlronc; tranaie 
naient: none; jtmd ivrbid. 

Odor, aiunt. dt ei dtd, ratntbline 

Sediment, compart, Jlvccvltnt, eranular, ftak 

L Hilk. 

Cleariax without coaculadon. 
Coaculatioa prompj, dtlaurd, obienf. 

Eitruaioa of whey becioa in day 

Coapilum ttowly peptonized, rapidly peptortist 
PeptonisaeiDn beginfl on. . . .d. complete on 
Reution. Id 2d 4d ...,10d .: 



m broumed, reddened, b 



Lab fen 



Elevation. jdU. tifiuc, n 
Edve, entire, bndb^f, lobai 



d, greened. 



artial WW r 



10. Afar ColoDiei. 



. . .b elov, rapid, (tempermture, .,.-.-.-. 
Fono. pvrtctiform, round, irregular, amdttoi 

Ivnd, Manrnlnue. rhitaid. 
Surface tmootk, rough, conctntrieallj/ firmed, 

Ed^, entire, undulate, tobate. eroee, iacer- 
bricte. floccoxe. curi/d. 

oranular, arumatt. fitamentaue, Jtoccoar, 
Starch JeUy. 
Growth, ecantu, copioue. 

Diaitoaic action, abtent. fetblr. profound. 
Medium stained 



..-owth. flliform, erhinulitte, beaded, epriad- 

int. plumoie. arborrerenl. rhUoid. 
Elevation of growth, flal. effuet. raited, conrei. 
Lultre. gtitteninff. dull, crrtaceau*. 
Topography, emootA, contoured, rutjoer. verrutote. 
ChromoienEaii (>l Pigment in water 

Odor. abetnt.da^did.raimbUna. ...'.'.'.'.'.'.'.'. .'''...'. 
CooBigtency, jfimv. bitt^roua, viecid. mrmbranout. 

Medium, ffrayed. browned, reddened, btued. greened. 
I. LocDei'i Blood-ienim. 

stroke inuieAtr, ecanty, madrratt. abundnnt. 

Form of growth. lUi/orm. erhinuiolr. beaded, tpread- 

Elevatuia of growth^ «al, tffute!"raieed, eonoex. 

LuetT. tflietenxnff, dull, cretaceoue. 

Topography, tmooth, contoured, rugoee, serrucoie. 

Chromogeneeis (•! 

Medium prayed, irsu-ned, reddmal, btued, ereened. 

Liquefaction bcgioi in. d. complete in d, 

I. Agai Bteb. 

(irowtfa uni/em, beM at lap, beet a( bottom; nirfase 

grwth ocanty. abundant; rretrieted, undo-epread. 

Line of puQctun. fUi/brm. beaded. ^piUate. villoue. 



Cohn'i SoluUoa. 



Cuhlnsky'i Solution. 

Fl^d i-i(SS!"M viKi/' 
Sodium Cbloiida in Bouillon. 



m. PHTSICAl AND BIOCaSMICAL PBAT 



plume 






la prodnctiaii, StAU, moderati, Mrong, (Amit, 



I, mtnta Id oltnle brotb. 
Redmed, Ml ttducid. 
Pntcnse of nitiilw 



It apply unleu both spply- 



«, TolnvUnn of HaOB, 
' " 7. Ontimiitti T«actlDn 






u teiml of FuUec'i seals 

8. Vitality on cullurs msdla, britS, i 

9. Tsmpecaturs relations. 



OptimuiD Mmparal 






1° C, 26° C, 30° C.,37° I 

i6=c.,so=c.,eo°c, 

MaidfDuia tempfirature for growth - _ 

MiniiDiim tempwatun for growth 

10. KUlsdrMdllTbydiTinai replant to dryinc. 

11. P«r '--'- --" - — ' 



L killed bj IrMiiof (salt and crusbed ii 



Per cenl. killed... 

13. Adds produced 

14. AlkallM prodoced 

15. Aleohala 

IS. FernuDli, jiiptin. 



. pernzidatt, iipaie. 



IS. BDMt olcB-micldes: 



•nil 



o-t- Nevdle-crawtli 



Starcfa deatroyed 

Om«i at 37° C. _ ^ 

<Jm«-8 in Cohn's Sol. ' 

Qiowt id Usehinsky's .Sol. 
; Gelatin (•) 



EBICITT. 

lie to Animals. 

cnHtaaant, fiihrt. repliler. Hrd». mice, nil. 

nes. rqbMU, doot. aUt, theep, foati, oUUe. 

lictoPUali; 

DiuUe, tndolaxint. 

a totmiac. 

r hacterlddal. 

y DoQ-bacMriddal. 

irultnce on cultiir* -media: prompts ffrdrfuoi, 

rted in. ...-.- - . . . .mobths. 



CHAPTER VII. 

THE SOIL BACTERIA AND THEIR FUNCTIONS— AIR BACTERIA 

—BACTERIA IN INDUSTRIES. 

The bacteria* in the soil belong to many varieties. Some varieties 
are only accidentally present, being due to the contamination of the 
earth with the bacteria contained in animal faeces and other waste 
products. The majority, however, pass their life and reproduce 
themselves chiefly or wholly in the soil. Many of these varieties have 
most important functions to perform in continuing the earth's food 
supply. Without them plant food; and, therefore, animal food, would 
cease to exist. Some make available for plants, the carbon, nitrogen, 
hydrogen, and other compounds locked up in the dead bodies of animals 
and plants. Others construct food for plants from the gases of the air 
and the inorganic elements of the earth which in their simpler forms 
were not available. 

The bacteria together with the other somewhat less important 
microscopic plants and animals, thus form a vital link in the earth's 
life cycle, plants and animals. The bacteria in the soil require for 
their activities food, moisture, and a proper temperature. They may 
be present to the extent of many millions in a single gram of rich 
loam, while in an equal quantity of sand they may be almost absent. 

The various species associated together in the soil flora influence 
each other. Thus anaerobic bacteria are enabled to grow because of 
associated aerobes using up the free oxygen, while other species make 
assimilable substances not usable by others. 

The Splitting up of Oarbon Oompounds. — ^The plants form starch, 
and from it cellulose, wood, fats, and sugar. These substances 
once formed cannot be utilized by other generations of plants. Some 
of these are transformed in the bodies of animals, but the largest 
percentage await the activities of the microorganisms. The sugars 
and starches usually undergo an alcoholic fermentation, excited by 
the yeasts and moulds with the production of alcohol and carbon-di- 
oxide, or an acid fermentation excited by bacteria with the production 
of acids and frequently of carbon-dioxide. 

Cellulose which is so resistant to decay is attacked by certain varieties 
of bacteria which are abundant in the soil. They act both in the 
presence and absence of free oxygen. Moulds also act on cellulose. 
Carbon-dioxide, marsh gas, and other products are produced. Wood 
IS apparently first attacked by the fungi and only later by the micro- 
organisms. These bacteria are carried into the intestines and act 
upon cellulose and other substances. 

• L. H. Bailey. "Bacteria in Relation to Country Life." 

95 



96 PATHOGENIC MICRO-ORGANISMS. 

The Decomposition of Nitrogenous Oompounds.— Plants obtain 
their nitrogen chiefly in the form of nitrates. The small amount of 
usable nitrogen in the soil must be constantly replenished. This 
must either come from the nitrogen forming a part of proteid materials 
or from the free nitrogen in the air. 

The animals utilize the plant proteids and reduce them to much 
simpler compounds, such as urea, but even these are not suitable for 
plant use. We now know that microorganisms are employed to break 
compounds into simpler compounds and also to utilize the nitrogen 
of the air. 

Decomposition. — This process is to some extent carried out through 
the agency of yeasts, moulds, and fungi, but it is chiefly due to the 
activities of bacteria. When this process is carried on in the absence 
of oxygen it is incomplete giving rise to substances with unpleasant 
odors, such as H^S, NHj and CH^. This is called putrefaction. 
When oxygen is freely accessible more complete decomposition occurs 
with such end products as COjjN and H2O. These two processes, 
putrefaction and complete decay, cannot be sharply separated as the 
second usually follows the first. The varieties of organisms causing 
these changes are many. Some groups will be found chiefly in decay- 
ing vegetable substances, others in animal tissues. They include all 
morphologic forms of bacteria as well as yeasts and higher fungi. 
These forms exist everywhere in nature, although in various degrees, 
so that every bit of dead organic matter is sure to be decomposed if 
only moisture and warmth are present. B, subtili'S and B. proteiis 
vulgarius are well known laboratory bacteria that are commonly found 
among decomposing materials. B. proteus is described under patho- 
genic Jbacteria. B, subtilis (hay bacillus) has the following char- 
acteristics (Fig. 62). 

Source and Habitat. — Hay, straw, soil, dust, milk, etc. 

Morphology. — Short, thick rods with round ends, sometimes form threads. 
Sometimes also chains of long rods, short rods, and coccus forms. 0. 8 to 1 . 2^ 
broad, 1 . 3 to S^t long. Often united in strings and threads. 

Staining Reaction. — Stains by Gram's method. 

Gapsule, Flagella, Motility* — Bacillus posseses a thin capsule and many 
flagella which are long and numerous; short forms actively motile; threads 
immotile. 

Spore Formation. — Oval spores formed in presence of air germinating at 
right angles to long diameter. Spores are set free in about 24 hours, size 1.2 
by 0.6m; widely distributed in nature, dust, air, excreta, etc., (see Fig. 62). 

Biology: Galtural Gharacters (Including Biochemical Features). — Bou- 
illon. — Uniformly cloudy growth with marked pellicle, wrinkled and thick; 
copious spore formation. 

Gelatine Plates and Tubes. — Saucer-like depressions ; colonies have granu- 
lar centres and folded margins. Surface growth in stab cultures is whitish- 
gray; colonies sink on liquefaction of medium; liquefaction progresses in a 
cylindrical form, and a thick white scum is formed. 

Agar Plates and Tubes. — Small, irregular, grayish-white colonies; moist 
glistening growth along needle track in stab cultures. 

The bacteria in taking certain atoms from the molecules utilized 
in their growth leave the other atoms to enter into new relations and 




THE SOIL BACTERIA AND THEIR FUNCTIONS. 97 

form new compounds. The actual products will depend on the 
decaying substance, the variety of bacteria and the conditions present. 
mtrification. — This is a process of oxidation by which through 
bacterial activities ammonia compounds are changed to nitrates and 
thus rendered utilizable by plants. This change is accomplished in 
two stages; first, the ammonia is oxidized to nitrite and second to 
nitrate. The nitrates are taken up by the plant roots from the soil. 
The bacterial nature of these changes were discovered in 1877 by 
two French investigators, Schlosing and Muntz. They noted that 
fermenting sewage after a time lost its ammonia and gained in nitrates, 
but that if the sewage was treated with antiseptics, so that fermentation 
ceased, no such change occurred. War- ^^^ ^2 

rington first and Winogradsky later more 
thoroughly investigated the bacterial cause 
of these changes. The latter by means of 
silica jelly, which contained no organic 
matter, was able to isolate two varieties of 
cocci, one in Europe and the other in 
America, which were able to change 
ammonia to nitrites. He called the one 
nitrosomonas and the other nitrosococcus. 
They are capable of acting on almost 
any ammonia salt. One variety of organ- 
isms capable of chan&^ine: nitrites to nitrates Bacillus subtiUs with sporee. Agar 

. *- , , 1 ^1 • 1 Ml 1 11 1 culture. Stained withKentian violet. 

was isolated, and this bacillus he called X lOOO diameters. (Fraenke.) 

nitrobacter. These are small slightly 

elongated bacilli. These bacteria are remarkable in that in pure 
cultures very small amounts of organic matter in the media act as anti- 
septics. They appear to be able to depend on mineral substances for 
their food. These bacteria are extremely important, for the plants 
take up most of their nitrogen in the form of nitrates. These changes 
are mostly produced in the surface soil. If the reaction of thf. soil 
becomes acid growth ceases. Soil bacteriologists are studying the 
nitrifying power of different types of soil under identical conditions. 
The process being one of oxidation, the access of air is necessary. 

Denitrification. — ^This is a reducing process. The nitrate is made 
to yield up a part or all of its oxygen and thus becomes changed to 
nitrites and to ammonia and even to free nitrogen. The partial change 
does not rob the soil of its available nitrogen as does the total change, 
for the nitrites and ammonia may be changed by the nitrifying bac- 
teria to nitrates. These bacteria exist normally in most soils and are 
especially abundant in manure. There are three different types of 
nitrogen reduction: 1. The reduction of nitrates to nitrites and am- 
monia. 2. The reduction of nitrates and nitrites to gaseous oxides of 
nitrogen. 3. The reduction of nitrites with the development of free 
nitrogen gas. 

mtrogen Fixing Bacteria.— Helbrigel in 1886 demonstrated that 
certain plants were able to use the nitrogen of the air and this ap- 
7 



98 • PATHOGENIC MICRO-ORGANISMS. 

parently through the aid of bacteria growing in their roots. These 
root bacteria are named B. radicicola. They produce enlargements 
(tubercles) on the roots. 

According to Ball/ there is no reasonable doubt but that B. 
radicicola can and usually does remain active for very long periods in 
soil devoid of leguminous vegetation. Furthermore, the bacterium 
diffuses at a very considerable rate through soils that are in proper 
condition; therefore, if a soil should be found lacking the organism, 
it is illogical to attempt to introduce it artificially without having first 
made the soil fit for the development of the bacteria. 

It has not been shown by anyone that increased powers of resistance 
to unfavorable conditions of certain varieties are at all correlated 
with their enhanced *' greed for nitrogen." Moreover, it is far from 
being proven that any one race or *' physiologic species" is really more 
virile than another. Greig-Smith^ has shown that as many as three 
races are sometimes present in one and the same tubercle. Possibly, 
therefore, fixation of nitrogen may occur most rapidly only when two 
or more of these races are growing together. 

Buchanan * has recently made a minute morphologic study of 5. 
radicicola. Some of his conclusions are as follows: 

1. Considerable variation in the morphology. of B. radicicola may 
be induced in artificial media by the use of appropriate nutrients. 
Of the salts of the organic acids, sodium succinate brings about the 
most luxuriant development and the production of the greatest 
variety of bacteroids. 

2. B. radicicola in the roots of the legumes shows the same type of 
bacteroids as may be found in suitable culture media. On the other 
hand, there is little or no correspondence between the type of bacteroid 
produced in culture media by a certain organism and that produced 
in the nodule by the same form. 

3. It is probable that the term B, radicicola includes an entire group 
of closely related varieties or species which differ from each other to 
some degree in morphological characters. 

4. The nodule organism resembles morphologically both the yeasts 
and the bacteria. . The difference between this form and those ordi- 
narily included under the terms Bacillus and Pseudomonas justify the 
use of a separate generic name, Rhizobium, 

In 1893 Winogradsky furnished proof that there are in the soil bac- 
teria which are outside of the plant roots performing the same func- 
tion as those within the roots.. These bacilli he called Clostridium 
pasteurianum. They are anaerobic and produce spores. Their 
power to fix nitrogen is increased in presence of sugar and lessened in 
presence of nitrogenous substances. 

Beyerinck in 1901 described two aerobic species of nitrogen fixing 

*Ball, O. M. A contribution to the Life History of B. radicicola Beij. 
Centralbl. f. Bakt., etc., 1909, II. Abt., xxiii, 47. 

^Greig-Smith. Journ. Soc. Chem. Indust., 1907, No. 7. 

^Buchanan, R. E. The Bacteroids of Bacillus radicicola. Centralbl. f. Bakt., 
etc., 1909, II. Abt., xxiii, 59. 



THE SOIL BACTERIA AND THEIR FUNCTIONS. 99 

bacteria. Later Bailey described three additional species. These 
were called Azotobacter. These studies have already led to the inocu- 
lation of soils and to the investigation of the kind of soils and crops best 
fitted for the growth of these bacteria. Many impoverished soils have 
already been greatly improved. There are probably many other 
varieties of bacteria capable of fixing nitrogen, because one can hardly 
examine the roots of any leguminous plants, without finding tubercles 
different. The use of seed inoculated with the special variety of 
bacteria suitable for the plant and the soil is already largely practised. 

Bacteria and Soil Minerals.— Some of the bacterial products act 
upon the inorganic constituents of the soil. The carbonic dioxide and 
the organic acids act up>on compounds of lime and magnesia, practically 
insoluble in water, to form more soluble substances. The same is true 
of the rock phosphates, the silicate of potassium, sulphates, etc. 

Scientific farming is beginning to make use of the knowledge already 
acquired, and there is reason to hope that great practical advantages 
will flow from the investigation of the relation of bacteria to soil 
exhaustion and replenishment. 

The effect of excessive bacterial development appears at times to 
be harmful to the soil. Each crop seems to favor the growth of 
certain varieties, and the exhaustion of the soil which follows the con- 
stant raising of the same crop is now suspected to be due in part at 
least to the continuance of a few restricted species of bacteria io the 
soil, which failing to produce all the necessary substances for the 
nutrition of the special crop, vegetation suffers, or again the bacteria 
finally entirely dissipate substances already in the soil necessary to 
growth. 

The application of manure not only adds food for plant life, but also 
countless numbers of bacteria which make the food more available. 
The greatest number of bacteria are contained a little below the sur- 
face of the soil, where they are protected from drying and sunlight and 
are in contact with oxygen and with the roots and other food of the 
superficial soil. 

Bacteria in Sewage. — The materials which flow from our sewers are 
a menace to public health, mainly because they so frequently contain 
pathogenic bacteria. The other products of men and animals are 
offensive but rarely concentrated enough in drinking water to be 
appreciably deleterious. Sewage can be made harmless by being 
sterilized, but can be freed from offense only by the destruction of 
organic matter. This, except when chemical precipitants are used, is 
almost wholly obtained through bacterial processes. The purifying 
value of soil has long been recognized. This is largely due to the 
action of the soil bacteria. 

In 1895, the Englishman, Cameron, introduced the ** septic tank" 
which was a covered cemented pit. The sewage admitted at the bottom 
flowed out at the top, after about twenty-four hours' subjection to 
anaerobic conditions. The anaerobic bacteria during this time ferment 
the organic matter energetically, liquefy it, and develop abundant gas. 



100 PATHOGENIC MICRO-ORGANISMS. 

The knowledge that soil and sand filters act not only mechanically, 
but also and perhaps chiefly bacteriologically, having been acquired, 
intermittent soil filtration was established as one of the best means of 
bacteriologically purifying sewage. The sewage is conducted to the 
beds, allowed to pass through, and then after a few hours again poured 
on. This purification is based chiefly on the action of the aerobic 
bacteria in the upper layers of the soil or sand. The best practical 
results are obtained by combining the two processes, first the anaerobic 
treatment is used to break down the solid materials, and then the 
intermittent sand filtration, to oxidize the compounds and render 
these products harmless. With low temperatures the chemical 
changes are very much lessened and the filter beds act more as pure 
mechanical filters. The anaerobic bacteria change the proteid sub- 
stances into simple chemical compounds, among which is ammonia. 
The carbohydrates are changed into gaseous compounds, acids, etc. 
The gases are mainly nitrogen, carbon-dioxide and marsh-gas. The 
bacterial changes produced in sewage poured on contact beds made of 
coarse coke, clinkers, or other material act much as in the sand filters 
after the filtration. 

Varieties ot Baetetia in Filter Beds and Septic Tanks. — ^Tbe septic 
tanks all contain spore-bearing bacilli, which destroy cellulose, others 
that attack nitrogenous compounds. The cocci are in a minority. 
The filter beds have a number of small non-spore-bearing bacilli, some 
of these change ammonia into nitrites and nitrates. There are also 
denitrifying bacteria. As before mentioned, the bacterial efficiency 
of the bed is increased with suitable temperature and much lessened 
with low temperature. 

Sewage Farming. — The action of bacteria is availed of in disposing 
of sewage over fields. The amount of sewage which can be poured on 
a certain area is limited. One acre of land can usually take care of 
the sewage from one hundred persons. If too much is poured on, it 
runs off impurified or clogs the soils and prevents the access of oxygen 
to aerobic bacteria. In warm weather evaporation and bacterial 
activities are much greater than in cold weather. So far as experience 
shows, those who eat vegetables from these small farms contract no 
disease from them. 

Bacteria in Atmospbere. — ^The air is kept constantly in motion by 

winds so that fine particles are constantly being carried into it from 

the ground, especially in an inhabited area with its dusty streets. 

The rays of sunlight visibly reveal these particles to us. The bacteria 

in the dust of the fields and streets are carried along with these dust 

isually the harmless soil bacteria or the almost 

itinal bacteria of animals. Pathogenic human 

r to be carried in harmful numbers except under 

nces and usually as spores, such as those of 

le du.st from the wool and hides of infected ani- 

cilli from the infected manure. After a storm 

' air, while on a dry windy day many thousands 



THE SOIL BACTERIA AND THEIR FUNCTIONS. 101 

exist in a cubic meter. In warm weather rain carries down the bac- 
teria of the air. The bacteria in the air of the country are much 
less than in the city air. Forests decrease the number of bacteria. 

On high mountains and on the sea far from land bacteria are very 
scarce. The bacteria that multiply in the soil of street and country 
are almost entirely saprophytic types. Sunhght and drying rapidly 
destroy bacteria. In dwellings the bacterial content depends on many 
factors, of which the chief are the opening of windows to the outside 
dust-laden air, the cleanliness of the dwelling, and the amount of 
stirring up of the dust by sweeping. It is almost impossible to separate 
the effect of the bacteria which we inhale from that of the dust particles 
which they accompany. Both probably act as slight irritants and so 
predispose to definite infections. 

Bacteria in Industries. — The curing of tobacco is apparently due 
partly to bacterial processes and partly to the action of leaf enzymes. 

The preservation of foods against decomposition by bacteria, yeasts, 
moulds, and higher fungi is obtained by using processes which will 
prevent the growth of microorganisms. Drying, exposure to wood 
smoke with consequent absorption of creosote, the addition of salt 
and sugar, of acids such as vinegar, spices, germicides such as boracic 
acid, formaldehyde, all are familiar methods of making foods unsuit- 
able for bacterial growth. Instead of using food preserved by drying 
or chemicals, products may be kept at temperatures too low for bac- 
terial growth. Cold storage of meats, eggs, vegetables, etc., is now 
common. 

The sterilization of food substances by heat with protection from 
infection afterward is made use of extensively in the canning of fruits 
and vegetables. Care must be taken that absolutely all bacteria are 
killed, for otherwise decomposition will finally occur. 

Vinegar Making. — ^Vinegar is made from some weak alcoholic solu- 
tion by the union of alcohol with oxygen. This oxidation can be 
brought about by a purely chemical process. When vinegar is formed 
in the usual way bacteria are essential. The scum on the surface of 
the fermenting alcohol is a mass of microorganisms. The mother of 
vinegar was named mycoderma by Pearson. Kutzing showed that 
this was composed of living cells. Hansen proved these to be bacteria. 
We now know there are many varieties of bacilli capable of producing 
this fermentation. Each variety has its own optimum temperature and 
differs in the amount of acid it produces. Most of these have the pecu- 
liarity of growing at high temperatures into long threads without any 
traces of division. At low temperatures they produce long threads with 
swollen centres. The usual vinegar is made by using the variety of 
bacilli prevalent in the surroundings, but the custom is growing of 
adding to the pasteurized alcoholic solution the special variety desired 
in pure culture. 

Sauerkraut. — This is cabbage leaves shredded, slightly fermented, 
and prevented from decay by the lactic acid bacteria. At first both 
yeasts and bacteria increase together, but with the increase in acidity 



102 PATHOGENIC MICRO-ORGANISMS. 

all growth ceases. Putrefaction is prevented by the same cause. The 
lactic acid bacteria are the same as those found in sour milk. 

Ensilage. — ^The fermentation here is believed to be due partly to en- 
zymes in the corn tissues and partly to bacterial action. The first 
changes are due chiefly to the enzymes. 

The Bacterial Disease of Plants.— These are probably as serious and 
varied for plants as for animals. The pear blight, the wilt disease 
of melons, the brown rot of tomatoes, the black rot of cabbages are 
examples. These plant diseases can be communicated by means of 
the pathogenic pure cultures of bacteria experimentally just as readily 
as animal diseases by their specific bacteria. 

Bacterial Fermentation in Relation to Miscellaneous Products. — 

Pasteur in 1857 explained the process of fermentation as due to the 
action of microorganisms. He demonstrated that the change of 
sugar into lactic acid only occurred when living bacilli were present. If 
the fluid was steriUzed the fermentation ceased. He stated that "or- 
ganic liquids do not alter until a living germ is introduced into them." 
When the action is direct we speak of an organized fern/ent; when it 
is indirect, that is, due to the cell product, we call it an unorganized 
soluble ferment or enzyme. Similar enzymes are produced by the 
cells of the animal tissues, such as ptyalin, pepsin, and trypsine. Pas- 
teur's work led to the conclusion that the different fermentations were 
due to different varieties of organisms. The major part of fermenta- 
tion is due to yeast.^ Some important fermentations are due to bacteria 
and a few to the moulds. 

Wines and Beers. — Alcoholic Fermentation. — If there is a develop- 
ment of the yeast cells in a solution of grape-sugar we have a fermenta- 
tion of the sugar with a final development of alcohol and carbon-dioxide. 
It is thus that beers and wines are developed. When the carbohydrate 
is in the form of starch this is" first converted into sugar and then later 
into the final products. If the sugar is in the form of saccharose, it is 
first changed by the yeast ferments to glucose. In all these three 
forms of fermentation the sugar is changed into alcohol and carbonic 
acid. When the alcohol reaches about 13 per cent, it stops further 
fermentation. These yeasts called saccharomyces comprise a number 
of distinct varieties, some of which are cultivated while others, called 
**wild yeasts," propagate themselves. The distillery, brewery, and 
wine industries each make use of special yeasts and special conditions. 
The rising of bread is one of the most common uses of fermentation 
by yeast. The yeast acts upon the sugar made by the diastase from 
the starch. The resulting COj and alcohol creates myriads of little 
bubbles in the dough. 

Diseases in Beer and Wines. — Hansen, Pasteur, and others demon- 
strated that the spoiling of beers and wines was due to the development 
of varieties of bacteria .and yeasts which produce different kinds of 
fermentation from that desired. These produce alterations in flavor, 
bitterness, aciditv. 

*For further study of yeasts see Sec. II. 



CHAPTER VIII. 

THE DESTRUCTION OF BACTERIA BY CHEMICALS— PRACTICAL 

USE OF DISINFECTANTS. 

Many substances, when brought in contact with bacteria, combine 
with their cell substance and destroy the life of the bacteria. While 
in the vegetative stage bacteria are much more easily killed than when 
in the spore form, and their life processes are inhibited by substances 
less deleterious than those required to destroy them. 

Bacteria, both in the vegetative and in the spore form, differ 
among themselves considerably in their resistance to the poisonous 
effects of chemicals. The reason for this is not wholly clear, but it 
is connected with the structure and chemical nature of their cell 
substance. 

Chemicals in suflScient amount to destroy life are more poisonous 
at temperatures suitable for the best growth of bacteria than at lower 
temperatures, and act more quickly upon bacteria when they are sus- 
pended in fluids singly than when in clumps, and in pure water rather 
than in solutions containing organic matter. The increased energy of 
disinfectants at higher temperatures indicates in itself that a true 
chemical reaction takes place. In estimating the extent of the de- 
structive or inhibitive action of chemicals the following degrees are 
usually distinguished: 

1. The growth is not permanently .interfered with, but the patho- 
genic and zymogenic functions of the organism are diminished — 
attenuation. This loss of function is usually quickly recovered. 

2. The organisms are not able to multiply, but they are not destroyed 
— antiseptic action. When transferred to a suitable culture fluid free 
of the disinfectant these bacteria are capable of reproduction. 

3. The vegetative development of the organisms is destroyed, but 
not the spores — incomplete or complete sterilization or disinfection, 
according as to whether spores are present in the organisms exposed 
and as to whether these spores are capable of causing infection. 

4. Vegetative and spore formation are destroyed. This is com- 
plete sterilization or disinfection,^ 

The methods employed for the determination of the germicidal 
action of chemical agents on bacteria are, briefly, as follows: 

If it is desired to determine the minimum concentration of thechemi- 

* Disinfection strictly defined is the destruction of all organisms and their 
products which are capable of producing disease. Sterilization is the destruction 
of all saprophytic as well as parasitic bacteria. It is not necessary in most cases 
to require disinfectants to be capable of sterilizing infected materials containing 
spores, for there are but few varieties of pathogenic bacteria which produce spores. 

103 



104 PATHOGENIC MICRO-ORGANISMS. 

cal substance required to produce complete inhibition of growth we 
proceed thus: A 10 per cent, solution of the disinfectant is prepared 
and 1 C.C., 0.5 c.c, 0.3 c.c, 0.1 cc, etc., of this is added to 10 c.c. of 
liquefied gelatin, agar, or bouillon, or, more accurately 10 cc. minus 
the amount of solution added, in so many tubes. The tubes then 
contain 1 per cent., 0.5 per cent., 0.3 per cent., and 0.1 per cent, of 
the disinfectant. The fluid media in the tubes are then inoculated with 
a platinum loopful of the test bacteria. The melted agar and gelatin 
may be simply shaken and allowed to remain in the tubes, and 
watched a3 to whether any growth takes place, or the contents of the 
tubes may be poured into Petri dishes, where the development or lack of 
development of colonies and the number can be observed. If no 
growth occurs in any of the dilutions, higher dilutions are tested. 
Bacteria that have l>een previously injured in any way will be inhib- 
ited by much weaker solutions of chemicals than will vigorous cells. 
The same test can be made with material containing only spores. 

If it is desired to determine the degree of concentration required 
for the destruction of vegetative development, the organism to be 
used is cultivated in bouillon, and into each of a series of tubes is 
placed a definite amount of diluted culture from which all clumps of 
bacteria have been filtered; to these a definite amount of watery solution 
of different percentages of the disinfectant is added. At intervals of 
one, five, ten, fifteen, and thirty minutes, one hour, and so on a small 
platinum loopful of the mixture is taken from each tube and inoculated 
into 10 c.c. of fluid agar or gelatin, from which plate cultures are made. 
Whenever it is possible that the antiseptic power of the bacteria ap- 
proaches somewhat the germicidal, it is necessary to inoculate a second 
series of tubes from the first so as to decrease still further the amount 
of antiseptic carried over. The results obtained are signified as follows : 
X per cent, of the disinfectant in watery solution and at x temperature 
kills the organism in twenty minutes, y per cent, kills in one minute, 
and so on. If there be any doubt whether the trace of the disinfectant 
carried over with the platinum loops may have rendered the gelatin 
unsuitable for growth, thus falsifying results, control cultures should 
be made by adding bacteria which have been somewhat enfeebled by 
slight contact with the disinfectant to fluid to which a similar trace of 
the disinfectant has been added. If the strength of the disinfectant 
is to be tested for different substances it must be tested in these sub- 
stances or their equivalent, and not in water. 

The disinfectant to be examined should always be dissolved in an 
inert fluid, such as water; if, on account of its being difficultly solu- 
ble in water. It ta necessary to use alcohol for its solution, control experi- 
equired to determine the action of the alcohol on the 
etimes, as in the case of corrosive sublimate, the cbemi- 
le cell substance to form an unstable compound, which 
vth of the organism for a time before destroying it. 
id is not broken up in the media, it will probably 
y. In some tests it i.s of interest to break up this union 



DESTRUCTION OF BACTERIA BY CHEMICALS. 



105 



and note then whether the organism is alive or dead. With corrosive 
sublimate the bacteria die in fifteen to thirty minutes after the union 
occurs. 

In the above determinations the absolute strength of the disinfect- 
ant required is considerably less when culture media poor in albumin 
are employed than when the opposite is the case. Cholera spirilla 
grown in bouillon containing no peptone or only 0.5 per cent, of pep- 
tone are destroyed in half an hour by 0.1 per cent, of hydrochloric 
acid; grown in 2 per cent, peptone-bouillon, their vitality is destroyed 
in the same time on the addition of 0.4 per cent. HCl. In any case 
the organisms to be tested should all be treated in exactly the same 
way and the results accompanied by a statement of the conditions 
under which the tests were made. It is becoming the custom to 
state the power of a disinfectant in terms of comparison with pure 
carbolic acid. A substance which had the same destructive power 
in a 1 to 1000 solution as carbolic acid in a 1 to 100 solution would 
be rated as of a strength ten times that of carbolic acid. 

The following table gives the results and methods used in an actual 
experiment to test the effect of blood serum upon the disinfecting ac- 
tion of bichloride of mercury and carbolic acid upon bacteria: 

Tmt for the DirFKRENCE OF ErrEcT OF Bichloride of Mebcuky and Car- 
bolic Acid Solutions on Typhoid Bacilli in Sebum and 
IN Bouillon. 



A. Serum 2,5 c.c, 1 

HfCl,Bol.l:10002,5c.c. [+ + + 
Typhoid broth culture. ) 

B. Bouillon.. ..2,5 c.c. | ' 






' Solution 






bichloride. 


Typhoid broth culture, j ^ 

C. Serum 2, 5 c.c. 1 1 

C«rbolicsol.5%2.5c.c. \,+ +,- 
Typhoid broth culture. J 1 | i 

D. Bouillon, .2 5 c.c. 1 1 

C»rbolicBol. 5%2,5c.c. ^ + 

Typhoid broth culture. J 


- 


1 

! 


1 1 ( Solution 

- 1 - :< equals 24% 

\ carbolic acid. 

- - 1 Same. 



Many substances which are strong disinfectants become altered 
under the conditions in which they are used, so that they lose a portion 
or all of their germicidal properties; thus, quicklime and milk of lime 
act by means of their alkali and are disinfecting agents only so long as 
sufficient calcium hydroxide is present. If this is changed by the car- 
bon dioxide of the air into carbonate of lime it becomes harmless. 
Bichloride of mercury and many other chemicals form compounds 



106 PATHOGENIC MICRO-ORGANISMS. 

with many organic and inorganic substances, which, though still 
germicidal, are much less so than the original substances. Solutions of 
chlorine, peroxides, etc., when in contact with an excess of organic 
matter soon become inert because of the chemical compounds formed. 

The Disinfecting Properties of Inorganic Oomponnds.— Bichloride 
of Mercury. — This substance, which dissolves in 16 parts of cold 
water, when present in 1 part in 100,000 in nutrient gelatin or 
bouillon, inhibits the development of most forms of bacteria. In 
water 1 part in 50,000 will kill many varieties in a few minutes, but 
in bouillon twenty-four hours may be needed. With organic substances 
its power is lessened, so that 1 part to 1,000 may be required. Most 
spores are killed in 1 500 watery solution within one hour. Corrosive 
sublimate is less effective as a germicide in alkaline fluids containing 
much albuminous substance than in watery solution. In such fluids, 
.besides loss in other ways, precipitates of albuminate of mercury are 
formed which are at first insoluble, so that a part of the mercuric salt 
does not really exert any action. In alkaline solutions, such as blood, 
blood serum, pus, sputum, tissue fluids, etc., the soluble compounds 
of mercury are converted into oxides or hydroxides. 

For ordinary use, where corrosive sublimate is employed, solutions 
of 1 :500 and 1 : 2000 will suffice, when brought in contact with bacteria 
in that strength, to kill the vegetative forms within from one to twenty 
minutes, the stronger solution to be used when much organic matter 
is present. 

Mercuric chloride volatilizes slowly and it is better to wash off 
walls after use of bichloride solutions. Solutions of tliis salt should 
not be kept in metal receptacles. Mercuric chloride solution has dis- 
advantages in that it corrodes metals, irritates the skin, and forms 
almost inert compounds with albuminous matter. In order to avoid 
accidents, solutions of this odorless disinfectant should be colored bv 
some dye. 

Biniodide of Mercury. — This salt is very similar in its effect to the 
bichloride. 

Nitrate of Silver. — Nitrate of silver in watery solution has about 
one-fourth the value of the bichloride of mercury as a disinfectant, but 
nearly the same value in inhibiting growth. In albuminous solutions 
it is equal to bichloride of mercury. Compounds of silver nitrate and 
albuminous substances have been used because of the absence of 
irritative properties combined with moderate antiseptic power. 

Sulphate of Oopper. — This salt has about 50 per cent, of the value 
of mercuric chloride. It has a quite remarkable affinity for many 
species of algoe, so that when in water 1:1,000,000 it destroys many 
forms; 1:400,000 destroys typhoid bacilli in twenty-four hours when 
the water has no excessive amount of organic material. It is not known 
to be poisonous in this strength, so that it can be temporarily added 
to water supplies. 

Sulphate of Iron. — This is a much less powerful disinfectant than 
sulphate of copper. A 5 per cent, solution requires several days to 



DESTRUCTION OF BACTERIA BY CHEMICALS. 107 

kill the typhoid bacilli. It can only be considered as a mild antiseptic 
and deoderant. 

Zinc Ohloride. — This is very soluble in water, but is a still weaker 
disinfectant than copper sulphate. 

Sodium Oompounds. — A 30 per cent, solution of NaOH kills anthrax 
spores in about ten minutes, and in 4 per cent, in about forty-five 
minutes. One per cent, kills vegetative forms in a few minutes. 
Sodium carbonate kills spores with difficulty even in concentrated 
solution, but at 85° C. it kills spores in from eight to ten minutes. 
It is used frequently to cover metallic instruments. A 5 per cent, so- 
lution kills in a short time the vegetative forms of bacteria. Even 
ordinary soapsuds have a slight bactericidal as well as a marked 
cleansing effect. The bicarbonate has almost no destructive effect on 
bacteria. 

Oaldnm Oomponnds. — Calcium hydroxide, Ca(OH)j, is a powerful 
disinfectant; the carbonate, on the other hand, is almost without eflFect. 
The former is prepared by adding one pint of water to two pounds 
of lime (quicklime, CaO). Exposed to the air the calcium hydrate 
slowly becomes the inert carbonate. A 1 per cent, watery solution 
of the hydroxide kills bacteria which are not in the spore form within 
a few hours. A 3 per cent, solution kills typhoid bacilli in one hour. 
A 20 per cent, solution added to equal parts of faeces or other filth and 
mixed with them will completely sterilize them within one hour. 

Effect of Adds. — An amount of acid which equals 40 c.c. of normal 
hydrochloric acid per litre is sufficient to prevent the growth of all 
varieties of bacteria and to kill many. Twice this amount destroys 
most bacteria within a short time. The variety of acid makes little 
difference. Bulk for bulk, the mineral acids are more germicidal than 
the vegetable acids, but that is because their molecular weight is so 
much less. A 1 : 500 Solution of sulphuric acid kills typhoid bacilli 
within one hour. Hydrochloric acid is about one-third weaker, and 
acetic acid somewhat weaker still. Citric, tartaric, malic, formic, 
and salicylic acids are similar to acetic acid. Boric acid destroys the 
less resistant bacteria in 2 per cent, solution and inhibits the others. 

Oaseoos Disinfectants. — The germicidal action of gases is much 
more active in the presence of moisture than in a dry condition. 

Sulphur Dioxide (SOj). — Numerous experiments have been made 
with this gas owing to the fact that it has been so extensively used for 
the disinfection of hospitals, ships, apartments, clothing, etc. This gas 
is a much more active germicide in a moist than in a dry condition; 
due, no doubt, to the formation of the more active disinfecting agent — 
sulphurous acid (H2SO3). In a pure state anhydrous sulphur dioxide 
does not destroy spores, and is not certain to destroy bacteria in the 
vegetative form. Sternberg has shown that the spores of the Bacillus 
onthracis and Bacillus subiilis are not killed by contact for some time 
with liquid SO, (liquefied by pressure). Koch found that various 
species of spore-bearing bacilli exposed for ninety-six hours in a dis- 
infecting chamber to the action of SO,, in the proportion of from 4 to 



108 PATHOGENIC MICRO-ORGANISMS, 

6 per cent, by volume, were not destroyed. In the absence of spores, 
however, the anthrax bacillus in a moist condition, attached to silk 
threads, was found by Sternberg to be destroyed in thirty minutes in 
an atmosphere containing 1 volume per cent. 

As the result of a large number of experiments with SO, as a disin- 
fectant it has been determined that an "exposure for eight hours to 
an atmosphere containing at least 4 volumes per cent, of this gas in 
the presence of mmsture" will destroy most, if not all, of the patho- 
genic bacteria in the Absence of spores. Four pounds of sulphur 
burned for each 1000 cubic feet will give an excess of gas. 

Peroxide of Hydrogen (HjOj). — This is an energetic disinfectant, and 
in 2 per cent, solution (about 40 per cent, of the ordinary commercial 
article) will kill the spores of anthrax in from two to three hours. A 
20 per cent, solution of a good commercial hydrogen peroxide solution 
will quickly destroy the pyogenic cocci and other spore-free bacteria. 
It combines with organic matter, becoming inert. It is prompt in its 
action and not poisonous, but apt to deteriorate if not properly kept. 

Ohlorine. — Chlorine is a powerful gaseous germicide, owing its 
activity to its aflSnity for hydrogen and the consequent release of 
nascent oxygen when it comes in contact with microorganisms in 
moist condition. It is, therefore, a much more active germicide in 
the preisence of moisture than in a dry condition. Thus, Fischer and 
Proskauer found that dried anthrax spores exposed for an hour in 
an atmosphere containing 44.7 per cent, of dry chlorine were not 
destroyed; but if the spores were previously moistened and were 
exposed in a moist atmosphere for the same time, 4 per cent, was 
eflFective, and when the time was extended to three hours 1 per cent, 
destroyed their vitality. The anthrax bacillus, in the absence of 
spores, was killed by exposure in a moist atmosphere containing 1 part 
to 2500 for twenty-four hours. 

In watery solutions 0.2 per cent, kills spores within five minutes and 
the vegetative forms almost immediately. 

Ohlorinated Lime (Galled "Ohloride of Lime")* — Chlorinated lime 
is made by passing nascent chlorine gas over unslaked lime. It should 
not contain less than 10 per cent, of available chlorine, and can now be 
obtained containing 30 per cent. It should have a strong odor of 
chlorine. Its efficacy depends on the chlorine it contains in the form of 
hypochlorites. The calcium hypochlorite is readily broken up into 
hypochlorous acid. A solution in water of . 6 to 1 per cent, of chlorin- 
ated lime will kill most bacteria in one to five minutes, and 1 part 
in 100,000 will destroy typhoid bacilli in twenty-four hours. A 6 per 
cent, solution usually destroys spores within one hour. Chlorinated 
lime not only bleaches, but destroys fabrics. 

The Hypochlorites (Labarraque's Solution). — Solutions of hypo- 
chlorites are practically the same as solutions of chlorinated lime and 
are much more expensive. 

Bromine and iodine are of about the same value as chlorine for 
gaseous disinfectants, in the moist condition; but, like chlorine, they 



DESTRUCTION OF BACTERIA BY CHEMICALS. 109 

are not applicable for general use in house disinfection, owing to their 
poisonous and destructive properties; they have a use in sewers and 
similar places. 

Trichloride of iodine in . 6 per cent, solution destroys the vegetative 
forms of bacteria in five minutes. 

Organic Disinfectants. — Alcohol in 10 per cent, solution inhibits 
the growth of bacteria; absolute alcohol kills bacteria in the vegetative 
form in from several to twenty-four hours. According to Epstein, 50 
per cent, alcohol (in water) has more germicidal power than any other 
strength, the power gradually diminishing with both stronger and 
weaker solutions. 

Formaldehyde. — Formaldehyde, or formic aldehyde, was isolated 
by von HoflFmann in 1867, who obtained it by passing the vapors of 
methyl-alcohol mixed with air over finely divided platinum heated to 
redness. The methyl-alcohol is oxidized and produces formaldehyde 
as follows : 

CH,OH -f O -CH,0 + H^O. 

Formaldehyde is a gaseous compound possessed of an extremely 
irritating odor. At a temperature of 68° F. the gas is polymerized — 
that is to say, a second body is formed, composed of a union of two 
molecules of CHjO. This is known as a paraformaldehyde, and is a 
white, soapy body, soluble in boiling water and in alcohol. Formal- 
dehyde is sold in commerce as a clear, watery liquid containing from 
33 to 40 per cent, of the gas and 10 to 20 per cent, of methyl-alcohol, 
its chief impurity. If the commercial solution — ordinarily known in 
the trade as ** formalin*' — is evaporated or concentrated above 40 per 
cent., paraformaldehyde results; and when this is dried in vacuo over 
sulphuric acid a third body — trioxymethylene — is produced, consisting 
of three molecules of CHjO. This is a white powder, almost soluble 
in water or alcohol, and giving oflF a strong odor of formaldehyde. 
The solid polymers of formaldehyde, when heated, are again reduced 
to the gaseous condition; ignited, they finally take fire and bum with 
a blue flame, leaving but little ash. When burned they have no 
germicidal properties. 

Formaldehyde has an active aflBnity for many organic substances, 
and forms with some of them definite chemical combinations. It 
combines readily with ammonia to produce a compound called hexa- 
methylene tetramine, which possesses neither the odor nor the anti- 
septic properties of formaldehyde. This action is made use of in 
neutralizing the odor of formaldehyde when it is desired to dispel it 
rapidly after disinfection. Formaldehyde also forms combinations 
with certain aniline colors — viz., fuchsin and safranin — the shades of 
which are thereby changed or intensified. These dyes are tests for 
aldehydes. These are the only colors, however, which are thus af- 
fected, and as they are seldom used in dyeing, owing to their liability 
to fade, this effect is of little practical significance. The most deli- 
cate fabrics of silk, wool, cotton, fur, leather, etc., are unaflFected in 



no PATHOGENIC MICRO-ORGANISMS. 

texture or color by formaldehyde. Iron and steel are attacked, after 
long exposure, by the gas in combination with watery vapor; but copper, 
brass, nickel, zinc, silver, and gilt work are not at all acted upon. For- 
maldehyde unites with nitrogenous products of decay — fermentation 
or decomposition — forming true chemical compounds, which are odor- 
less and sterile. It is thus a true deodorizer in that it does not replace 
one odor by another more powerful, but forms new chemical compounds 
which are odorless. Formaldehyde has a peculiar action upon albu- 
min, which it transforms into an insoluble and indecomposable sub- 
stance. It renders gelatin insoluble in boiling water and most acids and 
alkalies. It is from this property of combining chemically with the 
albuminoids forming the protoplasm of bacteria that formaldehyde is 
supposed to derive its bactericidal powers. Formaldehyde is an excel- 
lent preservative of organic products. It has been proposed to make 
use of this action for the preservation of meat, milk, and other food 
products; but, according to Trillat and other investigators, formalde- 
hyde renders these substances indigestible and unfit for food. It has 
been successfully employed as a preservative of pathologic and 
histologic specimens. 

There are no exact experiments recorded of the physiologic action 
of formaldehyde on the human subject when taken internally. A 1 
per cent, solution has been taken in considerable quantity without 
serious results; and trioxymethylene has been given in doses up to 
90 grains as an intestinal antiseptic. The vapors of formaldehyde 
are extremely irritating to the mucous membrane of the eyes, nose, 
and mouth, causing profuse lacrimatton, coryza, and flow of sahva. 
Aronson reports that in many of his experiments rabbits and guinea- 
pigs allowed to remain for twelve .and twenty-four hours in rooms 
which were being disinfected with formaldehyde gas were found to 
be perfectly well when the rooms were opened. On autopsy the ani- 
mals showed no injurious effects of the gas. Others have noticed that 
animals, such as dogs and cats, which have accidently been confined 
for any length of time in rooms undergoing formaUlehyde disinfection 
occasionally died from the effects of the gas. Many observers, how- 
ever, have reported that insects, .such as roaches, flies, and bedbugs, 
are not, as a rule, affected. The result of these obser\'ations would seem 
to indicate that although formaldehyde is comparatively non-toxic to 
the higher forms of animal life, nevertheless a certain degree of caution 
should be obser\'ed in the use of this agent. It is important to remem- 
ber that formaldehyde is practically inert as an insecticide except in 
extremely great concentrations. 

The researches of Pottevin and Trillat have shown that the germi- 
of the gas depends not only upon its concentration, but 
■ temperature and the condition of the objects to be steril- 
th other gaseous disinfectants — -viz,, sulphur dioxide and 
las been found that the action is more rapid and complete 
leratures— i. e., at 35" to 45" C. (95" to 120" F.)— and 
t objects are moist than at lower temperatures and when 



DESTRUCTION OF BACTERIA BY CHEMICALS. Ill 

the objects are dry. Still, it has been repeatedly demonstrated by 
actual experiment in rooms that it is possible to disinfect the surface of 
apartments and articles contained in them, under the conditions of 
temperature and moisture ordinarily existing in rooms even in winter, 
by an exposure of a few hours to a saturated atmosphere of formalde- 
hyde gas. The results of numerous experiments have shown that in 
the air 2.5 per cent, by volume of the aqueous solution, or 1 percent, 
by volume of the gas, are sufficient to destroy fresh virulent cultures of 
the common non-spore-bearing pathogenic bacteria in a few minutes. 

Stahl has shown that bandages and iodoform gauze can be kept 
well sterilized by placing in the jars containing them pieces of a prepa- 
ration of paraformaldehyde in tablet form containing 50 per cent, 
of formaldehyde. The same experimenter has also succeeded in 
making carpets and articles of clothing germ-free by spraying them 
with O.o to 2 per cent, solution of formaldehyde for fifteen to twenty 
minutes without the color of the fabrics being in any way aflFected. 
The investigations of Trillat, Aronson, Pottevin, and others have 
shown that a concentration of 1/10000 of the aqueous solution (40 per 
cent.), equal to 1 / 25000 of pure formaldehyde, was safe and sufficiently 
powerful to retard bacterial growth. 

A 2 per cent, watery solution of formalin destroys the vegetative 
forms of bacteria within five to thirty minutes. In our experiments 
formalin has upon the vegetative forms about one-half the strength of 
pure carbolic acid. 

Ohloroform (CHCl,). — This substance, even in pure form, does not 
destroy spores, although it kills bacteria in vegetative form, even in 
1 per cent, solution. Chloroform is used practically as an antiseptic 
in antitoxic sera and in blood serum for culture purposes. The 
chloroform is expelled from the serum by heating it to 55° C. 

Iodoform (CHI3). — This substance has but very little destructive 
action upon bacteria; indeed, upon most varieties it has no appreciable 
effect whatever. When mixed with putrefying matter, wound dis- 
charges, etc., the iodoform is reduced into soluble iodine compounds, 
which act partly destructively upon the bacteria and partly by uniting 
with the poisons already produced. 

Carbolic Add (CgHjOH). — Pure phenol crj^stallizes in long, color- 
less crystals. In contact with air it deliquesces. It has a penetrating 
odor, a burning taste, and is a corrosive poison. It is soluble at 
ordinary temperatures in about 15 parts of water. Carbolic acid 
dissolves in water with some difficulty and should be therefore thor- 
oughly mixed. It is not destructive to fabrics, colors, metals, or wood, 
and does not combine as actively with albuminous matters as bichloride 
of mercury. It is therefore more suitable for the disinfection of faeces, 
etc. A solution having 1 part to 1000 inhibits the growth of bacteria; 
1 part to 400 kills the less resistant bacteria, and 1 part to 100 kills 
the remainder. A 6 per cent, solution kills the less resistant spores 
within a few hours and the more resistant in from one day to four 
weeks. A slight increase in temperature aids the destructive action; 



112 PATHOGENIC MICRO-ORGANISMS. 

thus, even at 37.5° spores are killed in three hours. A 3 per cent, 
solution kills streptococci, staphylococci, anthrax bacilli, etc., within 
one minute. Carbolic acid loses much of its value when in solution in 
alcohol or ether. An addition of . 6 HCl aids its activity. Carbolic 
acid is so permanent and so comparatively little influenced by albumin 
that it is rightly widely used in practical disinfection even in places of 
more powerful substances. 

Oresol. — Cresol [C^H^CCHj)©!!] is the chief ingredient of the 
so-called "crude carbolic acid." This is almost insoluble in water, 
and therefore of restricted value. Many methods are used for bring- 
ing it into solution so as to make use of its powerful disinfecting 
properties. With equal parts of crude sulphuric acid it is a powerful 
disinfectant, but it is, of course, strongly corrosive. An alkaline 
emulsion of the cresols and other products contained in ''crude" car- 
bolic acid with soap is called creolin. It is used in 1 to 5 per cent, 
emulsions. It is fully as powerful as pure carbolic acid. Lysol is 
similar to creolin, except that it has more of the cresols and less of the 
other products. It and creolin are of about the same value. 

Tricresol. — Tricresol is a refined mixture of the three cresols (meta- 
cresol, paracresol, and orthocresol). It is soluble in water to the 
extent of 2.6 per cent, and its disinfecting power is about three times 
as great as that of carbolic acid. 

Oreolin. — Creolin contains 10 per cent, of cresols held in solution by 
soap. 

LysoL — Lysol contains about 50 per cent, of cresols. It mixes 
with water in all dilutions. 

Oil of turpentine, 1:200, prevents the growth of bacteria. 

Oamphor has very slight antiseptic action. 

Oreosote in 1:200 kills many bacteria in ten minutes; 1:100 failed 
to kill tubercle bacilli in twelve hours. 

Essential oils: Cardiac and Meumir found that the essences of 
cinnamon, cloves, thyme, and others killed typhoid bacilU within one 
hour. Sandalwood required twelve hours. 

Thymol and eucalyptol have about one-fourth the strength of car- 
bolic acid (Behring). 

Oil of peppermint in 1 : 100 solution prevents the growth of bacteria. 

Tables of Antiseptic Values.* 

Alum 1 : 222 Mercuric chloride 1 : 14^00 

Aluminum acetate 1 : 6000 Mercuric iodide 1 : 40.000 

Ammonium chloride 1 : 9 Potassium bromide 1 : 10 

Boric acid 1 : 143 Potassium iodide 1 : 10 

Calcium chloride 1 : 25 Potassium permanganate 1 : 300 

Calcium hypochlorite 1 : 1000 Pure formaldehyde 1 : 25,000 

Cart>olic acid 1 : 333 Quinine sulphate 1 : 800 



Chloral hydrate 1 : 107 Silver nitrate 1 : 12.500 

Ferrous sulphate 1 : 200 Sodium chloride 1 : 6 



Cupric sulphate 1 : 2000 Sodium borate 1 : 14 



Formaldehyde (40^0 I : 10,000 Zinc chloride 1 : 500 

Hydrogen jjeroxide 1 : 20,000 Zinc sulphate 1 : 20 

* These figures are approximately correct, and represent the percentage of 
disinfectant required to be Padded to a fluid containing considerable organic 
material, in order permanently to prevent any bacterial growth. Solutions of 
half the given strength will inhibit the growth of most bacteria and prevent the 
growth of many varieties. 



CHAPTER IX. 

PRACTICAL DISINFECTION AND STERILIZATION (HOUSE, PER- 
SON, INSTRUMENTS, AND FOOD)— STERILIZATION OF 

MILK FOR FEEDING INFANTS. 

DISINFECTANTS AND METHODS OF DISINFECTION EMPLOYED 

IN THE HOUSE AND SICK-ROOM. 

Disinfection and Disinfectants. — Sunlight, pure air, and cleanliness 
are always very important agents in maintaining health and in protect- 
ing the body against many forms of illness. When, however, it 
becomes necessary to guard against such special dangers as accumulated 
filth or contagious diseases, disinfection and general cleaning up are 
essential. In order that disinfection shall afford complete protection 
it must be thorough; and perfect cleanHness is better, even in the 
presence of contagious disease, than filth with poor disinfection. 

Since all forms of fermentation, decomposition, and putrefaction, 
as well as the infectious and contagious diseases, are caused by micro- 
organisms, it is the object of disinfection to kill these. Decomposition 
and putrefaction should at all times be prevented by the immediate 
destruction or removal from the neighborhood of the dwelling of all 
useless putrescible substances. In order that as few articles as possible 
shall be exposed to the germs causing the contagious diseases, and thus 
become carriers of infection, it is important when conditions allow of 
it that all articles not necessary for immediate use in the care of the 
sick person, especially upholstered furniture, carpets, and curtains, 
should be removed from the room before placing the sick person in it. 

Agents for Cleansing and Disinfection. — Too much emphasis 
cannot be placed upon the importance of cleanliness, both as regards 
the person and the dwelling, in preserving health and protecting the 
body from all kinds of infectious disease. Sunlight and fresh air 
should be freely admitted through open windows, and personal clean- 
liness should be attained by frequently washing the hands and body, 
disinfecting linen fabrics infected by expectoration, bowel discharges, 
etc. 

Cleanliness in dwellings, and in all places where men go, may, 
under ordinary circumstances, be well maintained by the use of the 
two following solutions: 

1. Soapsuds Solution. — ^For simple cleansing, or for cleansing after 
the method of disinfection by chemicals described below, one ounce 
of common soda should be added to twelve quarts of hot soapsuds 
(soft soap and water). 

2. Strong Soda Solution. — This, which is a stronger and more effec- 
tive cleansing solution and also a fairly efficient disinfectant, is made by 

8 113 



114 PATHOGENIC MICRO-ORGANISMS, 

dissolving one-half pound of common soda in three gallons of hot 
water. The solution thus obtained should be applied by scrubbing 
with a hard brush. 

When it becomes necessary to arrest putrefaction or to prevent the 
spread of contagious diseases by surely killing the living germs which 
cause them, more powerful agents must be employed than those re- 
quired for simple cleanliness, and these are commonly called disin- 
fectants. The following are some of the most reliable ones: 

3. Heat. — Complete destruction by fire is an absolutely safe method 
of disposing of infected articles of small value, but continued high 
temperatures not as great as that of fire will destroy all forms of life; 
thus, boiling or steaming in closed vessels for one-half hour will 
absolutely destroy all disease germs. 

4. Oarbolic Add Solution. — Dissolve six ounces of carbolic acid in 
one gallon of hot water (200 grams in 4000 c.c). This makes approxi- 
mately a 5 per cent, solution of carbolic acid, which, for many purposes, 
may be diluted with an equal quantity of water. The commercial 
** soluble crude carbolic acid" which is cheaper and twice as eflFective 
as the carbolic acid, can be used for privies and drains.^ It makes 
a white emulsion on account of its not entering readily into solution. 
Care must be taken that the pure acid does not come in contact with 
the skin. 

5. Bichloride Solution (bichloride of mercury or corrosive subli- 
mate). — Dissolve sixty grains of pulverized corrosive sublimate and 
two tablespoonfuls of common salt in one gallon of hot water. This 
solution, which is approximately 1 : ICKX), must be kept in glass, earthen, 
or wooden vessels (not in metal vessels). For safety it is well to color 
the solution. 

The carbolic and bichloride solutions are very poisonous when 
taken by the mouth, but are harmless when used externally. 

6. BftUk of Lime. — This mixture is made by adding one quart of 
dry, freshly slaked lime to four or five quarts of water. (Lime is 
slaked by pouring a small quantity of water on a lump of quicklime. 
The lime becomes hot, crumbles, and as the slaking is completed a 
white powder results. The powder is used to make milk of lime.) 
Air-slaked lime (the carbonate) has no value as a disinfectant. 

7. Dry OUorinated lime, "OUoride of Lime."— This must be fresh 
and kept in closed vessels or packages. It should have the strong, 
pungent odor of chlorine. 

8. Formalin. — Add 1 part of formalin to 10 of water. This equals 
in value the 5 per cent, carbolic acid solution. 

9. Oreolin, Tricresol, and Lysol. — The first is of about the same 
value as pure carbolic acid, the latter two about three times as powerful. 

The proprietary disinfectants, which are so often widely advertised 

* The cost of the pure carbolic acid solution is much greater than that of most 
of the other solutions, but except for the disinfection of the skin, which in some 
persons it irritates, and of woodwork, it is generallj^ much to be preferred by 
those not thoroughly familiar with disinfectants, as it does not deteriorate, and 
is rather more uniform in its action than some of the other disinfectants. 



PRACTICAL DISINFECTION AND STERILIZATION. 115 

and whose composition is kept secret, are relatively expensive and often 
unreliable and ineflScient. It is important to remember that substances 
which destroy or disguise bad odors are not necessarily disinfectants, 
and that there are very few disinfectants that are not poisonous when 
taken internally. Their value should be stated in the circular in 
comparison with pure carbolic acid, so that their strength may be 
known. 

Methods of Disinfection in Infectious and Contagious Diseases. — 

The diseases to be commonly guarded against, outside of surgery, by 
disinfection are scarlet fever, measles, diphtheria, tuberculosis, small- 
pox, typhoid and typhus fever, bubonic plague, and cholera. 

1. Hands and Person. — Dilute the 5 per cent, carbolic solution with 
an equal amount of water or use the 1 : 1000 bichloride solution with- 
out dilution. Hands soiled in caring for persons suflFering from con- 
tagious diseases, or soiled portions of the patient's body, should be 
immediately and thoroughly washed with one of these solutions and 
then washed with soap and water, and finally immersed again in the 
solutions. The nails should always be kept perfectly clean. Before 
eating, the hands should be first washed in one of the above solutions, 
and then thoroughly scrubbed with soap and water by means of a 
brush. 

2. Soiled clothing, towels, napkins, bedding, etc., should be immedi- 
ately immersed in the carbolic solution, in the sick-room, and soaked 
for one or more hours. They should then be wrung out and boiled 
in the soapsuds solution for twenty minutes. Articles such as beds, 
woollen clothing, etc., which cannot be washed, should at the end of 
the disease be referred to the Health Department, if such is within 
reach, for disinfection or destruction; or if there is no public disin- 
fection, these goods should be thoroughly exposed to formaldehyde gas, 
as noted later. 

3. Food and Drink. — ^Food thoroughly cooked and drinks that have 
been boiled are free from disease germs. Food and drinks, after 
cooking or boiling, if not immediately used, should be placed when 
cool in clean dishes or vessels and covered. In the presence of an 
epidemic of cholera or typhoid fever, milk and water used for drink- 
ing, cooking, washing dishes, etc., should be boiled before using, and 
all persons should avoid eating uncooked fruit and fresh vegetables. 
Instead of boiling, milk may be heated to 80® C. for twenty minutes. 

4. Discharges of all kinds from the mouth, nose, bladder, and bowels 
of patients suffering from contagious diseases should be received 
into glass or earthen vessels containing the carbolic solution, or milk of 
lime, or they should be removed on pieces of cloth, which are immer 
diately immersed in one of these solutions or destroyed by fire. Special 
care should be observed to disinfect at once the vomited matter and 
the intestinal discharges from cholera patients. In typhoid fever the 
urine and the intestinal discharges, and in diphtheria, measles, and 
scarlet fever the discharges from the throat and nose all carry infection 
and should be treated in the same manner. The volume of the solu- 



1 



116 PATHOGENIC MICRO-ORGANISMS. 

tion used to disinfect discharges should be at least twice as great as 
that of the discharge, and should completely mix with it and cover 
it. After standing for an hour or more the disinfecting solution with 
the discharges may be thrown into the water-closet. Qoths, towels, 
napkins, bedding, or clothing soiled by the discharges must be at once 
placed in the carbolic solution, and the hands of the attendants disin- 
fected, as described above. In convalescence from measles and scarlet 
fever the scales from the skin are also carriers of infection. To prevent 
the dissemination of disease by means of these scales the skin should be 
carefully washed daily in warm soap and water. After use the soap- 
suds should be disinfected and thrown into the water-closet. 

Masses of feeces are extremely difficult to disinfect except on the 
surface, for it takes disinfectants such as the carbolic acid solution 
some twelve hours to penetrate to their interior. If fsecal masses are 
to be thrown into places where the disinfectant solution covering 
them will be washed off, it will be necessary to be certain that the 
disinfectant has previously penetrated to all portions and destroyed 
the disease germs. This can be brought about by stirring them with 
the disinfectant and allowing the mixture to stand for one hour, or 
by washing them into a pot holding soda solution which is already 
at the boiling temperature, or later will be brought to one. 

5. Spatom from Oonsumptive Patients. — ^The importance of the 
proper disinfection of the sputum from consumptive patients is still 
underestimated. Consumption is an infectious disease, and is al- 
ways the result of transmission from the sick to the healthy or from 
animals to man. The sputum contains the germs which cause the 
disease, and in a large proportion of cases is the source of infection. 
After being discharged, unless properly disposed of, it may become 
dry and pulverized and float in the air as dust. This dust contains 
the germs, and is a common cause of the disease, through inhalation. 
In all cases, therefore, the sputum should be disinfected when dis- 
charged. It should be received in covered cups containing the car- 
bolic or milk-of-lime solution. Handkerchiefs soiled by it should 
be soaked in the carbolic solution and then boiled. Dust from the 
walls, mouldings, pictures, etc., in rooms that have been occupied by 
consumptive patients, where the rules of cleanliness have not been 
carried out, contain the germs and will produce tuberculosis in ani- 
mals when used for their inoculation; therefore, rooms should be 
thoroughly disinfected before they are again occupied. If the sputum 
of all consumptive patients were destroyed at once when discharged a 
large prof)ortion of the cases of the disease would be prevented. 

6. Olosets, Kitchen and Hallway Sinks, etc. — The closet should never 
be used for infected discharges until they have been thoroughly dis- 
infected; if done, one quart of carbolic solution or of 5 per cent, solution 
of formalin should be poured into the pan (after it is emptied) and 
allowed to remain there. Sinks should be flushed at least once daily. 

7. Dishes, knives, forks, spoons, etc., used by a patient should, as a 
rule, be kept for his exclusive use and not removed from the room. 



PRACTICAL DISINFECTION AND STERILIZATION, 117 

They should be washed first in the carbolic solution, then in boiling 
hot soapsuds, and finally rinsed in hot water. These washing fluids 
should afterward be thrown into the water-closet. The remains of 
the patient's meals may be burned or thrown into a vessel containing 
the carbolic solution or milk of lime, and allowed to stand for one hour 
before being thrown away. 

8. Rooms and Their Contents. — Rooms which have been occupied 
by persons suffering from contagious disease should not be again occu- 
pied until they have been thoroughly disinfected. It is true that when 
the patient is freed from isolation most of the disease germs have 
already died, but a few may have survived. The danger from infection 
is much greater when cases are removed during the acute illness. For 
disinfecting rooms either careful fumigation with formaldehyde gas or 
sulphur should be employed, or this combined with the following 
procedure: Carpets, curtains, and upholstered furniture which have 
been soiled by discharges, or which have been exposed to infection in 
the room during the illness, will be removed for disinfection to cham- 
bers where they can be exposed to formaldehyde gas and moderate 
warmth for twelve to twenty-four hours, or to steam. Some carpets, 
such as many Wiltons, are discolored by moist steam. These must be 
put in the formaldehyde chamber. Woodwork, floors, and plain furni- 
ture will be thoroughly washed with the soapsuds and bichloride solu- 
tions. After disinfection is finished it is well to remove the dried 
bichloride of mercury from the walls. 

9. Rags, cloths, and articles of small value, which have been soiled 
by discharges or infected in other ways, should be boiled or burned. 

10. In case of death the body should be completely wrapped in 
several thicknesses of cloth wrung out of the carbolic or bichloride 
solution, and when possible placed in an hermetically sealed coflBn. 

It is important to remember that an abundance of fresh air, sunlight, 
and absolute clewiliness not only helps protect the attendants from 
infection and aids in the recovery of the sick, but directly destroys the 
bacteria which cause disease. 

Methods of Oleanliness and Disinfection to Prevent the Occurrence 
of Illness. — 1. Water-closet bowls and all receptacles for hnman excre- 
ment should be kept perfectly clean by frequent flushing with a large 
quantity of water, and as often as necessary disinfected with the car- 
boUc, bichloride, or other eflBcient solutions. The woodwork around 
and beneath them should be frequently scrubbed with the hot soapsuds 
solution. 

2. Sinks and the woodwork around and the floor beneath them 
should be frequently and thoroughly scrubbed with the hot soapsuds 
solution. 

3. School Smks. — School sinks should be thoroughly flushed with a 
large quantity of water at least twice daily, and should be carefully 
cleaned twice a week or oftener by scrubbing. Several quarts of the 
crude carbolic solution should be frequently thrown in the sink after it 
has been flushed. 



118 PATHOGENIC MICRO-ORGANISMS, 

4. Oesspools and Privy Vaults. — An adundance of milk of lime or 
chloride of lime should be thrown into these daily, and their contents 
should be frequently removed. 

5. Refrigerators and the surfaces around and beneath them, dumb- 
waiters, etc., may be cleaned by scrubbing them with the hot soapsuds 
solution. 

6. Traps. — All traps should be flushed daily with an abundance of 
water. If at any time they become foul they may be cleaned by 
pouring considerable quantities of the hot strong soda solution into 
them, followed by the carbolic or formalin solution. 

7. The woodwork in school -houses should be scrubbed daily with 
hot soapsuds. This refers to floors, doors, door-handles, and all 
woodwork touched by the scholars' hands. 

8. Spittoons in all public places should be emptied daily and washed 
with the hot soapsuds solution, after which a small quantity of the 
carbolic solution or milk of lime should be put in the vessel to receive 
the expectoration. 

9. Oars, Ferry-boats, and Public Oonveyances. — The floors, door- 
handles, railings, and all parts touched by the hands of passengers 
should be washed frequently with the hot soapsuds solution. Slat- 
mats from cars, etc., should be cleaned by scrubbing with a stiff 
brush in the hot soapsuds solution. 

Telephone receiver mouth-pieces should also be frequently cleansed. 

Use of Bromine Solution as a Deodorant.— iS/ati^A/ef-Aou^^^, 

butchers* ice-boxes and wagons, trencheSy excavations, stable floors, 
manure-vatdts, dead animals, offal, offal docks, etc., may be deodorized 
by a weak solution of bromine, which is a valuable agent for this 
purpose. The bromine solution, however, is only temporary in its 
action, and must be used repeatedly. It should be applied by sprin- 
kling. Although somewhat corrosive in its action on metals, it is 
otherwise harmless. 

The solution of bromine must be prepared with great care, as the 
pure bromine from which it is made is dangerous. It is very caustic 
when brought in contact with the skin; it is volatile and its fumes are 
very irritating when inhaled. To prepare the solution an ounce 
bottle of liquid bromine is dropped into three gallons of water, and 
broken under the water and thoroughly stirred. 

The Practical Emplojrment of Formaldehyde Gkts in the Surface 
Disinfection of Booms and the Disinfection of Ooods which would 
be Injured by Heat. — ^Formaldehyde gas has come into such general 
use, and is for many purposes so valuable, that the description of 
methods employed to generate and use it will be given in detail. 

If we consider now the practical application of formaldehyde gas 
for purposes of disinfection we find that its destructive action on 
microorganisms depends upon a number of factors, the chief of which 
are its concentration in the surrounding atmosphere, the length of the 
contact, the existing temperature, the accompanying moisture, and 
the nature of the organism. 



PRACTICAL DISINFECTION AND STERILIZATION. 119 

The necessary concentration of gas in the surrounding atmosphere 
to kill the microorganisms varies with each species, for some resist 
chemical agents much more than others, and also with the freedom of 
access of the gas to the bacteria, for if they are under cover or within 
fabrics a greater amount of gas must be generated than if they are 
freely exposed. 

For purely surface disinfection, when the less resistant bacteria or 
other microorganisms are to be destroyed, there will be required, 
according to the method used, ten to twelve ounces of formalin of full 
strength, or its equivalent, to 1000 cubic feet of air space. 

For the destruction of the more resistant but non-spore-bearing 
forms, such as typhoid fever or tubercle bacilli, at least twelve ounces 
of formalin should be used. The gas penetrates through fabrics with 
diflSculty, and to pass through heavy goods the concentration of the 
gas must be doubled and moderate heat added (45° C. or above). 

Value of Moisture. — At first it was thought that formaldehyde gas 
acted more eflFectually in a dry atmosphere, but further investigation 
has proved that, although it does destroy bacteria with the amount 
of moisture usually present in the air, and contained in their own 
substance, it acts much more powerfully and certainly when additional 
moisture is present, and best when present up to the point of saturation. 
The actual sprajdng with water of walls and goods to be disinfected 
is even more efficacious. 

A fairly high temperature — but one still below that which would 
injure delicate fabrics — increases not only the activity of formalde- 
hyde gas, but also its penetrative power, and for heavy goods it is 
essential. The production of a partial vacuum in the chambers be- 
fore the introduction of the formaldehyde gas still further assists its 
penetration. 

The length of exposure necessary for complete disinfection depends 
upon the nature of the disease for which it is carried out — the penetra- 
tion required, the concentration of the gas used, the amount of moisture 
in the air, the temperature of the air, and the size and shape of the room. 
For surface disinfection in rooms, when as much as twelve ounces of 
formalin are used for each 1000 cubic feet, five hours' exposure is amply 
sufficient, most bacteria being killed within the first thirty minutes. 
For the destruction of microorganisms protected by even a layer of 
thin covering, double the formalin and double the time of exposure 
should be allowed, and even then the killing of many species of non- 
spore-bearing bacteria cannot be counted upon in ordinary rooms. 
When absolutely complete disinfection is demanded, where penetra- 
tion of gas is required, the goods must be placed in chambers where 
moderate heat can be added and all leakage of gas prevented. 

Various forms of apparatus can be properly employed to liberate 
formaldehyde gas for purposes of disinfection. There are two essen- 
tials to any good method — namely, that the formaldehyde gas is given 
off quickly, and that there is no great loss by deterioration of the 
formalin. 



120 



PATHOGENIC MICRO-ORGANISMS. 



Wood Alcohol. — A number of lamps have been de\ased, all verv 
much on the same principle, though varying somewhat in mechaniciil 
construction, which bring about the incomplete oxidation of methyl- 
alcohol by passing the vapors mixed with air over the incandescent 
metal. Although disinfection can be carried out by the best of these 
lamps, in our experience none of them up to the present time are satis- 
factory or economical. They may be very useful as deodorizers in the 
sick-room or other places. 

The same principle is used efficiently in another form. The vapor 
of wood alcohol is passed over the surfaces of asbestos containing par- 
ticles of finely divided platinum. This apparatus has given very good 
Fio. 63 results, and for a given amount of disinfec- 

tion leaves less odor of formaldehyde gas 
in the room than any other. The appa- 
ratus is, however, bulky and expensive. 

Formalin by Boiling and Passing the 
Vapor through a Superheated Coil or 

Chamber. — This system consists in heating 
the ordinary commercial formalin to a high 
temperature in an incandescent copper coil 
or chamber, and allowing the vapors to 
pass off freely. It is claimed for this 
method that the degree of heat necessary to 
break up the polymerized products formed 
is supplied, and thus a loss of formaldehyde 
is prevented. A further action of the in- 
tense heat in the copper tube on the 
solution is partially to convert the methyl- 
alcohol contained in commercial formalin 
into formaldehyde gas by partial oxidation, 
thereby utilizing a part of the methyl- 
alcohol and increasing the amount of 
formaldehyde. 

In operation the desired quantity of formalin is placed in the receiver 
and the receiver is closed. The lamp is lighted and the coil brought 
to a red heat. The valve is then opened and the solution contained in 
the receiver is allowed to pass down and into the coil in a fine stream. 
Upon coming in contact with the heated metal the formaldehyde solu- 
tion is instantly decomposed, and the liberated gas is further purified 
as it progresses through the incandescent coil. The apparatus is liable 
to get out of order, in that the valve is apt to become clogged and so 
stop the flow of formalin until freed by a wire supplied for the purpose. 
In the apparatus (Fig. 63) the formalin is first boiled in the large 
chamber and passes as vapor through the tube connecting B and C 
In C it is superheated and passes out the tube D through a rubber tube 
into the room. In all forms of apparatus where formalin is used the 
large receiving chamber should be washed out from time to time with 
hot water, to remove any deposit there may be. 




Formaldehyde apparatus. 



PRACTICAL DISINFECTION AND STERILIZATION. 121 

Trioxymethylene by Schering's System. — ^This system consists in 
heating the solid polymer of formaldehyde (trioxymethylene) in a 
lamp specially constructed for the purpose. The trioxymethylene is 
used in the form of compressed tablets or pastilles, as being more con- 
venient for use. Each pastille contains the equivalent of 100 per 
cent, of formaldehyde gas, according to the manufacturers, and weighs 
1 gram. 

The mode of using the apparatus is very simple: The disinfector 
is placed upon a sheet of iron on the floor of the room to be disinfected. 
From 100 to 250 pastilles can be evaporated at a time in the apparatus. 
For the production of greater quantities of formaldehyde vapor several 
of these outfits may be used together. The lamp is filled with ordinary 
or wood alcohol, about twice as many cubic centimetres of the alcohol 
being employed as there are pastilles to be evaporated. The wicks 
should project but little above the necks of the burners, or the apparatus 
may get too hot and ignite the pastilles. The vessel is charged with 
formalin pastilles and the disinfector placed over the lighted spirit lamp. 
The lamp is then allowed to burn out in the closed room. One hundred 
pastilles are considered to be suflScient for the disinfection of 1000 cubic 
feet of space. Lately, a small steam boiler has been added to the appa- 
ratus, for the purpose of furnishing suflScient moisture with the gas. 
The results obtained by us in superficial disinfection, when from 
150 to 200 pastilles have been used to each 1000 cubic feet, have been 
good. The great advantage of the method is in the small cost of the 
apparatus, $3.00, and the avoidance of the danger of deterioration, 
which is present to some extent in formalin. Smaller lamps are very 
useful for the deodorization of rooms. 

From Pastilles Composed of a Top of Compressed Paraform and a 
Base of Prepared Charcoal, — This is a very neat but somewhat expensive 
method of liberating formaldehyde gas. Our results with it have been 
good. 

Formalin to which Glycerin has been Added. — To the formalin is 
added 10 per cent, of glycerin, and the mixture is simply boiled in a 
suitable copper vessel, the steam and formaldehyde gas passing off 
by a tube. This is a very serviceable apparatus. When it is attempted 
to vaporize the formalin too rapidly part of it bubbles over in fluid 
form. 

With 50 per cent, more of formalin than that used in the high tem- 
perature autoclave and heated tube or chamber methods, the results 
seem to be equally as good. The apparatus is very easy to use, and is 
not liable to get out of order. 

Similar forms of apparatus are also employed, when instead of gly- 
cerin the formalin is mixed with an equal quantity of water. The 
water is for the purpose of giving additional moisture to the air, and, at 
the same time, like the glycerin, to prevent the change of formaldehyde 
into inert substances. 

From Formalin in an Open Pan. — A very simple method, devised 
by Dr. R. J. Wilson, is to fill a tin pan with twelve ounces of formalin 



122 PATHOGENIC MICRO-ORGANISMS, 

for each 1000 cubic feet and put this on an upright sheet of tin, which 
is cut so as to allow of the entrance of air below and yet protect the 
formalin in the pan from the flame. For heating put under it a small 
tin can filled with asbestos packing which has been soaked with wood 
alcohol. A still simpler method is to pour on folded sheets sixteen 
ounces of formalin per 1000 cubic feet and then stretch them out over 
lines in a room and leave for ten hours. If the room is tightly sealed 
very fair surface disinfection will take place. 

Lime and Permanganate Method of Generating Formaldehyde 

Oas. — Satisfactory results in disinfection have been obtained from the 
following combination of chemicals. Two ounces of a quick-slaking, 
coarsely granular lime (calcium oxide) ; 5 ounces of permanganate of 
potash; ^ gram oxalic acid; 5 ounces formaldehyde solution, 40 per 
cent, strength; and 2^ ounces of water. This is suflScient in quan- 
tity to disinfect 1000 cubic feet of space in five hours. It is used as 
follows: The lime and permanganate of potash are mixed together 
in a pan at least 10^ inches in diameter and 3^ to 4 inches in depth. 
Over this is poured the freshly prepared mixture of formaldehyde 
solution, oxalic acid, and water. A rapid evolution of gas takes place. 

Another combination is lime 2.7 ounces; potassium permanganate, 
5.5 ounces; formaldehyde solution, 7.4 ounces; water, 2.7 ounces. 
The technic is as follows: The lime and permanganate are mixed 
in a wide, deep pan as above, and the freshly prepared formaldehyde 
and water mixture is poured over it. 

Permanganate of potash method. The following combination 
will also disinfect 1000 cubic feet of space in five hours: potassium 
permanganate, 10 ounces; formaldehyde solution, 40 per cent., 9 
ounces; water, 4.5 ounces. The formaldehyde and water are mixed 
together and rapidly poured over the permanganate of potash. The 
reaction is immediate and violent. This mixture requires a deep, wide 
pan or a pail at least 18 inches deep. The^ addition of the water is 
believed to increase the liberation of the formaldehyde gas. 

Lime Method of Generating Formaldehyde Oas. — To ten ounces 
of 40 per cent, formaldehyde solution slowly add one ounce of concen- 
trated sulphuric acid; pour this solution on to two pounds of quicklime 
that had previously been cracked into small lumps and placed in a 
dairy pan not less than twelve inches in diameter. The liberation of 
a large amount of gas in a short time more than compensates for the 
loss by polymerization, and disinfection is effected by a quick union 
of the gas and organisms to be destroyed. Saturated solution of 
aluminum sulphate may be used instead of concentrated sulphuric acid. 

Rapid Generation of Formaldehyde Oas for Large Chambers by the 
Method of Dr. B. J. Wilson. — The generator (Fig. 64) is made of ordi- 
nary iron steam pipe and can be manufactured in any pipe-cutting es- 
tablishment in a very few hours. It consists of an outer steam jacket 
of six-inch pipe, two feet long, and capped at both ends. Through 
the upper cap there is a four-inch opening, with a thread, through 
which projects an inner chamber for formalin. This chamber con- 



PRACTICAL DISINFECTION AND STERILIZATION. 



123 



FiQ. 64 




sists of a four-inch pipe, twenty-two inches long, capped at the upper 
end and welded or capped at the lower end. The upper end of this 
pipe is so threaded as to permit of its being screwed through the cap 
of the steam jacket before that cap is screwed on. The cap of the 
formalin chamber is fitted on the same thread that passes through 
the cap of the steam jacket. The in-take for steam is near the top 
of the steam jacket, through a half-inch pipe, and the steam is con- 
trolled by a globe valve. The outlet for steam or drip is through a 
half-inch pipe from the bottom cap of the chamber and is also con- 
trolled by a globe valve. The in-take for 
formalin is through the upper cap of the 
formalin chamber through a half-inch pipe 
controlled by a globe valve. The outlet 
for formaldehyde is a half-inch pipe through 
the upper cap of the formalin chamber. 

This generator is cheap and eflBcient, but 
considerable care should be observed in 
operating it, as there is a tendency to throw 
out some formalin before the gas begins to 
be evolved. This is easily avoided by using 
care in the proper application of the heat. 
These generators have now been in use 
for eight years by the New York Health 
Department, and have given complete 
satisfaction. 

As a result of the investigations under- 
taken in the Department of Health labora- 
tories on the use of formaldehyde as a 
disinfectant, and a consideration of the 
work of others, the conclusions reached by 

us may be summarized as follows: ^^^^^.^ Fonnaldehyde Generator. 

1. General Rules for Disinfection a, steam chamber; b, fonnaUn 
OF Infected Dwellings.— Exposed sur- E!'S^t%^r'"^C^t^^A^ 
faces of walls, carpets, hangings, etc., in ^o™»a**^e*»y*^e- 
rooms may be superficially disinfected by means of formaldehyde gas. 
All apertures in the rooms should be tightly closed and from ten to 
sixteen ounces of formalin or its equivalent used to generate the gas 
for each 1000 cubic feet. The time of exposure should be not less 
than four hours, and a suitable apparatus should be employed. The 
temperature of the apartment should be as high as possible, and cer- 
tainly not below 50° F. With even lower temperature surface disin- 
fection is possible, but larger amounts of formalin must be used. 
When generated very rapidly the formaldehyde gives much better 
results than when given off slowly. 

Under these conditions spore-free bacteria and the contagion of the 
exanthemata are surely destroyed when freely exposed to the action of 
the gas. Spore-bearing bacteria are not thus generally destroyed; 
but these latter are of such rare occurrence in disease that in house 




124 PATHOGENIC MICRO-ORGANISMS. 

disinfection they may usually be disregarded, and, if present, special 
measures can be taken. 

The penetrative power of formaldehyde gas in the ordinary room, 
at the usual temperature, even when used iff double the strength 
necessary for surface disinfection, is extremely limited, not passing, 
as a rule, through more than one layer of cloth of medium thickness. 
Articles, therefore, such as bedding, carpets, upholstery, clothing, 
and the like should, when possible, be subjected to steam, hot air, or 
formaldehyde disinfection in special chambers constructed for the 
purpose. If not, they must be thoroughly exposed on all sides. 

2, Disinfection of Bedding, Carpets, Upholstery, Etc. — 
Bedding, carpets, clothing, etc., which would be injured by steam, 
may be disinfected by means of formaldehyde gas in an ordinary 
steam disinfecting chamber, the latter to be provided nith a heating 
and if possible a vacuum apparatus and special apparatus for gen- 
erating the gas. Where penetration through heavy articles is required 
the gas should be used in the proportion of not less than the amount 
derived from thirty ounces of formalin for each 1000 cubic feet, the 
time of exposure to be not less than eight hours and the temperature 
of the chamber not below 100° F. 

In order to insure complete sterilization of the articles they should 
be so placed as to allow of a free circulation of the gas arouod them 
— that is, in the case of bedding, clothing, etc., these should either 
be spread out on perforated wire shelves or loosely suspended in the 
chamber. The aid of a partial vacuum facilitates the operation. 
Upholstered furniture and articles requiring much space should be 
placed in a lai^e chamber, or, better, in a room which can be heated 
to the required temperature. 

The most delicate fabrics, furs, leather, and other articles, which 
are injured by steam, hot air at 230" F,, or other disinfectants, are 
unaffected by formaldehyde. 

3. Disinfection of Books. — Books may be satisfactorily disin- 
fected by means of formaldehyde gas in a special room, or in the 
ordinary steam chamber, as above described, and under the same 
condition of volume of gas, temperature, and time of exposure. The 
books should be arranged to stand as widely open as possible upon 
perforated wire shelves, set about one or one and a half feet apart 
in the chamber. A chamber having a capacity of 200 to 250 cubic 
feet would thus afford accommodation for about one hundred books 
at a time. 

Books, with the exception of their surfaces, cannot be satisfactorily 

A;^i„ta^t.=A k,. ''^-"laldehyde gas in the bookcases of houses or libraries, 

)t in special chambers constructed for the purpose, 

ions required for their thorough disinfection cannot 

lied with. 

ustrations, and print of books are in no way affected 
rraaldehyde gas. 
fl OF Carriages, Etc. — Carriages, ambulances. 



PRACTICAL DISINFECTION AND STERILIZATION. 125 

cars, etc., can easily be disinfected by having built a small, tight 
building, in which they are enclosed and surrounded with formaldehyde 
gas. Such .a building is used for disinfecting ambulances in New 
York City. With the apparatus there employed a large amount of 
formalin is rapidly vaporized, and superficial disinfection is completed 
in sixty minutes. 

5. Method for Testing Efficacy of Room Disinfection. — 
The following method modified by Dr. Schroeder, working in the 
Research Laboratory, is now in use in the Department of Health. 

The main points of the system are as follows: 

No. 36 cotton is cut into inch lengths, placed in a Petri dish, and 
covered with a forty-eight-hour broth culture of pyocyaneus. 

They are left for two or three minutes or until they are thoroughly 
saturated, then removed to filter-paper in another covered Petri dish 
and left to dry. When dry, they are placed in tissue-paper envelopes, 
which are stamped with all necessary data. Each envelope is dated 
and sealed and sent to the disinfector who places it in the room which 
is to be disinfected. 

The driver who calls for the bedding takes up the tests, placing 
them in a manilla envelope and entering them upon his card. The 
envelopes are then returned to the laboratory where the tests and re- 
ceipt card are compared and any discrepancy noted. 

The test envelopes are then stamped with date of receipt, and the 
threads are removed and placed in a modified Ayer's medium, which 
is a synthetic medium and consists of the following: 

Asparagin 4 

Neutral NaPhos 2 

Sodium lact 6 

Sodium chlor 5 

Water 1000 



Add enough NaOH to render the medium alkaline to litmus. 
This culture medium may be depended upon to give bright green 
color reaction in twenty-four to forty-eight hours. 

The tubes are incubated for forty-eight hours and the color reac- 
tion noted and entered upon test envelope. 

UpK)n completion of this process the envelopes are sorted upon a 
table marked oflF into alphabetical spaces. They are then entered 
upon the disinfector's card. 

At the end of the week a bacteriologist's report is compiled which 
shows at a glance the work of each disinfector, the number of cases 
of each disease for which disinfection was performed, the number of 
successful disinfections, the number of tests lost, etc. 

6. Advantages of Formaldehyde Gas over Sulphur Dioxide 
FOR Disinfection of Dwellings. — ^Formaldehyde gas is superior 
to sulphur dioxide as a disinfectant for dwellings: first, because it is 
more eflBcient in its action; second, because it is less injurious in its 




126 PATHOGENIC MICRO-ORGANISMS, 

eflPects on household goods; third, because when necessary it can easily 
be supplied from a generator placed outside of the room and watched 
by an attendant, thus avoiding in some cases danger of fire. 

Apart from the cost of the apparatus and the greater time involved, 
formaldehyde gas, generated from commercial formalin, is not much 
more expensive than sulphur dioxide — viz., twelve to twenty cents per 
1000 cubic feet against ten cents with sulphur. Therefore, we believe 
that formaldehyde gas is the best disinfectant at present known for 
the surface disinfection of infected dwellings. For heavy goods it is 
far inferior in penetrative power to steam; but for the disinfection of 
fine wearing apparel, furs, leather, upholstery, books, and the like, 
which are injured by great heat, it is, when properly employed, better 
adapted than any other disinfectant now in use. 

Sulphur Dioxide in House Disinfection. — ^Four pounds of sulphur 

should be burned for every 1000 cubic feet. The sulphur should be 
broken into small pieces and put into a pan suflBciently large not to 
allow the melted sulphur to overflow. This pan is placed in a much 
larger pan holding a little water. The cracks of the room should be 
carefully pasted up and the door, after closing, also sealed. Upon 
the broken sulphur is poured three to four ounces of alcohol and the 
whole lighted by a match. The alcohol is not only for the purpose of 
aiding the sulphur to ignite, but also to add moisture to the air. An 
exposure of eight to twelve hours should be given. 

Sulphur fumigation carried out as above indicated is not as eflBcient 
as formaldehyde fumigation, but suflSces for surface disinfection for 
diphtheria and the exanthemata. All heavy goods should be removed 
for steam disinfection if there is any possibility of the infection having 
penetrated beneath their surface. If there is no place for steam 
disinfection their surfaces should be thoroughly exposed to fumigation 
and then to the air and sunlight. In many cases when cleanliness 
has been observed, surface disinfection of halls, bedding, and furniture 
may be all that will be required. 

There is always a very slight possibility of a deeper penetration of 
infection than that believed to have occurred; it is, therefore, better 
to be more thorough than is considered necessary rather than less. 

Sulphur dioxide without the addition of moisture has, as already 
stated under the consideration of disinfectants, very little germicidal 
value upon dry bacteria. 

Public Steam Disinfecting Ohambers.— These should be of sufficient 
size to receive all necessary goods, and may be either cylindrical 
or rectangular in shape, and are provided with steam-tight doors 
opening at either end, so that the goods put in at one door may be 
removed at the other. When large the doors are handled by con- 
venient cranes and drawn tight by drop-forged steel eye-bolts swinging 
in and out of slots in the door frames. The chambers should be able 
to withstand a steam pressure of at least one-half an atmosphere, and 
should be constructed with an inside jacket, either in the form of an 
inner and outer shell or of a coil of pipes. This jacket is filled with 




PRACTICAL DISINFECTION AND STERILIZATION. 127 

steam during the entire operation, and is so used as to bring the goods 
in the disinfecting chamber up to the neighborhood of 220° F. before 
allowing the steam to pass in. This heats the goods, so that the 
steam does not condense on coming in contact with them. It is an 
advantage to displace the air in the chamber before throwing in the 
steam, as hot air has far less germicidal value than steam of the same 
temperature. To do this, a vacuum pump is attached to the piping, 
whereby a vacuum of fifteen inches can be obtained in the chamber. 
The steam should be thrown into the chamber in large amount, both 
above and below the goods, and the excess should escape through an 
opening in the bottom of the chamber, so as more readily to carry off 
with it any air still remaining. The live steam in the chamber should 
be under a pressure of two to three pounds so as to increase its action. 
To disinfect the goods, we place them in the chamber, close tight 
the doors, and turn the steam into the jacket. After about ten minutes, 
when the goods have become heated, a vacuum of ten to fifteen inches 
is produced, and then the live steam is thrown in for twenty minutes. 
The steam is now turned off, a vacuum is again formed, and the 
chamber again superheated. The goods are now throughly disinfected 
and dry. In order to test the thoroughness of any disinfection, or any 
new chamber maximum, thermometers are placed, some free in the 
chamber and others surrounded by the heaviest goods. It will be 
found that, even under a pressure of three pounds, live steam will 
require ten minutes to penetrate heavy goods. 

The Disinfection of Hands, Instruments, Ligatures, and Dressings 
for Surgical Operations. — Instruments. — All instruments, except 
knives, after having been thoroughly cleansed, are boiled for three 
minutes in a 1 per cent, solution of washing soda. Knives, after 
having been thoroughly cleansed, are washed in sterile alcohol and 
wiped with sterile gauze and then put into boiling soda solution for 
one minute. This will not injure their edges to any great extent. 

Oaose. — Gauze is sterilized by moist heat either in an Arnold steam 
sterilizer for one hour or in an autoclave for thirty minutes. It is 
placed in a perforated cylinder or wrapped in clean towels before 
putting in the sterilizer, and only opened at the operation. 

Iodoform gauze is best made by sprinkling sterile iodoform on 
plain gauze sterilized as described above. 

Ligatures — Oatgat. — Boil for one hour in alcohol under pressure at 
about 97° C. It is often put in sealed glass tubes, which are boiled 
under pressure. These remain indefinitely sterile. The alcohol does 
not injure the catgut. If desired, the catgut can be washed in ether 
and then soaked a short time in bichloride before heating in alcohol. 
Boeckman, of St. Paul, suggested wrapping the separate strands of 
catgut in paraffin paper and then heating for three hours at 140° C. 
This procedure prevents the drying out of the moisture and fat from 
the catgut, so that it remains unshrivelled and flexible after its exposure. 
Darling, of Boston, tested this method and found it satisfactory. 
Dry formaldehyde gas does not penetrate sufficiently, and is not 



128 PATHOGENIC MICROORGANISMS. 

reliable. Silver wire, silk, silkworm gut, rubber tubiog, and catheters 
are boiled the same as the instruments. 

H&nd Bmibu. — These should be boiled in soda solution for ten 
minutes. 

The Skin of the Patient. — It is impossible absolutely to sterilize 
the deeper portions of the skin, but sufficient bacteiia can be removed 
to render infection rare. The skin is washed thoroughly with warm 
green soap solution, then with alcohol, and finally with 1:1000 bi- 
chloride. A compress wet with a 25 per cent, solution of green soap 
is now placed on, covered with rubber tissue, and left for three to 
twelve hours; and after its removal the skin is washed with ether, 
alcohol, and bichloride solution, and then covered with a gauze com- 
press previously moistened with a 1:1000 bichlorideof mercury solution. 
At the operation the skin is again scrubbed with green soap solution 
followed by ether, alcohol, and then with the bichloride of mercury 
solution. In some places the bichloride compress is replaced one hour 
before the operation by a pad wet in 10 per cent, solution of formalia. 
The Hands. — Ftirbinger's method, slightly modified, is now much 
used, and gives good results. The hands are washed in hot soap and 
water for five minutes, using the nail brush. They are (hen soaked 
in 85 per cent, alcohol for one minute and scrubbed with a sterile brush. 
They are finally soaked in a 1 : 1000 bichloride of mercury solution for 
two minutes. The alcohol and bichloride of mercury are sometimes 
combined and used together. Another method which gives good 
results is as follows: Skin of operator is scrubbed for five minutes 
with green soap and brush, then washed in chlorinated lime and 
carbonate of soda in proportions to make a good lather; washed off 
in sterile water, and then scrubbed with brush in warm bichloride 
solution 1:1000. 

Owing to the risk of leaving untouched bacteria under the nails and 
in cracks of the skin, sterilized rubber gloves are now being used 
more and more in operations. Some surgeons prefer sterilized cotton 
gloves frequently changed. The gloves can be sterilized by steam. 

Mucous Membranes. — Here absolute sterilization cannot be achieved 
without serious injury to the tissues. Those of the mouth and throat 
are cleansed by a solution consisting of equal parts of peroxide of 
hydrogen and lime-water. In the nostrils it is better to employ the 
milder solutions, such as diluted Dobell's or Listerine. These are also 
used in the mouth instead of the peroxide. Wadsworth' urges the use 
of preparations containing about 30 per cent, of alcohol as being very 
efficient. 

is swabbed out thoroughly with sterile warm soap and 
n irrigated with a 2 per cent- carbolic acid or a 1 : 1000 
lercury solution. 

and Other Srriuges. — These when not boiled are steril- 
ig up into them boiling water a number of times and 
) per cent, solution of carbolic acid, the acid after three 
ection. Jour. Infect. Dis., 1906, page 779. 



PRACTICAL DISINFECTION AND STERILIZATION. 129 

minutes to be washed out by boiling water. If cold water is used the 
carbolic solution should remain in the barrel for ten minutes. Great 
care should be taken to wash out all possible organic matter before 
using the carbolic acid or boiling to sterilize. Syringes made entirely 
of glass or of glass and asbestos can be boiled in soda solution. 

The Sterilization of Milk. — Complete sterilization destroys all the 
germs in milk, and so prevents permanently fermentative changes. 
This requires boiling for fifteen to forty-five minutes on two or three 
consecutive days, according to the presence or absence of certain 
sfK)res. By partial sterilization most of the germs which are not in 
the spore form may be destroyed, so that the milk will remain whole- 
some for at least twenty-four hours when kept under proper conditions. 

Milk is best sterilized by heat, for nearly all chemicals, such as boric 
acid, salicylic acid, and formalin, are not only slightly deleterious 
themselves but also make the milk less digestible, and, therefore, less 
fit for food. Formalin is the least objectionable of the three. Milk 
may be sterilized at a high or low temperature — that is, at the boiling 
temperature — or at a lower degree of heat, obtained by modifying the 
steaming process. 

Pasteurisation. — Milk sterilized at as high a temperature as 100° C. 
is not altogether desirable for prolonged use for infants, as the high 
temperature causes certain changes in the milk which make it less 
suitable as a food for them. These changes are almost altogether 
avoided if a temperature below 80° C. is used. It is recommended, 
therefore, that the lowest temperature be used for partial sterilization 
which will keep the milk wholesome for twenty-four hours in the 
warmest weather and kill the tubercle, typhoid, and other non-spore- 
bearing bacilli. Raising the milk to a temperature of 60° C. for 
twenty minutes, 65° C. for fifteen, 70° for five, 75° for two, or 80° 
for one will accomplish this. Exposure for even one minute at 70° 
destroys 98 per cent, of the bacteria which are not in the spore form. 
Fully 99 per cent, of tubercle bacilli are destroyed. This subject is 
considered more fully in the chapter on milk. One of the many 
forms of apparatus is the following: 

(a) A tin pail or pot, about ten inches deep by nine inches in diameter, 
provided with the ordinary tin cover which has been perforated 
with eight holes each an inch in diameter. 

(6) A wire basket, with eight nursing bottles (as sold for this pur- 
pose in the shops). 

(r) Rubber corks for bottles and a bristle brush for cleaning them. 

Directions (Koplik). — ^Place the milk, pure or diluted (as the physi- 
cian may direct), in the nursing bottles and place the latter in the wire 
basket. Put only suflScient milk for one nursing in each bottle. Do 
not cork the bottles at first. 

Having previously poured about two inches of water in the tin pail 
or pot and brought it to the boiling point, lower the basket of nurs- 
ing bottles slowly into the pot. Do not allow the bottles to touch the 
water or they will crack. Put on the perforated cover and let the 
9 



130 PATHOGENIC MICRO-ORGANISMS. 

steaming continue for ten minutes; then remove the cover and firmly 
cork each bottle. After replacing the cover, allow the steaming to 
continue for fifteen minutes. The steam must be allowed to escape 
freely or the temperature will rise too high. 

The process is now completed. Place the basket of bottles in a 
cool, dark place or in an ice-chest. The bottles must not be opened 
until just before the milk is to be used, and then it may be warmed 
by plunging the bottle in warm water. If properly prepared the milk 
will taste but little like boiled milk. 

The temperature attained under the conditions stated above will 
not exceed in extreme cases 87° C. (188° F.). 

A different but admirable method is the one devised by Dr. Free- 
man.^ Here a pail is filled to a certain mark with water, and then 
placed on the stove until the water boils. It is then removed, and 
immediately a milk-holder, consisting of a series of zinc cylinders, 
is lowered with its milk bottles partially full of milk. The cover is 
again applied. The heat of the outside water raises the temperature 
of the milk in ten minutes to about 65° C. (150° F.), and holds it nearly 
at that point for some time. After twenty minutes the milk is removed, 
placed in cold water, and quickly cooled. The milk is kept in the ice- 
chest until used. 

Milk should be pasteurized when it is as fresh as possible, and only 
sufficient milk for twenty-four hours should be pasteurized at one time. 
If after nursing the infant leaves some milk in the bottle this should be 
thrown away. 

Care of the Bottles. — After nursing, the bottles should be filled with 
a strong solution of washing soda, allowed to stand twenty-four hours, 
and then carefully cleaned with a bristle (bottle) brush. The rubber 
corks and nipples after using should be boiled in strong soda solution 
for fifteen minutes and then riuvSed and dried. 

After sterilizing milk should never be put into unsterilized bottles, as 
this will spoil it. 

* Agent for Pasteurizer, James Dougherty, 411 W. 59th St. 



CHAPTER X. 
THE RELATION OF BACTERIA TO DISEASE. 

In preceding chapters we have considered the growth of bacteria for 
the most part in dead organic substances. Now we have to consider the 
growth of bacteria and the production of their poisons in the living host 
and the results of such development. While it is true that there is a 
great difiFerence between living and dead matter, and that, therefore, 
the living animal cannot be looked upon as merely a quantity of special 
material to be used for food for bacterial growth, still, in a very real 
sense, we are warranted in considering the infected living body as a 
food mass more or less favorable for bacterial growth. The difference 
is that besides the chemical substances, temperature, and conditions 
inherent to the fluids of the living body and its tissues, microorganisms 
have also to reckon with the constant production of new substances by 
the living cells of the invaded organism, which may be antagonistic to 
them. In the production of lesions by microorganisms there are four 
main factors involved — viz., on the part of micoorganisms, the power 
to elaborate poison and the ability to multiply; on the part of the body 
the degree of sensitiveness to the poisons of the bacteria and the ten- 
dency to produce antitoxic or bactericidal substances. No known 
variety of bacterial cell has as a single organism the ability to produce 
enough poison to do appreciable injury in the body, nor is it probable 
that there is any variety which, if it multiplied in the body to the extent 
that some pathogenic bacteria are capable of, would not produce disease. 
As already mentioned, varieties of bacteria even under similar condi- 
tions differ enormously in the amount of poison which they produce 
and in their ability after gaining entrance to multiply in the body. 

To understand the bacterial factor in the production of disease we 
must recognize that both the body invaded and the bacteria which 
invade are living organisms, ^nd that the products of the cellular 
activity of the body act on the bacteria at the same time the bacterial 
products act upon the human cells. Just as there are different races 
and species of animals having dissimilar characteristics, there are dif- 
ferent races and species among bacteria, and just as the descendants 
of one animal species under changing conditions gradually become di- 
verse, so do the descendants of one bacterial species. In fact, the ra- 
pidity of the development of new generations of bacteria allow in 
them of much quicker changes under new conditions than are possible 
in the higher animals and plants. Considering these and other facts, 
we can readily understand how the diflFerent types of bacteria do not 
grow equally well in every variety of animal, and after discovering 
that there are variations in the bacterial properties of the blood from 

131 



132 PATHOGENIC MICRO-ORGANISMS. 

day to day we are not surprised that they do not find the body of the 
same animal always equally suitable. The study of bacteria in the 
more simple and known conditions of artificial culture media has 
shown us how extremely sensitive many bacteria are to slight chemi- 
cal, and other changes. We have also found that conditions which 
are favorable to multiplication may still be unfavorable for the pro- 
duction of poisons. 

If we take specimens of diphtheria bacilli from three different cases 
of diphtheria, we sometimes find that on growing them for several 
days in suitable bouillon one culture will have produced poison in the 
fluid to such a degree that a single drop suffices to kill a large guinea- 
pig; the second, grown in a similar manner, will kill another animal of 
the same size with half a drop; while the third will kill with one-tenth 
of a drop. This illustrates the important fact that different varieties 
of the same bacillus have different toxin-producing powers under the 
same conditions. 

Let us now cultivate these same strains in bouillon which is a little 
too acid or a little too alkaline for their maximum development, and 
we shall find that while all of them will grow, only one and probably 
that one which produced the most toxin under favorable conditions 
will continue to develop it, while the others will fail to produce any 
specific poison. This illustration makes clear one reason for the 
variation in severity among different cases in an epidemic, since the 
conditions in one throat may favor growth but not toxin production, 
while in another throat both are favored. The fact that growth of 
bacteria may occur in the body and yet but little poison be produced, 
and that, of the same species of bacteria, some varieties are capable 
of producing specific poisons under less favorable circumstances than 
others, is very important to remember. 

The cultivation of the tetanus bacillus also furnishes some inter- 
esting facts which illustrate the comphcated ways in which the growth 
of varieties of bacteria are hindered or assisted. The tetanus bacillus, 
when placed in suitable media, will not grow except in the absence of 
oxygen; but place it under the same conditions, together with a micro- 
organism which actively assimilates oxygen, and the two in association 
will grow in the presence of air. As a rule, when tetanus bacilli are 
driven into the flesh by a dirty nail or blank cartridge plug, aerobic 
bacteria are driven in also and so help to further infection by using up 
the free oxygen, thus introducing an anaerobic environment. 

The influenza bacillus is a striking example of the special require- 
ments of certain bacteria. On media it will thrive in pure culture 
ice of hcemoglobin. 

herefore, that for each variety of organism there are 
i re<|uisite for growth, and that a temperature, degree 
r food, supply of oxygen, etc., suitable for one may be 
; for another; that, still further, when two organisms 
e may so alter some of these conditions as to render 
uitable, and vice versa. 



RELATION OF BACTERIA TO DISEASE. 123 

Let us now consider some of the facts which have been observed 
concerning the growth of bacteria in the living body as contrasted 
with culture media. In the first place, it has been learned, as will be 
described in the latter part of the book, that each variety of bacteria 
can incite only certain types of infection. Indeed, because of this 
fact, the majority of bacteria which excite disease can be traced back 
for thousands of years by means of the records, these parasitic bacteria 
breeding true and keeping distinct from the great mass of bacteria 
occurring in the air, water, and soil. 

Parasitic bacteria have gradually adapted themselves not only to 
certain species of animals, but to certain circumscribed areas of the 
body. Thus the diphtheria bacilli grow chiefly upon the mucous 
membranes of the respiratory tract, but cannot develop in the blood 
or in the subcutaneous tissues. The cholera spirilla develop in the 
inflamed intestinal mucous membrane, but cannot grow in the respi- 
ratory tract, blood, or tissues. The tetanus bacilli develop in wounds 
of the subcutaneous tissues, but cannot grow on the intestinal mucous 
membranes or in the blood. 

Other bacteria find, indeed, certain regions especially suitable for 
their growth, but under conditions favorable for them are capable of 
developing in other locations. Thus, the typhoid bacillus grows 
most luxuriantly in the Peyer patches and mesenteric glands, but 
also invades the blood, spleen, and other regions. The tubercle bacil- 
lus often remains localized in the apex of a lung or a gland for 
years, but may at any time invade many tissues of the body. The 
gonococcus finds the mucous membrane of the genitourinary tract 
most suitable for its development, but also frequently is capa- 
ble of growth in the peritoneum and even sometimes in the gen- 
eral circulation. The prieumococcus develops most readily in the 
lungs, but also invades the connective tissues, serous membranes, and 
the blood. 

All these bacteria, although ordinarily increasing only in the body 
of man, can be grown on suitable dead material. 

There are organisms which, in so far as we know, find the bodies of 
human beings or animals the only fit soil for their growth. These 
are strictly the true parasites. The bacillus of leprosy until recently 
classed with these has just been made to grow on artificial culture 
media (see Sec. II, under leprosy). 

Adaptation of Bacteria to the Soil upon which They are Grown.— 

Those bacteria which grow both in living and dead substances vary 
from time to time as to their readiness to develop in either the one 
or the other. As a general rule, bacteria grown in any one medium 
become more and more accustoitied to that and other media more or 
less analogous to it, while, on the other hand, they are less easily culti- 
vated on media widely different from that in which they have developed. 
Thus we had a culture of tubercle bacilli which, after having grown 
for three years in the bodies of guinea-pigs, would grow only with great 
difficulty on dead organic matter, while a bacillus which was obtained 



134 PATHOGENIC MICRO-ORGANISMS. 

from the same stock, but grown since on bouillon, will no longer in- 
crease in the animal body. From the same stock, therefore, two 
varieties have developed, the one having lost and the other gained in 
ability to develop as a parasite. 

Local Effects Produced by Bacteria and Tbeir Products. —After 
the bacteria gain entrance to a suitable part of the body and find con- 
ditions favorable for growth, there is a certain lapse of time before 
sufficient bacterial poisons have accumulated to cause by the action 
on the tissue noticeable disturbance. This is called the period of in- 
cubation. Its length depends on the amount, kind, and virulence 
of the microorganisms introduced and the tissue invaded. The incuba- 
tion period over, we note the course of the local and general lesions 
excited by the specific and general poisons. The extent to which this 
will progress depends, on the one hand, on the characteristics of 
the invading microorganisms; on the other, on the characteristics of the 
tissues invaded. 

The local effects of the bacterial poisons upon the cells give rise to 
the various kinds of inflammation, such as serous, fibrinous, puru- 
lent, croupous, hemorrhagic, necrotic, gangrenous, and, finally, pro- 
liferative. Some bacteria incite specific forms of inflammation along 
with those common to many bacteria; others produce, so far as we 
can detect, no peculiar form of lesions. 

Thus inflammation and serous exudation into the subcutaneous 
tissues follow injections of the pneumococcus or anthrax bacillus. 
The development of the streptococcus or pneumococcus in the endo- 
cardium or pleural cavity is followed by a serous exudation, frequently 
with more or less fibrin production. The formation of pus results 
more especially from the streptococcus, pneumococcus, and staphylo- 
coccus; but nearly all forms of bacteria, when they accumulate 
in one locaUty, may produce purulent inflammation. The colon, 
typhoid, and influenza baciUi frequently cause the formation of 



Catarrhal inflammation, with or without pus, follows the absorp- 
tion of the pruducts of many bacteria, such as the gonococcus, pneu- 
mococcus, streptococcus, and influenza bacillus, etc. The hemor- 
rhagic exudation seen in pneumonia is usually due to the pneumo- 
coccus; it is observed also in other infections. Cell necrosis is pro- 
duced frequently by the products of the diphtheria and of the typhoid 
bacilli and by those of other bacteria. Specific proliferative inflam- 
mation follows the localization of the products derived from the tubercle 
bacillus and the leprosy bacillus. 

*' ' ' the poisons of one species of bacteria, according to 

ked, produce several forms of inflammation, but the 
will vary as to its mode and extent of invasion; this 
upon its own characteristics, at the time, as to viru- 
seconil, upon the conditions in the infected animal, 
ih and power of resistance, the location of infection, 
tances under which the animal is kept. Such varia- 



RELATION OF BACTERIA TO DISEASE. 135 

tions, therefore, are in no case specific, for different poisons will pro- 
duce changes which appear identical. 

Manner in which Bacteria Produce Injury.— Bacteria produce 
serious mechanical injury only when they exist in such enormous 
numbers or bunched together as to interfere mechanically with the 
circulation or cause minute thrombi, and later emboli, which finally 
produce infarction and abscesses in different parts of the body. Even 
these dangerous effects are almost wholly due to the chemical sub- 
stances given off, which are more or less directly poisonous. Some 
portion of the protoplasm of almost every variety of bacteria acts as an 
irritant to tissues and combines with some of the substance of some 
of the body cells, and the protoplasm of most exerts a positive 
chemotaxis. 

These poisonous products, as already described in the previous 
chapter, can often be separated from the culture fluid in which the 
bacteria have grown, or they can be extracted from the bacteria. In- 
jected into animals these products cause essentially the same cellular 
lesions as are produced by the bacteria when they develop in the 
animal body. The. substances contained in or produced by the bac- 
teria, with few exceptions, attract the leukocytes, and when great 
masses of bacteria die suppuration usually follows. 

General Symptoms Gaused by Bacterial Poisons Absorbed into 
the Girculation. — ^Fever is produced, under favorable conditions, by 
all bacterial poisons. A prime requisite is that suflScient poisons be 
absorbed; on the other hand, they must not be absorbed with such 
rapidity as to overwhelm the infected host, for a moderate dose may 
raise the temperature, while a very large dose lowers it, as occurs 
sometimes when a very large surface, such as the peritoneum, is sud- 
denly involved. The fever itself has no known antibacterial effect, 
but this effect may be some part of the reaction of the tissues which 
in other portions gives rise to the antitoxins and bactericidal sub- 
stances. It is also a sign that the body cells as a whole are not yet 
overwhelmed by the infection. 

With few exceptions the bacterial poisons produce an increase in 
the number of leukocytes and a lessening in the amount of haemoglobin 
in the blood. In uncomplicated infection with typhoid bacilli there is 
a hypoleukocytosis. The different varieties of leukocytes are increased 
in varying proportions in different infections. The red-blood cells 
are directly injured by a number of bacterial substances. The 
deleterious effects on the nutrition are partly due to the direct effect 
of the poison and partly to the diseased conditions of the organs of 
the body, such as the spleen, kidney, and liver. Degeneration of the 
nerve cells is frequently noticed after infectious diseases; especially 
is this true of diphtheria. Several bacterial poisons have been found 
to produce convulsions; the best example of this is the tetanus toxin. 

Influence of Quantity in Infection. — With pathogenic bacteria the 

number introduced has an immense influence upon the probability 
of infection taking place. 



136 PATHOGENIC MICRO-ORGANISMS. 

If we introduce into a culture luediuiu containing some fresh human 
blood or serum a few bacteria it is probable that they will all die 
because of the presence of sufficient bactericidal substance in the blood 
to destroy them; whereas if a greater number are introduced, while 
there will at first be a great diminution of these, those that die, having 
combined with the bactericidal substances in the serum, neutralize 
them; then those bacteria which survive begin to increase, and soon 
they multiply enormously. The same is true for parasitic bacteria 
in the body. A few only gaining entrance, they may die; a larger 
number being introduced, some may or may not survive; but if a still 
greater quantity is injected it is almost certain unless the animal is 
immune that there will be some surviving members, which will begin 
to proliferate and excite disease. 

Variation in Degree of Virulence Poasesaed by Bacteria. — Bacteria 
differ, as has already been stated, as to the ease and rapidity with 
which they grow in any nutritive substance and the amount of poison 
they produce. Both of these properties not only vary greatly in 
different members of the same species, but each variety of bacteria 
may to a large extent be increased or diminished in virulence. The 
septioemic class of bacteria when grown in the body fluids seem to 
gradually develop the power to elaborate protective substances in their 
own bodies or produce cells with less substance having affinity for the 
bacjericidal bodies of the blood, and thus become less vulnerable. 

With those bacteria whose virulence is great a very few organisms 
will produce disease almost as quickly as a million, allowance only 
being made for the short time required for the few to become equal 
in number to the million. At the other extreme of virulence, however, 
many millions may have to be introduced to permit of the development 
of any of the organisms in the body. With these bacteria we are 
thus able to produce either no effect whatever, or a local effect, or in 
some cases a general septicemia, by regulating the amount of infection 
introduced. 

Somewhat distinct, again, from that class of bacteria which multiply 

rapidly are those which, like the tubercle and leprosy bacilli, which 

while surely developing infection, increase more slowly. Here increase 

of virulence is shown, as before, by the production of disease through 

the introduction of very small numbers into the body, but increase in 

rapidity of development cannot progress except to within certain limits. 

A single streptococcus may, through its rapid multiplication, produce 

. single tubercle bacillus, on the other hand, 

numbers in less than two weeks. The 

ic cla.ss of bacteria is not at all the same 

nt animals, and it is largely for this reason 

nimals does not usually correspond with the 

hich the organism was derived. 

and Decrease in Toxicity and Virulence.— 

(tin can be taken from bacteria by growing 

istances, such as cultivation at the maximum 



RELATION OF BACTERIA TO DISEASE. 137 

temperature at which they are capable of development. Some bacteria 
are easily attenuated; others are robbed of their virulence only with 
great difficulty. Increase of toxin production is more difficult, and 
it is only possible to obtain it to a certain extent. The means usually 
employed are the frequent replanting of cultures. But with all our 
efforts we are usually only able to restore approximately the degree of 
toxin formation which the cultures originally possessed. The adap- 
tation of bacteria to any nutritive substance, living or dead, so that 
they will grow more readily, is more easily brought about, provided 
they will grow at all. The streptococcus from erysipelas and the 
pneumococcus from pneumonia are typical of this class of bacteria. 
Inoculate a rabbit with a few streptococci obtained from a case of 
human sepsis, and, as a rule, no result follows; inject a few million, 
and usually a local induration or abscess appears; but if one hundred 
million are administered septicaemia develops. From this rabbit now 
inoculate another, and we find that a dose slightly smaller suffices to 
produce the same effect; in the next animal inoculated from this, still 
less is required, and so on, until in time, with some cultures, a very 
minute number will surely develop and produce death. With other 
cultures this increase does not take place. The same increase in 
virulence can be noted when septic infection is carried in surgery or 
obstetrics from one human case to another. By allowing bacteria to 
continue to develop under certain fixed conditions they become accus- 
tomed to these conditions, and less adapted to all that differ. 

Blixed Infection. — The combined effects upon the tissues of the 
products of two or more varieties of pi^thogenic bacteria, and also of 
the influence of these different forms on each other, are of great im- 
portance in the production of disease. The infection from several 
different organisms may occur at the same time, or one may follow 
the other or others — so-called secondary infection. Thus, an abscess 
is often due to several forms of pyogenic cocci. If a fresh wound is 
infected from such a source the inflammation produced will probably 
be caused by all the varieties present in the original infection. Peri- 
tonitis following intestinal injuries must necessarily be due to more 
than one variety of organism. Thus, whenever two or more varieties 
of bacteria are transferred to a new soil, mixed infection takes place 
if more than one is capable of developing in that locality. 

Forms of infection which are allied to both mixed and secondary 
infection are those occurring in the mucous membranes of the respi- 
ratory and digestive tract. In these situations pathogenic bacteria 
of slight virulence are always present even in health. Thus, in the 
upper air passages there are usually found streptococci, staphylococci, 
and pneumococci. When through a cold, or the invasion of another 
infective agent, as the diphtheria bacillus or the virus of smallpox or 
scarlet fever, the epithelium of the mucous membrane of the throat 
is injured or destroyed, the pyogenic cocci already present are now 
enabled in this diseased membrane to grow, produce their poison, and 
even invade deeper tissues. The intestinal mucous membrane is 



138 PATHOOENIC MICRO-ORGANISMS. 

invaded in a similar way by the colon bacilli and other organisms 
after injury by the typhoid or dysentery bacilli or cholera spirilla. 
Generally speaking, all inflammations of the mucous membranes and 
skin contain some of the elements of mixed infection. Blood infection, 
on the other hand, is usually due to one form of bacteria, as even when 
several varieties are introduced, only one, as a rule, is capable of 
development. The same is true to a somewhat less extent of inflam- 
mation of the connective tissue. The additional poison given off by 
the associated bacteria aid infection by the primary invaders by 
causing a lowering of the vital resistance of the body. In some cases 
the secondary infection is a greater danger than the primary one, as 
pneumococcic bronchopneumonia in laryngeal diphtheria or strep- 
tococcic septicaemia in scarlet fever and smallpox. 

The bacteria are also at times directly influenced by the products 
of associated organisms. These may affect them injuriously, as, for 
example, the pyogenic cocci in anthrax; or they may be necessary to 
their development, as in the case of anaerobic bacteria. Not infre- 
quently the tetanus bacilli or spores would not be able to develop in 
wounds were it not for the presence of aerobic bacteria introduced 
with them. This is shown outside the body, where tetanus bacilli 
will not grow in the presence of oxygen unless aerobic bacteria are 
associated with them. Again, it is found that the association of one 
variety with another may increase its virulence. Streptococci are 
stated to increase the virulence of diphtheria bacilli, but here it is 
probably the loss of resistance of the tissues because of the strepto- 
coccic poison. On the other hand, the absorption of the products of 
certain j bacteria immunizes the body against the invasion of other 
bacteria, as shown by Pasteur that attenuated chicken-cholera cultures 
produce immunity against anthrax. In intestinal putrefaction harm- 
less varieties of bacteria may be made to crowd out dangerous ones. 

Tissae Gharacteristics Influencing the Entrance and Growth of 

Bacteria. — The Skin. — The skin is a poor soil for bacteria and is a 
great protection against the penetration of microorganisms. When 
they do penetrate, it is through the diseased glands, or more often 
through some unobserved wound. The bacterial toxins are, when at 
all, absorbed to a slight extent through the skin. 

There is an apparent exception to the above statements in the fact 
that the pyogenic staphylococci and sometimes the streptococci exist 
upon the skin or in it between its superficial horny cells, some excep- 
tional circumstances, such as wounds or burns, being required to 
allow the organisms to penetrate deeper. The cutaneous sweat 
glands, and the hair follicles with their appended sebaceous glands, 
may allow entrance of infection, as various incidents may lead to the 
introduction and retention of virulent microorganisms. WTien this 
occurs the retained products may lead to necrosis of the epithelium 
and thus allow the bacteria to penetrate to the deeper tissues. The 
secretion of the sebaceous glands appears to be little, if at all, bac- 
tericidal, but the perspiration, on account of the aciditv, is slightly so. 



RELATION OF BACTERIA TO DISEASE, 139 

Subenteneoas Oonnective Tissaes. — Many bacteria cannot develop 
in the connective tissues and others produce a milder infection there 
than elsewhere. Others develop readily. 

The Macoos Membranes. — The' moist condition of the surface of the 
membranes and their frequent contact with irritating substances 
render them liable to bacterial infection. Bacteria, such as the pneu- 
mococci and streptococci, reproducing themselves in it become some- 
what attenuated. The mucous membranes are protected by the 
cleansing produced by the flow of the secretion and by its slight germ- 
icidal action. In infancy the membranes are readily infected by 
gonococci and later by pneumococci, by the Koch-Weeks bacillus and 
others. The mucous membranes of the nasal cavity are somewhat 
cleansed by the nasal secretion. The deeper portions of the nasal 
cavity are usually the seat of streptococci and other bacteria, while 
the extreme anterior portion contains saprophytic bacteria from the 
air. The mouth in a person in health is cleansed by the feebly bac- 
tericidal saliva. When the teeth are decayed many varieties of bacteria 
abound. Many of these are difficult to cultivate. The bacteria, 
such as the diphtheria bacilli, streptococci, etc., rarely invade the 
mucous membrane of the tongue or mouth. 

The tonsils with their crypts are usually the seat of the pyogenic 
cocci and are readily infected by the diphtheria bacilli and others. 
Whether the absolutely intact epithelium allows the passage of these 
bacteria is disputed, but the probability is that it does. With the 
slight pathological lesions usually present it undoubtedly does. 

The Lungs. — Most inhaled bacteria which pass the larynx are caught 
in the bronchi. Many of these are gradually removed by the ciliated 
epithelium. Both the alveolar epithelial cells and the leukocytes which 
enter the air sacs and bronchioles have been shown to take up bacteria. 
The normal lung is, therefore, rapidly freed of saprophytic and many 
parasitic bacteria. When subjected to deleterious influences, such as 
exposure to cold, the lung tissues seem to lose their protective defences 
and become subject to infection. 

The Stemach. — ^The pure gastric juice, through the hydrochloric 
acid it contains, is able to kill most non-spore-bearing organisms in a 
short time, but because of neutralization through food, or because the 
bacteria are protected in the food, many of them pass into the intes- 
tines. Tubercle, typhoid, colon, and dysentery bacilli, when fed by 
the mouth with food, readily pass beyond the stomach. Certain 
acidophilic germs, as well as yeasts and torute, seem to grow in the 
gastric secretion; these are largely non-pathogenic. Perforation of 
the stomach is usually followed by peritonitis, because of the irritant 
effect of the gastric juice and the presence of bacteria which are tem- 
porarily retained. The gastric juice alters tetanus and diphtheria 
toxins. The toxicity of some poisons, such as occur in decayed meat, 
are not destroyed. The stomach is exceptionally free from bacterial 
inflammations. 

Intestines. — The bile is feebly bactericidal for some bacteria, but. 



140 PATHOGENIC MICRO-ORGANISMS. 

on the whole, the intestinal secretions have little or no germicidal 
power. The number of bacteria increases steadily from the duode- 
num to the head of the colon, and diminishes slightly from the upj>er 
to the lower end of the colon. The pancreatic juice destroys many 
of the toxic bacterial products. The presence of the bacilli of the 
colon group, of streptococci, etc., does not often lead to any inflamma- 
tory condition in the normal intestines of healthy persons. In chil- 
dren suffering from the prostrating effects of heat they are apt to excite 
inflammatory changes. Even pathogenic bacteria, such as the 
typhoid, dysentery, and tubercle bacilli, may pass through the whole 
length of the healthy intestines without inciting inflammations. Slight 
lesions aid the passage of bacteria to the deeper structures. Tubercle 
bacilli and other pathogenic bacteria may pass through the intestinal 
wall to the lymph and cause distant infection without leaving any 
trace of their passage. 

Importance of Location of Point of Entry of Bacteria. — Most 

bacteria cause infection only when they gain access to special tissues 
and must, therefore, enter through certain portals. This fact is of 
immense importance in the transmission or prevention of disease. 
Thus, for example, let us rub very virulent streptococci, typhoid bacilli, 
and diphtheria bacilli into an abrasion on the hand. The typhoid 
bacillus produces no lesion, the diphtheria bacillus but a very minute 
infected area, but the streptococcus may give rise to a severe cellulitis 
or fatal septicaemia. Now place the same bacteria on an abrasion 
in the throat. The typhoid bacillus is again harmless; the diphtheria 
bacillus produces inflammation, a pseudomembrane, and toxaemia, 
and the streptococcus causes an exudate, an abscess, or a septiceemia. 
Finally, introduce the same bacteria into the intestines, and now it is 
the typhoid bacillus which produces its characteristic lesions, while 
the streptococcus and diphtheria bacillus are usually innocuous. 

It we tried in this way all the parasitic bacteria we would find that 
certain varieties are capable of developing, and thereby exciting disease, 
only on the mucous membrane of the throat, others of the intestine, 
others of the urethra; some develop only in the connective tissues or in 
the blood, while others, again, under favorable conditions, seem able 
to grow in or upon most regions of the body. 

The Dissemination of Disease. — The spread of infection is influ- 
enced by: 1. The number of species of animals subject to infection. 

Many human infectious diseases do not occur in animals, and many 
animal infections are not found in man. Thus, so far as we know, 
gonorrhoea, syphilis, measles, smallpox, typhoid fever, etc., do not 
occur in animals under ordinary conditions; while tuberculosis, an- 
thrax, glanders, hydrophobia, and some other diseases are common to 
both man and animals. 

2. The quantity of the infectious material and the manner in which 
it is thrown off from the bodv. 

In diphtheria, typhoid fever, cholera, pulmonary tuberculosis, septic 
endometritis, influenza, and gonorrhoea, enormous numbers of infec- 



RELATION OF BACTERIA TO DISEASE. 141 

tious bacteria are east off through the discharges from the mouth, 
intestines, and genitourinary secretions, causing great danger of infec- 
tion. On the other hand, in tuberculous peritonitis, streptococcic 
meningitis, and endocarditis, gonorrhoeal rheumatism, and the like 
there is little or no danger of infecting others, as few or no bacteria are 
cast off. 

3. The resistance of the infectious bacteria to the deleterious effects 
of drying, light, heat, etc. 

In this case the presence or absence of spores is of the greatest 
importance. The spore-bearing bacilli such as tetanus, anthrax, 
etc., being able to withstand destruction for a long time, retain their 
power of producing infection for months or even years after elimination 
from the body. The bacteria which form no spores show great varia- 
tion in their resistance to outside influences. Some of these, such 
as the influenza bacilli and the gonococci, the virus of syphilis and 
hydrophobia, are extremely sensitive; the pneumococci, cholera spirilla, 
glanders bacilli, etc., are a little hardier; then follow the diphtheria 
bacilli, and after them the typhoid and tubercle bacilli and the 
staphylococci. 

4. The ability or the lack of ability to grow outside of the infected 
tissues. 

Such bacteria as the pneumococcus, tubercle, influenza, diphtheria, 
glanders, and leprosy bacilli do not develop, as far as we know, outside 
of the body under ordinary conditions. Under exceptional circum- 
stances, as in milk, some may develop. Others, again, such as the 
streptococcus and staphylococcus, typhoid and anthrax bacillus, the 
cholera spirillum, and some anaerobes, may develop under peculiar 
conditions existing in water or soil. 

While for the pathogenic bacteria, as a rule, the saprophytes met 
with in the soil and water are antagonistic, yet in some cases — and 
especially is this true of the anaerobic bacteria — they are helpful. 
Such bacilli as tetanus are believed to require the association of aerobic 
bacteria to permit of their development in the soil in the presence of 
oxygen. 

5. Bacteria Garriers. — Human bacteria develop in these cases in or 
upon some portion of the skin or mucous membrane, either after or be- 
fore disease, and without causing infection. As complete a knowledge 
as possible of this saprophytic development in man of parasitic bacteria 
is necessary if we are to combat the spread of infection. In the super- 
ficial layers of the epithelium and on the surface of the skin we find 
the different pyogenic cocci, which are capable of infecting a wounded 
or injured part or causing inflammation in the glands. Acne, the 
pustules in smallpox, the pus on a burned surface, boils, etc., all 
come from these pyogenic cocci. In surgical cases the skin has to 
l)e as thoroughly disinfected as possible, to prevent the formation of 
stitch-hole abscesses and wound suppuration. 

In the secretion of the mucous membrane covering the pharynx 
and nasopharynx there is always an abundance of bacteria. In throats 



142 PATHOGENIC MICRO-ORGANISMS. 

examined in New York City, streptococci, staphylococci, and pneu- 
mococci are found in almost every instance, and even in the country 
they are often present. In the anterior nares there are fewer parasitic 
bacteria than in the posterior portions. The nasal secretion is only 
very slightly, if at all, bactericidal. Many other varieties of bacteria, 
such as the meningococci and the influenza bacilli, are probably often 
present in small numbers. In those constantly in contact with cases 
of diphtheria, and in those convfilescent from diphtheria, virulent 
diphtheria bacilli are frequently found in the throat. 

After convalescence from typhoid fever, from one to three per cent, 
remain bacillus carriers for months or years, the bacilli continuing to 
develop in the bile passages and are passed with the feces. 

Lower Animals. — ^The lower animals, as a rule, do not retain in 
their bodies bacteria pathogenic for human beings, but, as direct 
carriers of infection, they are important factors. Biting insects, es- 
pecially, such as fleas, ticks, bedbugs, lice, flies, and mosquitoes are 
a source of danger (see under Protozoa for these insects acting as in- 
termediate hosts for certain Protozoa). Flies and other insects may 
convey organisms which are simply attached to their feet or other 
surfaces of their bodies. 

Bacterial Autoinfection. — When the intestinal canal is injured, or 
its circulation hindered by strangulation, etc., BacUltLS coli and some 
other bacteria may penetrate through the injured walls and cause 
peritonitis or general infection. Under certain conditions, as during 
the debility due to hot weather, the bacteria in the intestines cause, 
through their products, irritation, and in children even serious intes- 
tinal inflammation. Long after an acute gonorrhoea has passed 
gonococci may remain in sufficient numbers to cause a new inflam- 
mation or produce infection in others. A cystitis may run on chron- 
ically for years, and then suddenly become acute or spread infection 
to the kidneys. A persistent gonorrhoeal vaginal infection may lead 
to a gonorrhoeal endometritis, or peritonitis or salpingitis, under 
suitable conditions. The staphylococci in the skin and the colon 
bacilli and pyogenic cocci in the fecal discharges may also be carried 
into the bladder and uterus and produce septic infection. Persons 
carrying diphtheria bacilli in their throats or typhoid bacilli in their 
gall-bladder may, under predisposing conditions, develop diphtheria 
or typhoid fever. 

In nearly all cases of infection the products of bacterial growth are 
absorbed into the blood, and along with them. a few bacteria also, even 
when they do not reproduce themselves in it. The greater the extent 
of the infection and the more deep-seated it is, the greater is the amount 
of absorption. The bacteria enter the blood, according to Kruse, by 
(1) passive entrance through the stromata of the capillary walls; (2) 
carriage into the blood in the bodies of leukocytes; (3) growth of the 
bacteria through the walls of the vessels; (4) transmission of the bac- 
teria through the lymph glands placed between the lymph- and blood- 
vessels. 



RELATION OF BACTERIA TO DISEASE. 143 

When bacteria are abundant in the blood they become fixed in the 
capillaries of one or all of the organs, especially of the liver, kidneys, 
spleen, and lungs, and then directly or by means of the leukocytes, 
which penetrate the capillary walls, they pass into the tissues and 
substance of the organs. They thus reach the lymph channels and 
glands, or gain entrance into the gall-bladder, saliva, etc., or press 
through the epithelium, as in the alveoli of the lungs; more rarely 
they pass through the kidney tissue into the urine, as in typhoid 
fever, though some deny that this can happen unless there is a pre- 
vious inflammation of the kidneys. 

EUmination of Bacteria through the BCilk. — The passage of bac- 
teria through the breast is important, from the fact that milk is so 
largely used as food. Observers have reported the finding of tubercle 
bacilli in cow's milk when the gland itself was intact and the ani- 
mal tuberculous. Some authorities have put its presence in milk, 
under these circumstances, as high as 50 per cent, of the cases. This, 
in our experience, is undoubtedly too high. The fact that tubercle 
bacilli swallowed with the sputum are passed alive in the faeces ex- 
plains the frequent occurrence of bacilli in the milk of cows without 
udder tuberculosis because of the contamination of the milk with 
manure. They are undoubtedly present, however, in the milk of 
some animals in which tuberculous disease of the gland could not be 
demonstrated. In these cases lymph glands adjacent to the udder 
are usually infected. The finding of streptococci and staphylococci 
is due probably in the majority of cases to the infections taking place 
as the milk is voided, for the epithelium at the outlet of the lacteal 
ducts is always infected with staphylococci, and frequently with 
streptococci, which have often been received from the mouth of the 
sucking infant. 

EUnSnation of Bacteria by the Skin and Mucous Membranes. — 

WTiether bacteria pass from the blood by the sweat is a mooted point. 
The skin is always the seat of the staphylococcus and frequently of 
other bacteria, so that it is difiicult to determine in any given case 
the origin of the bacteria found in the sweat. Many observers have 
reported the passage of bacteria from the blood through the mucous 
membrane. These, as shown by Hess, are few in number, however. 
Bacteria are sometimes eliminated through the urine, but here, as a 
rule, when great numbers of organisms are found, it is due to devel- 
opment in the bladder. The removal of the poisonous products of 
bacteria by the kidneys, intestines, etc., on the contrary, is of great 
advantage to the organism. 



CHAPTER XL 

THE ANTAGONISM EXISTING BETWEEN THE FLUIDS AND 
CELLS OF THE LIVING BODY AND MICRO-ORGANISMS. 

That certain races of animals and men, and certain individuals 
among these, are more refractory to disease than others is a fact 
which has long been known. Experience and observation have 
taught us, further, that the same individuals are at one time more 
resistant to disease than at another. This inborn or spontaneous 
refractory condition to an infectious disease is termed natural im- 
munity, in contradistinction to that acquired by recovery from 
infection. 

In regard to variations in susceptibility, certain known facts have 
been accumulated. Thus, cold-blooded animals are generally insus- 
ceptible to infection from those bacteria which produce disease in 
warm-blooded animals, and vice versa. This is partly explained by 
the inability of the bacteria which grow at the temperature of warm- 
blooded animals to thrive at the temperature commonly existing in 
cold-blooded animals. But differences are observed not only between 
warm-blooded and cold-blooded animals, but also between the several 
races of warm-blooded animals. The anthrax bacillus is very infec- 
tious for the mouse and guinea-pig, while the rat is not susceptible 
to it unless its body resistance is reduced by disease and the amount 
of infection is great. The inability of the microorganism to grow 
in the body of an animal does not usually indicate, however, an insus- 
ceptibility to its poison; thus, for instance, rabbits are less suscep- 
tible than dogs to the effects of the poison elaborated by the pneumo- 
cocci, but these bacteria develop much better in the former than in 
the latter. In animals, as a whole, it is noticed experimentally that 
the young ones are less resistant to infection than the older and larger 
ones. 

The difficulty experienced by the large majority of bacteria in 
developing in the tissues of the healthy body can be to a great extent 
removed by any cause which lowers the general or local vitality of 
the tissues. Among the causes which bring about such lessened 
resistance of the body are hunger and starvation, bad ventilation and 
heating, exhaustion from over-exertion, exposure to cold, the delete- 
rious effects of poisons, bacterial or other, acute and chronic diseases, 
vicious habits, drunkenness, etc. Purely local injuries, such as 
wounds, contusions, etc., give a point of entrance for infection, and 
tissue of less resistance, where the bacteria may develop and through 
their poison produce adjacent injury and so predispose to further 
bacterial invasion in much the same way as the heat of the forest fire 

144 



ANTAGONISM BETWEEN FLUIDS AND CELLS. 145 

dries the green trees in front of it and so prepares them to ignite. 
Local affections, such as endocarditis, may also afford an area of 
lessened resistance. The presence of foreign bodies in the tissues in 
like manner predisposes them to bacterial invasion. Interference with 
free circulation of blood and retention in the body of poisonous sub- 
stances which should be eliminated also tend to lessen the vitality. 
In these and other similar ways animals which are otherwise refractory 
may acquire a susceptibility to disease. 

Increase of Resistance by Non-specific Means. — All conditions 

w^hich are favorable to the health of the body increase its resistance, 
and thus aid in preventing and overcoming infection. The internal 
use of antiseptics against bacteria is so far unsuccessful, for the reason 
that an amount still too small to inhibit bacterial growth is found 
to be poisonous to the tissue cells. The efficacy of quinine in malaria 
and mercury in syphilis are, possibly, exceptions to the rule, but in 
both cases we are dealing with animal parasites, not with bacteria. 
Such substances as leukocytic extract, nuclein, and similar organic 
substances contained in blood serum, when introduced into the body 
in considerable quantity, aid somewhat in inhibiting or preventing 
the growth of many bacteria. Even bouillon, salt solution, and small 
amounts of urine have a slight inhibitory action. The hasten- 
ing of elimination of the bacterial poisons by free intestinal evacuation 
and encouragement of the functions of the skin and kidneys are also 
of some avail. The enzymes formed by certain bacteria have been 
found to exert a slight bactericidal action not only on the germs which 
have directly or indirectly produced them in the body, but also on other 
varieties. None of these enzymes are sufficiently protective to be of 
practical value, nor are they equal in power to the protective substances 
formed by the tissues from the bacterial products. 

Use of Local Treatment in Limiting Bacterial Invasion. — The 

total extirpation of the infected area by surgical means, if thoroughly 
carried out, removes the bacteria entirely; but, unfortunately, this 
procedure is rarely possible. When incomplete it is frequently help- 
ful; but it may be harmful, for by creating tissue injury and expos- 
ing fresh wounded surfaces to infection it may lead to the further 
development of the disease. In some cases, however, like anthrax 
and infection from bites of rabid animals, almost complete removal 
of the virus, either by the knife or thorough cauterization, will prevent 
a general infection or so lessen the number of bacteria in the body as 
to allow the bactericidal element of its fluids to exterminate them. So 
also in tetanus, the invasion being limited, surgical interference may be 
of great use by removing not only the bacilli themselves, but also that 
portion of their poison which has not as yet been absorbed from the 
tissues. The beneficial effects of opening an abscess, or cleansing and 
draining the pleural, peritoneal or uterine cavities are well known. 
The retention of the poisonous products of the bacteria leads to 
their absorption, and then through their combining with some of the 
tissue cells and with the protective substances of the adjacent fluids 
10 



146 PATHOGENIC MICRO-ORGANISMS. 

the tone of the tissues is lowered at the same time that bactericidal 
substances have been neutralized. This enables the gems to penetrate 
into tissues which would otherwise resist them. The mechanical 
effect of pressure on the walls of an abscess by its contents also aids 
absorption of toxins and bacterial progress. Local bleeding and the 
application of cold probably act by lessening absorption. The ap- 
plication of warmth increases the block! flow to the part, and so, when 
the general blood supply is bactericidal, as it often is, it acts favorably 
on the inflammation. A similar effect of operative interference is 
noticed in the frequently observed beneficial result of laparotomy 
in tuberculous peritonitis. 

Antiseptic solutions have the power of cleansing and rendering 
sterile the surfaces of a wound^ — that is, of lessening the introduc- 
tion of infection. After infection has taken place, however, it is doubt- 
ful whether antiseptic washing has much more direct influence than 
simple cleansing,^ and it certainly can have no bactericidal effect 
at any distance from the surface, either direct or indirect. Certain 
infectious diseases which are comparatively superficial are probably 
benefited by antiseptic solutions; such are gonorrhoea, diphtheria, and 
other inflammation of the mucous membranes. Even here, how- 
ever, it is impossible to do more than disinfect superficially, and in 
some cases any irritation of the tissues is apt to do more harm than 
good. In the superficial lesions of syphilis and tuberculosis the 
local use of antiseptics is sometimes of great value. In these dis- 
eases the irritant effects of the antiseptics which stimulate the tissues 
may also be beneficial. 

Specific Immunity, or a Gondition of the Body which Prevents 
the Development in it of One Variety of Microdrganisms or Benders 
it Unaffected by Their Bacterial Poisons. — ^The invasion of the body 
by almost every variety of microorganism is followed, if death does 
not quickly ensue, by conditions which for a variable period and 
to a variable degree are deleterious to the further growth of that 
variety. This more or less pronounced specific immunity may be 
created in various ways: 

1. Through recovery from disease naturally contracted or from 
infection artificially produced. According to the nature of the in- 
vading microorganism this immunity may be slight, as after recovery 
from erysipelas or pneumonia, marked for a limited period of time, 
as in diphtheria and typhoid fever, or prolonged, as after scarlet 
fever or syphilis. 

2. By inoculation with microorganisms attenuated by heat, chem- 
icals, or other means. In this case an infection of the animal is pro- 
duced, of moderate severity, as a rule, and the immunity is not quite as 
marked and lasting as after recovery from a more serious attack; but 
it is, nevertheless, considerable. The inoculation of sheep with the 
attenuated anthrax bacillus and the use of vaccination with cow-pox 
in man are examples of this method. 

3. By the injection of the living organisms into tissues where develop- 



ANTAGONISM BETWEEN FLUIDS AND CELLS. 147 

ment will not take place, as the injection of diphtheria bacilli, typhoid 
bacilli or cholera spirilla into the subcutaneous tissues. Here the de- 
struction of the bacteria with the absorption of their products causes a 
mild chemical poisoning, with considerable resulting immunity. 

4. By the injection of the dead bodies of bacteria or of the chemical 
products which they elaborate and discharge into the surrounding 
culture media during their life. This produces a less marked immunity 
than when the living culture is used, but the method is a safer one. 

5. By the injection of the blood serum of animals which have pre- 
viously passed through a specific disease or have been inoculated with 
the bacterial products. The first, probably, to think of the possibility 
of effecting this was Raynaud, who in 1877 showed that the injection 
of large quantities of serum derived from a vaccinated calf into an 
animal prevented its successful vaccination. The results obtained by 
Behring and Kitasato upon diphtheria and tetanus, where the serum 
neutralized the poisons rather than the direct development of the bac- 
teria, gave a still greater impetus to these investigations. 

Suitable animals after repeated infections gradually accumulate 
in their blood considerable amounts of these protective substances, 
so that very small amounts of serum inserted in another animal will 
inhibit the growth of the bacteria or neutralize their products. Thus, 
. 1 c.c. of a serum from a horse frequently infected by the pneumococcus 
will prevent the development in the body of a rabbit of many thousand 
times the fatal dose of very virulent pneumococci, and a few times a 
fatal dose of less virulent ones, the actual number as well as the virulence 
of the bacteria affecting the protective value of the serum. 

These protective substances are found also in other fluids of the 
body than in the blood; they occur, indeed, in the substance of many 
cells to a greater or less extent. 

The immunity produced by these five methods affects the entire 
body, as is natural, since the blood into which they are absorbed is 
distributed everywhere. The protective substances pass from the 
blood through the walls of the capillaries and finally find their way to 
the lymph and back to the blood. When the immunity is but slight, 
infection may take place in the more sensitive regions or where a 
large number of bacteria have gained access, and still be impossible 
in those tissues having more natural resistance or slighter infection. 

Passive as Gontrasted with Active Immunity. — After the immune 

serum is injected into man the immunity is greatest at the time of its 
reception into the blood. This, of ,course, is instantaneous after an 
intravenous injection, but only after eight to sixteen hours when given 
subcutaneously, and then declines, being rather quickly (in several 
months or weeks, according as to whether or not the serum is injected 
into the same species of animal as the one from which it was drawn) 
almost entirely lost, so that repeated injections are required to main- 
tain the immunity. This passive immunity is distinctly in contrast 
to the active immunity acquired after the introduction of bacteria or 
bacteria products, where the tissues of the organism, in ways to us 



148 PATHOOENIC MICRO-ORGANISMS, 

unknown, throw out, in response to the bacterial stimulus, inhibitory 
or antitoxic substances. Here immunity is actually lessened for one 
or two days, and then is increased, and reaches its height a week or 
ten days after the injection, and then continues for a week or two, 
when it slowly declines again and is lost after several months 
or years. 
Testing of Protective Power of Antibacterial and Antitoxic Sera. 

— ^The serum is tested by mixing it with a certain number of times 
the fatal dose of a culture or its toxins whose virulence or toxicity 
is known, and then injecting this under the skin, in the vein, or into 
the peritoneum, according to the nature of the substance to be tested. 
The main point is that some definite method be carried out by which 
the relative value of the serum can be judged in comparison with other 
serums. As a rule, the value is stated in the number of fatal doses of 
culture or toxin which a fraction of a cubic centimetre of serum will 
prevent from destroying the animal. It is well to remember that with 
a living germ a multiple of a fatal dose is not as much more severe 
than a single dose as the figure would suggest. One thousand times 
a fatal dose of a very virulent microorganism will be neutralized by 
several times the amount of serum which a single fatal dose requires, 
since in the case of very virulent bacteria, whose virulence is due to 
their ability to increase, it is not the organisms which are introduced 
that kill, but the millions that develop from them. 

Limitation of Curative Power of Serums which act Directly Against 
Blicrodrganisms. — As a rule, the serum has to be given before the 
bacteria introduced into the body have multiplied greatly. After that 
period has elapsed the serum usually fails to act. This is partly because 
the bactericidal and antitoxic substances of the serum are insufficient 
in amount and partly because suitable antibodies develop for only a 
portion of the varied types of poison produced by bacterial cells. 

Practical Therapeutic Value of Bactericidal Sera. — The use of 

serums having specific protective properties has been tried practically 
on a large scale in man as a preventive of infection. In susceptible 
animals injections of some of the very virulent bacteria, as pneumo- 
cocci, streptococci, meningococci, and typhoid bacilli, can be robbed 
of all danger if small doses of their respective serums are given 
before the bacteria have increased to any great extent in the body. 
If given later they are usually ineffective. For some bacteria, such as 
tubercle bacilli, no serum has been obtained of suflScient power surely 
to prevent infection. Through bactericidal serums, therefore, we can 
immunize against many infections, and even stop some just commencing; 
but as yet we cannot cure an infection which is already fully developed, 
though even here there is reason to believe that we may possibly 
prevent an invasion of the general system from a diseased organ, 
as by the pneumococcus from an infected lung in pneumonia. On 
the whole, the serums which simply inhibit the growth of bacteria 
without neutralizing the toxins have not given, as observed in practice, 
conclusive evidence of great value in already developed disease. 



ANTAGONISM BETWEEN FLUIDS AND CELLS. 149 

Relative Development of Antitoxins and Bactericidal Substances 
in the Different Infections. — Although the serum of animals which 
have been infected with any one of many varieties of bacteria is usually 
both antitoxic and bactericidal, still one form of these protective 
substances is usually present almost alone; thus antitoxic substances 
are present almost exclusively in animals injected with two species 
of bacteria which produce powerful specific poisons — viz., the bacilli 
of diphtheria and tetanus. When the toxins of either of these are 
injected in small amounts the animals after complete recovery are 
able to bear a larger dose without deleterious effects. To Behring and 
Kitasato we owe the discovery that this protecting substance accumu- 
lates to such an extent in the blood that very small amounts of serum 
are sufiicient to protect other animals from the effects of the true 
extracellular toxins. 

Except the diphtheria and tetanus bacilli, a few only of the impor- 
tant parasitic bacteria attacking man produce these extracellular toxins 
in any considerable degree and thus become capable of causing the 
production in the body of antitoxins, and even these do it to a far less 
extent than those of tetanus and diphtheria. Following them are the 
dysentery and plague bacilli, and then the cholera spirilla, the typhoid 
bacilli, the gonococci, meningococci, streptococci, etc. These latter 
bacteria when injected excite more of the substances which inhibit 
bacterial growth than of those which neutralize their toxins. The 
bacillus of symptomatic anthrax and of botulismus and the vegetable 
poisons ricin, crotin, and abrin also produce specific antitoxins. 

Antitoxin a Preventive. — Antitoxin prevents the poisonous action 
of toxin. It does not restore the cells after they have been injured 
by the toxin: it is, therefore, like the bactericidal substances, a pre- 
ventive rather than a cure. We find, experimentally, that a very 
much smaller amount of antitoxin will neutralize a fatal dose of toxin 
in an animal, if given before or at the same time, than if given only 
shortly after it. An animal already fatally poisoned by the toxin is 
unaffected by any amount of antitoxin. 

Stability of Antitoxins. — Kept cool, and protected from access of 
light and air, the more resistant antitoxins may be preserved some- 
times for a year or two with very little deterioration in strength. 
At other times, however, from unknown causes, they are gradually 
destroyed, so that there may be a loss of about 2 per cent, per month. 
Preservatives, such as chloroform, carbolic acid, tricresol, etc., alter 
antitoxins only very slightly when in dilute solution, but in strong solu- 
tion they partially destroy them. Heat up to 62^ C. does not injure 
them greatly, but higher temperatures alter them. 

Method of Administration. — Antitoxins and bactericidal sub- 
stances are absorbed by the gastrointestinal tract to a very slight 
extent only — certainly less than 2 per cent. They must, therefore, be 
introduced subcutaneously or intravenously to enter the body in ap- 
preciable amounts. 



CHAPTER XII. 

NATURE OF THE PROTECTIVE DEFENCES OF THE BODY AND 
THEIR MANNER OF ACTION— EHRLICH 'S "SIDE 
CHAIN" AND OTHER THEORIES. 

The fluids and tissues of the animal body under the normal con- 
ditions of life are, as we have seen, not only unsuitable for the growth 
of the great majority of the varieties of bacteria, but even bactericidal 
to the living organisms. 

In seeking to account for the bactericidal property of the blood, 
which to a greater or less extent affects all bacteria, we cannot find 
it either in the insuflScient or excessive concentration of the nutritive 
substances, or in the temperature, or in the reaction. We are thus 
driven to the conclusion that the body fluids and cells contain substances 
which are deleterious to bacteria. 

Bactericidal Properties of the Blood.— The bactericidal eflFect upon 
most bacteria of the blood serum, noted by Nuttall in 1888, is now 
undisputed, and is readily shown by the fact that moderate numbers 
of bacteria when inoculated into freshly drawn blood usually die soon, 
and this destruction may be so rapid that in a few hours none of 
millions remains alive. Even when some of the bacteria survive there 
is for a time a decrease in the number living. Buchner in 1889 showed 
that serum heated to 55° lost its destructive power. He believed that 
in serum there was but a single bactericidal substance and called it 
alexin. 

PfeiflFer in 1894 showed that when an excessive number of cholera 
spirilla were injected into the peritoneal cavity of a guinea-pig, which 
had not been immunized to cholera spirilla, they increased and caused 
death, while in an immunized animal they rapidly disintegrated. He 
discovered further that if a little of the serum of an immunized animal 
is injected into the peritoneum of an untreated one, destruction of 
bacteria takes place. He thus showed that there was a great increase 
in the bactericidal power of a serum after immunization for the 
species of bacteria used in immunization. Metchnikoff then showed 
that the immunized serum added to peritoneal fluid in the test-tube 
would have the same effect on the spirilla. 

Bordet in 1895 reported that defibrinated blood filtered free of blood 
cells could be used to replace the peritoneal fluid and that if to a serum 
from an immunized animal, which had lost through age its bactericidal 
power, fresh serum from an untreated animal was added, the serum re- 
gained its destructive powers, ?*. e., it was activated, although the fresh 
serum by itself had almost no effect. These observations of Pfeiffer and 
Bordet indicated clearly that two types of substances were required 

150 



THE PROTECTIVE DEFENCES OF THE BODY, 151 

to destroy cells. Both of these were present in fresh immune serum, 
one of which was stable and more or less specific, and the other un- 
stable and non-specific. The latter was proven to be present in all 
blood, while the former existed, except in minute amount, only in the 
blood of the immunized. The number of bacteria introduced in a 
germicidal test is of great importance, for the serum with its contained 
substances is capable of destroying only a certain number, and after 
that it has lost its bactericidal properties. 
Thus the following test illustrates tliis: 

Approximate number alive after being kept at 37^ C. 



No. of bacteria 


Amount of 








— ' 


in 1 c.c. fluid 


Serum added 


One hour 


Two hours 


Four houra 




30,000 

100.000 

1.000.000 


0.1 c.c. 
0.1 c.c. 
0.1 c.c. 


400 

5.000 

400.000 


2 

1.000 

2.000.000 




2.000 

20.000.000 





Haas found that the circulating blood is not always bactericidal for 
any given variety of bacteria to the same extent the serum is. 

During the testing of the bactericidal power of the serum on dif- 
ferent bacteria it was discovered that numerous varieties were not 
destroyed by the serum alone, but only when exposed to both serum 
and leukocytes. 

During these earlier years MetchnikoflF perceived that the infected 
host was too little considered, and he drew attention to the r6le of 
the leukocytes. He noted that in inflammation there is an active mi- 
gration of leukocytes through the walls of the vessels toward the 
infecting bacteria. If the bacteria are very virulent they continue to 
increase, destroying the leukocytes. If the bacteria are not suflSciently 
virulent to set up a progressive inflammation they are themselves dis- 
integrated. Later it was discovered that bacteria after being acted 
upon by the serum from the body after infection were much more 
susceptible to the leukocytes. (See chapter on Opsonins.) 

Buchner made many experiments on the nature of the process. He 
showed that bacteria absorbed these bactericidal substances. Later, 
Bordet, Ehrlich, and others established that the alexin of Buchner was 
really a mixture of two types of substances of which one, named 
"immune body," "sensitizer," or "opsonin" is developed as the re- 
sult of the injection of foreign cell substance, and the other, named 
"complement" or "alexin," is present in the blood of normal animals, 
and is not increased by injection. Neither one of these types of sub- 
stances alone destroys bacteria, while together they destroy certain 
varieties. Other bacteria require the action of the complement-like 
ferment in the leukocytes also. 

During the investigations on the bactericidal power of the blood 
the discovery of the antitoxins which combine with the toxins, but 
leave untouched the bacteria, was made by Behring and Kitasato, 
and the nature of the union was investigated by Bordet, Ehrlich, and 
others. The facts developed by these studies became the basis for 
EhrUch's side chain theory. 



152 



PATHOGENIC MICRO-ORGANISMS. 



Ehriich's Theories Upon Antitoxin Production.— Ehrlich began 

by observing that of the many poisonous substances known to us 
only a comparatively small number existed against which we could 
truly immunize t. c, obtain specific antibodies in the blood serum 
of the immunized ojganism. Let us look at two poisons which are 
very similar in their physiologic action, for example, strychnine and 
tetanus poison, both of which excite spasms through the central nervous 
system. One, strychnine, produces no antibody whatever in the 
serum, while the injection of the other, the tetanus poison, causes 
the formation of the specific tetanus antitoxin. Ehrlich says that 
this is because these substances enter into entirely different relations 
with the cells of the living organism. The one substance, strychnine. 



Fio. 65 






--B 



— F 



Graphic representation of receptors of the first and third orders and of complement as conceived 
by Ehrlich: A, complement: B, intermediary or immune body; C, cell receptor; D, part of 
cell; E, toxophorouA group of toxin; /*, haptophorous group. 

merely enters into a loose combination with the cells of the central 
nervous system, so that it can again be abstracted from these cells 
by all kinds of solvents — e, gr., by shaking with ether or chloroform. 
The combination, therefore, is a kind of solid solution, such as has 
been shown in the staining with aniline dyes. The tetanus poison, 
on the contrary, Ehrlich says, is firmly bound to the cell; it enters 
the cell itself, becoming a chemical part of the same, so that it can- 
not again be abstracted from the cell by solvent agents. Ehrlich 
says that the first requirement for every substance against which we 
can obtain a specific serum must be its power to enter into such a 
combination with one or more types of cells in the li\ing animal. 
The substance must possess a definite chemical aflBnity for certain 
parts of the organism. Hence, in each substance against which we 
can specifically immunize, Ehrlich assumes a group of atoms which 
effects the specific binding to certain cells, the haptophore group (Fig. 



THE PROTECTIVE DEFENCES OF THE BODY. 153 

65, F). Corresponding to this is a group in the cell of the living 
organism C, the receptor groups with which the haptophore group 
combines. The haptophore group is distinct from that part of the 
substance which exerts the physiologic or pathologic eflFect, in toxins, 
for example, from the group which is the carrier of the poisonous ac- 
tion, the so-called toxophore group E, or in ferments, from the group 
which exerts the ferment action, the zymophore group. Both groups, 
haptophore and functional, are independent of each other, and their 
separate presence can easily be demonstrated because the functional 
group — e, g,f in poisonous toxins the toxophore group — is more readily 
destroyed by heat than the haptophore group. Thus by heating a 
toxin for some time to 60° to 65° C. a product will be obtained which 
is much less poisonous, but which still possesses largely its power to 
bind antitoxins. In the case of toxins such substances are called 
toxoids. Ehrlich conceived the finer mechanism of the formation of 
specific substances to be somewhat as follows: The haptophore group 
is bound to the receptor of the living organism owing to a specific 
aflSnity. As a result of this the receptor is lost to the living organism, 
disposed of, and a biological law formulated by Weigert now comes 
into action, the law of supercompensation; that is, the organism seeks 
to replace this defect, but in doing so, not merely replaces the receptors 
in question, but, according to Weigert, produces more of them than 
were previously present. The conditions are somewhat like those seen 
in the callus after a fracture, in which the organism likewise does not 
produce just the amount of bone previously present; there is always 
an overproduction. 

In this way, Ehrlich states, such a large number of one type of 
receptors are produced by certain cells, that these become excessive; 
they are then thrust oflF into the blood, and these free receptors cir- 
culating in the blood constitute the specific antibodies. Ehrlich 
therefore believes that the specific antibodies in the serum are nothing 
else than all receptors for which the substance employed in immuniza- 
tion possesses specific aflSnity. Hence, the same substance which, so 
long as it remains in the cell, attracts the toxin and makes it possible 
for that to exert its poisonous action on it, now when it circulates free 
in the blood or tissue fluids acts as a protection by satisfying the 
aflinity of the poison's haptophore group while still in the blood, and 
thus preventing the poison molecule from reaching the cell itself. 

In the formation of the specific antibodies we must therefore dis- 
tinguish three stages (Fig. 66) : 

The binding of the haptophore group to the receptor (2). 

The increased production of the receptors following this bind- 
ing (3). 

The thrusting oflF of these increased receptors into the blood (4). 

One objection against the Weigert-Ehrlich hypothesis of overpro- 
duction of antitoxin by the specifically attacked cells is that while 
the animals are still showing tetanic symptoms the receptors of the 
still diseased cells are supposed to have been reproduced, as shown 



154 PATHOGENIC MICRO-ORGANISMS. 

by antitoxin production. This is answered by Weigert that while 
the more important cell atom groups are still suffering, the groups 
producing the receptors may have recovered. This supposition is 
difficult to prove or disprove. 

The idea of Weigert, that the cells are biologically altered so as 
to continue to make receptors (antitoxin) after the cessation of the 
injections, and that they increase in capacity to produce antitoxin 



X ..m\.-''w^' 






K^ 



d tfae neutraliiAtioii 



receplon CBSl aS in (he blood. 

blood with frn mepton bad bwn tn 



tomed to forming it through the stimulus of re- 
ot in accord with the observations made by us. 
)roved since there is uniformly a great drop in 
;wo weeks after the cessation of the fresh stimur 
ons. The second point is, we believe, rendered 
ict that by partially neutralizing toxin before 
lis, we have found it possible to excite the cells 
mtitoxin from the first as from any later injec- 
into a previously untreated horse of one litre 
had been neutralized just sufficient not to poison 



THE PROTECTIVE DEFENCES OF THE BODY. 155 

a guinea-pig was followed by the development of antitoxin -during 
the following seven days so that each c.c. of serum contained 60 units 
of antitoxin. 

It is true that by the ordinary methods of immunizing the first 
injections of toxin produce a very small response in antitoxin, but 
this is because it is possible to give only minute amounts of toxin 
without causing the death of the animal. Very few cells are thus 
brought in contact with the toxin. 

The Nature of Bacterioljrtic, Hsemolytic, Cytolytic Sera.— Bordet, 

through his own researches and those of Gruber and Durham was able 
to show that the same type of reaction took place in the animal body 
when cells of any kind were injected. He showed, for instance, that 
there was a close similarity between bacteria and the cells of the blood. 
By immunizing an animal, species A, with red blood cells of animal, 
species B, he found that the blood of A became hsemolytic for the cells 
of fi, just as if immunized with cholera spirilla it would have been 
bacteriolytic for cholera spirilla. Since then truths obtained from 
investigation with any type of cells have been applied equally to all 
others. This allowed the nature of these processes to be studied by 
Ehrlich, Bordet, and others upon blood cells instead of bacteria. 

Experiments Devised by Ehrlich to Show the Nature of Gytolsrtic (Bac- 
teriolytic, Hamolytic, etc.) Substances in the Blood. — Ehrlich asked 
himself two questions: (1) What relation does the hsemolytic serum 
or its two active components, immune body and complement, bear 
to the cell to be dissolved ? (2) On what does the specificity of this 
hemolytic process depend? He made his experiments with a hsemo- 
lytic serum that had been derived from a goat treated with the red cells 
of a sheep. This serum, therefore, was hsemolytic specifically for sheep 
blood cells — i. e,, it possessed increased solvent properties exclusively 
for sheep blood cells. Ehrlich argued as follows: **If the hsemolysin 
is able to exert a specific solvent action on sheep blood cells, then either 
of its two factors, the immune body or the alexin (complement) of 
normal serum, must possess a specific affinity for these red cells." 
To show this he devised in conjunction with Morgenroth the following 
series of experiments : 

Experiment 1. — The serum that was specifically hsemolytic for 
sheep blood cells was made inactive by heating to 55° C, so that then 
it contained only the heat resistant substance (immune body). To 
this was then added a sufficient quantity of sheep red blood cells, and 
after a time the mixture was centrifuged. Ehrlich and Morgenroth 
were now able to show that the red cells had combined with all the 
heat resistant substances, and that the supernatant clear liquid was 
free from the same. In order to prove that such was the case they 
proceeded thus: To some of the clear centrifuged fluid they added 
more sheep red cells; and, in order to reactivate the serum, a sufficient 
amount of alexin in the form of normal serum was also added. The 
red cells, however, did not dissolve — there was no sensitizing sub- 
stance. The next point to prove was that immune body had actually 



156 PATHOGENIC MICRO-ORGANISMS. 

combined with red cells. The red cells which had been separated 
by the centrifuge were mixed with a little normal salt solution after 
freeing them as much as possible from fluid. Then a little alexin in 
the form of normal serum was added. After remaining thus for two 
hours at 37° C. these cells had all dissolved. 

In this experiment, therefore, the red cells had combined with all 
the sensitizing substance, entirely freeing the serum of the same. 

The second important question solved by these authors was this: 
What relation does the alexin bear to the red cells? They studied 
this by means of a series of experiments similar to the preceding. 

Experiment 2. — Sheep red blood cells were mixed with normal — 
i, e,f not hsemolytic goat serum. After a time the mixture was cen- 
trifuged and the washed red cells tested with the addition of sensi- 
tizing substance to determine the presence of alexin. It was found 
that in this case the red cells, in direct contrast to their behavior toward 
the sensitizing substance in the first experiment, did not combine 
with even the smallest portion of alexin, and remained unchanged. 
This experiment showed that the sensitizing substance first combined 
with the cell and then only could the alexin unite with the combined 
cell-immune body complex. 

Experiment 3. — The third series of experiments was undertaken 
to show what relations existed between the blood cells on the one hand 
and the sensitizing substance and the alexin on the other, when both 
were present at the same time, and not, as in the other experiments, 
when they were present separately. This investigation was compli- 
cated by the fact that the specific immune serum very rapidly dissolves 
the red cells for which it is specific, and that any prolonged contact 
between the cells and the serum at ordinary temperatures, in order 
to eflFect union, is out of the question. Ehrlich and Morgenroth found 
that at 0° C. no solution of the red cells by the haemolytic serum takes 
place. They therefore mixed some of their specific haemolytic serum 
with sheep blood cells, and kept this mixture at 0° to 3° C. for several 
hours. No solution took place. They now centrifuged and tested 
both the sedimented red cells and the clear supernatant serum. It 
was found that at the temperature 0° to 3° C. the red cells had com- 
bined with all of the sensitizing substance, but had left the alexin prac- 
tically untouched. 

The addition of red cells in the experiments was always in the form 
of a 5 per cent, mixture or suspension in .85 per cent. — f. f., isotonic- 
salt solution. 

The significance of the last of the above-cited experiments is, ac- 
cording to Ehrlich, at once apparent. It is that the sensitizing sub- 
stance possesses one combining group with an intense affinity (active 
even at 0° C.) for the red cell, and a second group possessing a weaker 
aflSnity (one requiring a higher temperature) for the alexin. 

Names Attached to Substances Producing Bacterioljrsis.— Dif- 
ferent investigators have applied to them different names. The one 
which is resistant to heat, which attaches itself directly to bacteria. 



THE PROTECTIVE DEFENCES OF THE BODY. 



157 




even at low temperatures, and is increased during immunization, is 
called sensitizing substance, interbody, amboceptor, or immune 
body. The other, which is sensitive to heat, which is present in the 
healthy normal serum, is not increased during immunization, and 
which unites with the bacterial protoplasm only at temperatures con- 
siderably above the freezing point, is called alexin, or complement. 

The immune body attaches itself to the bacterial substance, but does 
not appreciably harm the cells. The complement destroys the cells 
after the immune body has made the cell vulnerable. 

According to Ehrlich, the immune body first F'q- «7 

unites with the protoplasm of the cell and this 
develops in the immune body an aflSnity for 
the complement and the two unite. (See Fig. 
67.) He believes that it is through the im- 
mune body that the complement exerts its 
action on the cell. Very similar to the im- 
mune body is the substance called opsonin. 
This unites with the cell, but instead of mak- 
ing it sensitive to the complement it makes 
it sensitive to some ferment contained in the 
leukocytes. The destruction of bacteria by 
the opsonins and leukocytes will be considered 
in detail in a special chapter. 

Bordet's Theory. — Bordet supposes that in- 
stead of the tissue cell receptors which have 
combined with the toxin or foreign cell sub- 
stance (antigen, haptine) producing an excess 
of similar receptors, that the body of the animal 
that is immunized instead of reproducing old 
receptors in large amount without changing them, builds up substances 
which in their character resemble, but are not identical with pre-existent 
principles. These new substances have become endowed with a 
more marked aflSnity for the specific antigen in question. Bordet 
considers that Ehrlich in oflFering explanations which seem definitive 
has come to make certain problems which have scarcely been touched 
upon regarded as worked out. According to Bordet, Ehrlich is wrong 
in attributing such special properties to the immune body rather than 
at least equally to the antigen. He states that **as a matter of fact, 
these phenomena should be related, not as regards antigen or antibody 
considered separately, but as regards the complexes which result from 
their union, and it is evident that the special properties of the antigen 
must aflFect markedly and perhaps to a preponderating degree the 
qualities of such complexes. Just as the union of agglutinins with 
bacteria produces in them a remarkable sensitivity to the agglutinat- 
ing effect of electolytes by modifying their property of molecular 
adhesion, in a similar way sensitizers confer on their antigens a 
similar modified property of adhesion, namely, alexin absorption." 
In his opinion, antibodies, whatever their nature, act very much 



Graphic representation of 
amboceptor or receptors of the 
third order and of complement, 
showing on left the immune 
body uniting complement to 
foreign cell and on risht the 
action of anticompTement, 
binding complement: A, com- 
plement; B, intermediary 
body; C, freceptor; D, cell; 
E, anticomplement. 



158 PATHOGENIC MICRO-ORGANISMS. 

alike; but the effects which they produce differ with the antigen in 
question. 

Muir has shown that when cells are saturated with both immune body 
and complement, the addition of fresh cells causes a splitting off of im- 
mune body, but not of complement. This throws further doubt upon 
the direct union of immune body and complement. 

There are exceptional normal sera, the complement of which may be fixed 
by certain cells without the presence of an immune serum, MalvoE ' showed 
that this is the case with dog serum mixed with B. anthracls. This serum 
acts however as if it contained a true, sensitizer, because in the presence of 
this organism it will cause the fixation of the complement of the aera of 
rabbits and guinea-pigs. 

Most of the experiments which have been made with the purpose 
of clearing up these difficult problems have been made upon red blood 
cells. Here the absorption of the immune bodies at low temperatures 
and the lack of noticeable injury until the complement is added, at a 
temperature of 20° to 30° C, is very striking. 

Multiplicity of Inunune Bodies and Oomplementa. — The immune 
bodies are very numerous and fairly specific in their action. The 
complement substance is much less specific and, although probably 
multiple, when chemically considered each variety acts upon widely 
different bacteria and cells after they have united with the immune 
body. There is little reason to think that the complement of one ani- 
mal is any more capable of attacking bacteria prepared by immune 
bodies developed in its blood than by immune bodies developed in 
some other species. 

Relation Between Vimlence and the Building of Inunune Bodies. 
— It is believed by most to take place the more rapidly the more viru- 
lent the infecting organisms. In our experiments this has not been 
evident. It must be remembered that increase of virulence for one 
species of animal does not mean increase for all animals; so that in 
order to draw conclusions, the animal upon which the virulence is 
tested must be the same variety as the one being immunized. 

Ori^nn of Immune Bodies. — Their source must undoubtedly be at- 
tributed to the cells, but probably only certain cells produce them. 
The red blood cells, for instance, seem rather to destroy than to 
increase them. Injections into the lung and into the subcutaneous 
tissues of toxins and bacterial substances give rise to the formation of 
antibodies which are certainly formed partly, it not wholly, locally, and 
later find their way to the blood. The nuclein derived from the cells, 
alt)iniin>l> \t haa a non^fal bactcricidal actiou, and may enter into the 
as different properties, and so cannot itself be 

lent (Alexin). — The cells which have abun- 
;e, such as the leukocytes and lymph cells, 
a source, and Metchnikoff asserts their pre- 



THE PROTECTIVE DEFENCES OF THE BODY. 159 

eminent r6le as the producers of both complements and immune 
bodies. Buchner and others have found that through the irritation 
of bacterial filtrates the leukocytes were attracted in great numbers 
to the region of injection, and that the fluid here, which was rich in 
leukocytes, was more bactericidal than that of the blood serum else- 
where. Some claim to have demonstrated that along with increased 
leukocytosis there is a general increase in the complement in the 
blood; still, it has not yet been positively established that the com- 
plement is derived solely from the leukocytes, nor from all leuko- 
cytes, and a mere increase in them does not always mean an increase 
in the complement. 

Deflection of the Oomplement. — It frequently happens that when 
the addition of a small amount of immune serum renders a normal 
serum more bactericidal, or an animal immune, a greater addition 
robs it of most, and sometimes, all of its bactericidal power. This 
is explained by Neisser and Wechsberg to be due to a locking up of 
complement by excess of immune body. In Fig. 67 if we substitute 
an additional immune body molecule (B) for the anticomplement 
(E) it would theoretically lock up the complement (A) and prevent its 
union with the immune body which had attached itself to the cell. 
This is no evidence that amounts of serum even as large as 100 c.c. 
in an adult, given for therapeutic purposes, have produced deflection 
of complement. These large injections certainly seem to give the 
best results. The subject is in need of further study. 

Multipartial or Polsrvalent Sera. — Bacteria are not homogeneous 
masses of protoplasm, but are made up of various molecules which 
differ biologically from one another. Conforming to this, the anti- 
substances, immune bodies (antitoxins, opsonins, etc.), which ap- 
pear in a serum are made up of the sum of the antibodies which 
correspond to these partial elements in the bacterial body. These 
separate groups are called "partial groups." An immune serum, 
therefore, consists of the partial groups which correspond to the 
separate partial elements of the bacterial body. We are further able 
to show that these partial elements in one and the same bacterial 
species are not the same for all the bacteria of that species. Thus 
one culture of streptococci or of Bacillus coli may have a few par- 
tial elements which differ from those of another culture. What is 
the consequence of this? The consequence will be that when we 
immunize with a culture a of such bacteria we shall obtain a serum 
which acts completely on this culture, for in this serum all the par- 
tial elements present in culture a are represented. If, however, we 
employ culture b, c, or d, which perhaps possesses other partial ele- 
ments, we shall find that the serum does not completely affect these 
cultures. As already stated, such a condition of things is met with 
in inflammations due to streptococci and other bacteria, 'and is, there- 
fore, of considerable practical importance. It is because of this 
fact that a serum from an animal immunized to one culture acts best 
only in a certain percentage of cases. In order to overcome this 



160 PATHOGENIC MICROORGANISMS. 

difficulty in persons infected with these bacterial species we have no 
choice but to make sera, not by means of one culture, but by means 
of a number of different strains of the same species. The result of 
this will be that, corresponding to the various partial elements in 
these different cultures, we shall obtain a serum containing a large 
number of the partial groups. Such a serum will then exert a 
specific action on a large number of different cultures, but not quite as 
great an influence on any one as if only that variety had been injected. 

In other words, the development and the closer analysis of the 
problem of immunity, especially during the past few years, have 
shown us that we must make use, more than heretofore, of so-called 
polyvalent or multipartial sera. In the serum therapy of strepto- 
coccus infections, of dysentery, etc., the production of such multi- 
partial sera is an advantage in practice. Owing to these partial 
groups also, a serum — e. g., anti-typhoid serum — can specifically 
affect to a very slight degree a closely allied species of bacterium, like 
Bacillus coll, for example. For it is known that closely related species 
of bacteria possess certain partial groups in common, and a serum is 
thus produced which to a certain extent acts on such allied species. 
This constitutes what is known as the "group reaction." 

Aggressins. — A further contribution has recently been made to 
the problems of virulence and immunity in the form of the "aggres- 
sin theory" of Bail.* Apparently it grew out of an attempt to explain 
the so-called "phenomenon of Koch" — an observation made years 
ago by Koch — to the effect that tuberculous animals when inoculated 
intraperitoneally with a fresh culture of tubercle bacilli succumb 
quickly to an acute attack of the disease, the resulting exudate contain- 
ing almost exclusively lymphocytes. Bail found that if tubercle bacilli, 
together with sterilized tuberculous exudate, were injected into healthy 
guinea-pigs, the animal died very suddenly — t. c, in twenty-four hours 
or thereabouts. The exudate alone had no appreciable effect on the 
animal, while inoculation with tubercle bacilli alone produced death 
in a number of weeks. He therefore concludes that there is something 
in the exudate that allows the bacilli to become more aggressive, and 
hence has called this hypothetical substance "aggressin." He thinks 
it is an endotoxin liberated from the bacteria as a result of bacteriolysis 
and that it acts by paralyzing the polynuclear leukocyte, thereby 
preventing phagocytosis. Heating the exudate to 60*^ C. increases its 
aggressive properties rather than diminishes them and small doses act 
relatively more strongly than larger ones. These facts he explains 
by assuming the presence of two properties, one that prevents rapid 
death, is thermolabile and acts feebly in small doses, and one that 
favors rapid death and is thermostabile. He assumes that in a tuber- 
culous animal the tissues are saturated with the aggressin and w^hen 
fluid collects in the body cavities, as it does on injection of tubercle 

« Wiener klin. Woch., 1905, No. 9. Ibid., 1905, Nos. 14, 16, 17. Berliner 
klin. Woch., 1905, No. 15. Zeit. f. Hyg., 1905, vol. i.. No. 3. Arch. f. Hyg., 
vol. Hi., pp. 272 and 411. 



THE PROTECTIVE DEFENCES OF THE BODY. 161 

bacilli, it contains large quantities of aggressin, which prevents migra- 
tion of the polynuclear leukocytes, but not of the lymphocytes, and 
hence allows the bacilli to develop freely, producing acute symptoms. 
In the peritoneal cavity of the normal animal injected with tubercle 
bacilli, on the other hand, are large numbers of polynuclear leukocytes 
which engulf the bacilli, thus inhibiting their rapid development, there 
being here no aggressin to prevent phagocytosis. 

This theory has been applied to a number of infections, including 
typhoid, cholera, dysentery, chicken cholera, pneumonia, and staphylo- 
coccus infections. In all, similar results have been obtained as with 
tubercle bacilli. When exudates, produced by virulent cultures of 
these various organisms and properly sterilized, are injected with fresh 
cultures into an animal, death occurs in much shorter time than when 
the organisms alone are injected. 

Moreover, it has been possible to immunize animals against these 
various infections by repeated injections of the aggressin in the form 
of exudates. This results in the formation of an *'antiaggressin," 
which opposes the action of the aggressin, thereby enabling the leuko- 
cytes to take up the bacteria and thus to protect the animal. This 
has been done in staphylococcus, dysentery, typhoid, cholera, pneu- 
mococcus, and chicken cholera infection in animals. In addition, a 
very marked agglutinative property of the bloocLis acquired for the 
bacteria in the animals so immunized. 

The Fixation of Oomplement by Sensitized Cells and Its Prac- 
tical Application. — Bordet * and Gengou showed that the existence of 
a sensitizer, or specific immune body in an antimicrobial serum, by 
uniting with its specific antigen (bacterium or other cell or proteid 
material), absorbs alexin (complement). 

This experiment of Bordet is usually spoken of as the "Bordet- 
Gengou phenomenon." 

In order to demonstrate this phenomenon of fixation of comple- 
ment with sensitized antigen (cell protoplasm or soluble proteid), an 
experiment similar to the following should be made. 

Mixtures are prepared into six test-tubes, respectively, as indicated 
in the accompanying table. The mixture of sensitized blood (sixth 
column), which is prepared by adding twenty drops of defibrinated 
rabbit's blood to 2 c.c. of serum from a guinea-pig immunized against 
rabbit's blood and previously heated to 55° C, is added to each tube 
after the rest of the mixture has stood at room temperature for about 
five hours. Hemolysis, which is indicated by the -I- sign in the table, 
takes place quickly in tubes 2, 3, 4, because they contain no sensi- 
tized antigen to which the complement may become fixed, while in 
tube 1, which contains such antigen and in tubes 5 and 6 to which no 
complement serum has been added, no hemolysis occurs. 

* Annales de I'Institut Pasteur, xv, 1901, 290. 



11 



162 



PATHOGENIC MICRO-ORGANISMS. 



I Amount of comple- 
No of I ment serum (fresh 
tube I normal guinea-pig 

{ serum) 



Emulsion 
of antigen 



Specific 

serum 

heated to 



Normal 

horse Sensi- 
serum i tized 
heated to blood 
56° C. I 



Hemolysis 



1 
2 
3 
4 
5 
6 



0.1 c.c. 
0.1 c.c. 
0.1 c.c. 
0.1 c.c. 



0.2 c.c. 
0.2 c.c. 



0.2 c.c. 
0.2 c.c. 



0.2 c.c. 
0.2 c.c. 



0.2 c.c. 



0.2 c.c. 

|0.2 C.C' 

0.2 c.c. 10.2 c.c. 

0.2 c.c. i0.2 C.C.I 

0.2 c.c. 0.2 C.C.; 

0.2 c.c. 






Wasserman^ and others applied this method in measuring the am- 
boceptor content of specific sera, the most important practical applica- 
tion of its use being in the diagnosis of syphilis. This complicated 
subject is considered under the subject of syphilis (see under Sec. III). 

H3rper8iisceptibilit7 or Anaphylaxis. — By introducing any strange 
proteid into the body of an animal, a condifion of exaggerated sus- 
ceptibility to the foreign substance may develop in that animal. 
This condition is now generally known by the name of Anaphylaxis, 
a term introduced by Richet (1905) in his studies on the poison of 
Actiniae.' 

The best studied instance of anaphylaxis is that produced in the 
guinea-pig by the injection of a foreign proteid, such as horse serum, 
egg white, milk, etc. For example, if a guinea-pig is injected with a 
small quantity (about 0.01 c.c.) of horse serum, and, after a certain 
interval (ten to twelve days), is again injected with horse serum, but 
with a comparatively large amount (3-5 c.c. subcutaneously; 0.25 c.c. 
intravenously), it will probably die in a short time (within ten min- 
utes symptoms appear in a very sensitive animal and death occurs 
within an hour). The chief symptoms are respiratory failure, clonic 
spasms and paralysis. If a smaller dose of serum be given to the 
sensitized animal it may show only slight symptoms and recover with 
the production of immunity. This phenominon was noticed simul- 
taneously in several laboratories but was first definitely described by 
Theobald Smith. 

Rosenau and Anderson, who have studied many phases of the sub- 
ject,' found that the young of sensitized animals are also sensitive. 

Gay and Southard* think that anaphylaxis depends upon the pres- 
ence of a substance which is contained in the injected proteid and is re- 
tained in the sensitized animal. They call this substance anaphylactin. 

Tuberculin and malein reactions are well-known instances of ana- 
phylactic manifestations (see special subjects). The so-called serum 
sickness and a further discussion of animal sensitiveness are described 
in the chapter on diphtheria. 

* Wassermann, Neisser and Bruck, Deutsche med. Wochenschr., 1906; Wasser- 
mann and Plaut, ibid. 

'Richet. Compt. rend. Soc. Biol., 1905, Iviii, 109. 
'Rosenau and Anderson. Jour. Inf. Dis., 1908, v, 85. 
*Gay and Southard. Journ. Med. Res., 1907 \i, 143. 




CHAPTER XIII. 

THE NATURE OF THE SUBSTANCES CONCERNED IN 

AGGLUTINATION. 

The discovery of agglutinins in the serum of those passing through 
many infections was made by Gruber and Durham in 1896, and their 
characteristics were studied by Bordet. Several months later Widal re- 
ported that in typhoid fever the development of agglutinins could be 
used for diagnostic purposes. Later it was demonstrated that through 
agglutinins a new means was available for the identification of bacteria. 
See pages 42-46 for a description of the 
phenomenon of agglutination and the 
technique of investigation. 

As to the nature of these phenomena a 
number of theories have been advanced. 
As in the case of the immune body, there 
is positive proof that the agglutinin com- 
bines directly with substances in the 
bacterial body. Bordet believes that all 
the antibodies act very much alike in 

. i_ • aL J * • J j.1. X xi_ Receptors of the Second Order are 

their metnod of Umon and that the pictured in c. Here « repieseiita the 

effects produced vary with the antigen phSregroS^57hJ*ieSptolfbeiS?Sbe 

in question and the characteristics which, ^bri!"'8uTh';^'^p'^^^ 

on account of its own nature, it can pro- ItlTtoLTotedXtThlSJSl^^^^^ 

duce as soon as it unites with the ap- group »» an integral part of the re- 

propriate antibody. He does not be- 
lieve in different families of antibodies, but in an infinite variety 
of antigens. Ehrlich, on the other hand, considers that the 
agglutinin consists of a haptophore or combining atom group 
which is stable and of a ferment group which is labile. The latter 
causes the phenomenon of agglutination. Bordet draws attention 
to the fact that bacteria do not agglutinate when they have com- 
bined with the agglutinin unless they are in the presence of salt. 
A^lutinin does not dialyze through animal membranes. In diluted 
solution agglutinin slowly deteriorates. Dried it lasts longer. It is 
precipitated with the globulins by ammonium sulphate. When a 
solution containing agglutinin is passed through a stone filter the first 
few cubic centimeters contain no agglutinin. The next contain a 
moderate amount and the remainder the same as the solution. 

In some types of infection there is a great accumulation of agglu- 
tinins in the blood. Thus in typhoid patients and convalescents distinct 
agglutination has been observed in dilutions of 1:5000, and this action 
persisted for months, though not, of course, in the same degree. Even 

163 



164 PATHOGES'lC MICRO-ORGASISMS. 

normal blood serum, when undiluted, often produces agglutination 
through group agglutinins. But the spiecific agglutinins, which are 
formed only in conse<]uence of an infection, are characterized bv this, 
that they produce agglutination even when the serum is highly diluted, 
and, furthermore, that after this dilution the action is specific — i. e., the 
high dilutions of cholera immune serum agglutinate only cholera 
bacilli, of typhoid immune serum only typhoid bacilli, etc. This speci- 
ficity, however, as will be shown later, is not always absolute. 

The agglutinating substances when mixed with bacteria are bound 
to their agglutinable substances, the two bodies effecting a loose com- 
bination very like toxin and antitoxin. By chemical means it is 
possible again to separate a portion of the agglutinin from bacteria 
saturated with it anil use it to agglutinate bacteria anew. 

It was formerly assumed that agglutination was a prerequiaiie for 
bacteriolysis. This, however, is not so, for both in cholera and in 
typhoid immunity bacteriolvtic substances have been observed with- 
out agglutinins, and agglutinating sulwtances without bacterid y sins. 

Oh^actemtics of Agglutinins. — Agglutinins changed by heat, 
acids, and other influences become agglutiaoids, which are comparable 
to toxoids, complementoids, etc. 

The union of agglutinin with receptors in bacteria is a chemical 
or physical reaction, and is quantitative. Before agglutination occurs 
sodium chloride or a similarly acting compound must be present. 
The amount of bacteria in the emulsion used to test the amount 
of a^lutinin must, therefore, be known. An emulsion one hundred 
times as dense as another would require one hundred times as much 
agglutinin to give an equally complete reaction. Agglutinin acts 
upon dead bacteria. 

Heat diminishes the agglutinability of bacteria when above 60° C. 
Dreyer found that if a twenty-four-hour bouillon culture of Bacillus 
coli required 1 part of agglutinin to agglutinate it, then if heated to 
60° C. it required 2.3 parts; if to 80° C, 18 parts; if to 100° C, 24.6 
parts. He found the surprising fact that long heating of the culture 
restored to some extent its ability to be agglutinated by smaller amounts 
of agglutinins. 

Heated thirteen hours to 100° C, the culture was agglutinated by 
4 parts. Dreyer's explanation of this result is that agglutinin-fixing 
substance is dissolved out by the prolonged heating. 

Heating the serum above 60° C. injures the agglutinin slightly, 
above 70° C. greatly, and above 75" C. destroys it. Weak and strong 
— :,!.. «~„i..<:„«.„ '-icteria, while me<iium acidity does not. Alkalies 
n. Agglutinin which has lost its power to agglu- 
^ffect of heating to 65" C. or through the action of 
lly retains its affinity for the bacterial protoplasm, 
utinins are called agglutinojds. 
} remember that in low dilutions of serum agglu- 
hile in higher dilutions of the serum agglutination 



THE SUBSTANCES CONCERNED IN AGGLUTINATION. 165 

The growth of bacteria in fresh blood containing agglutinins inhibits 
the development of agglutinable substance in bacteria or causes 
them to produce substances which prevent the union of agglutinin 
with them. Bacteria should therefore not be grown on serum media 
when they are to be used in agglutination tests. Even the addition 
of ascitic fluid to broth has some effect. 

Group Agglutination. — Many varieties of bacteria have among the 
diflFerent substances composing their bodies some that are common 
to other bacteria which are more or less allied to them (Fig. 69). These 
substances all exciting agglutinins, we have from an immunized animal 
a serum acting on the different bacteria somewhat in proportion to the 
amount of protoplasm which they have in common with the infecting 
organisms. These agglutinins are called, therefore, group agglutinins. 
If a typhoid or paratyphoid serum possess a high degree of activity — 
t. e.y abiUty to agglutinate even in large dilution — it may happen that 
with lesser dilution it may also agglutinate the two related bacilli. 
Thus, in a case, the infecting paratyphoid bacilli type B were aggluti- 
nated 1:5700; typhoid bacilli, however, only 1:120, while para- 
typhoid bacilU type A were agglutinated only 1 : 10. In a case of 
typhoid fever an agglutination of paratyphoid type B occurred with a 
dilution 1 : 40, while typhoid bacilli were agglutinated with 1 : 300. 
Since it is found that in a paratyphoid infection the serum possesses a 
fairly strong agglutinating action on typhoid bacilli, Korte advises 
that in every case of typhoid all three bacteria be tested for agglutina- 
tion, so that, according to the strongest agglutinating action, one can 
decide which infection is present. If in practice it is immaterial whether 
this point be decided, the agglutination with paratyphoid need only 
be undertaken when the typhoid agglutination is absent. 

The bacteria which are agglutinated by one and the same serum need 
not at all be related in their morphological or other biological charac- 
teristics, as at first assumed. Conversely, microorganisms which, 
because of the characteristics mentioned, are regarded as entirely 
identical or almost so, are sharply differentiated by means of their 
agglutination. In other words, the ** groups'* arrived at by means of a 
common agglutination have no necessary relation to species as the 
term is usually employed, but only of chemical similarity. This is 
indicated by the diagrams in Fig. 69. The letters indicate chemical 
substances capable of stimulating the production of agglutinin and of 
combining with it when made. Thus both the typhoid and colon will 
stimulate and react to B agglutinins. Because of this lack of absolute 
specificity the diagnons of the type of infection or the absolute identifi- 
cation of bacteria through the agglutination or bacteriolytic tests can 
only be determined with a certain degree of accuracy. This suflBces 
for some infections such as those caused by the typhoid bacillus and 
the cholera spirillum, but not for others as those due to the colon 
group of bacilli. 

The Davalopmant of Agglutinin. — Experimental or natural infection 
of animals and men is followed in seven to ten days by an appreci- 



166 



PATHOGENIC MICRO-ORGANISMS. 



\ 



able development of agglutinin. This development is much greater 
for some bacteria than for others. 

The Relative Development of Specific and Oronp Agglutinins. — 

The study of a large number of series of agglutination tests obtained 
from young goats and rabbits injected chiefly with typhoid, dysentery, 
paradysentery, paracolon, colon, and hog-cholera cultures has shown 
that there is considerable uniformity in the development of the specific 
and group agglutinins. The specific agglutinins develop in larger 
amount in the beginning, being in the second week usually from five to 
one hundred times as abundant as the group agglutinins. Later the total 
amount of the group agglutinins tends to approach more nearly to 



FiQ. 69 



A 


B 


C 


(<^_L-7I-— ^—JL— _L.TJL— _'— ^ 







B 






' ■•''/ 



Typhoid BaciUos 



: ■..•..•:...■• V ^.•■-:l||it|.i':-; 



E F 



) 



E H 



Colon BaciUus 





^■: :.^.:-..x:^ 



<X\^N|.lliil[l|i|!llll^ 



Dysentery BaciUus 
Specific and common agglutinums. 

that of the specific, and reach as high at 50 per cent. In a number of 
tests carried out by us we found that many group agglutinins supple- 
ment specific ones in their action, causing by their addition an increased 
agglutinating strength. In our experience the variety of micoorganism 
used for inoculation is, if equally sensitive, agglutinated by the combined 
specific and group agglutinins produced through its stimulus in a higher 
dilution than any microorganism affected merely by the group agglu- 
tinins. It is true that bacteria not injected were at times agglutinated 
in higher dilutions than the variety injected; this, if not due to greater 
sensitiveness, was on account of normal group agglutinins present in the 
animal before immunization. In horses and adult goats it was found 
that before injections were commenced there was often a great ac- 
cumulation of agglutinins for bacteria and especially for members of the 
dysentery, paradysentery, and colon groups, so this comes about through 
the absorption from the intestines of bacteria of the colon group and 
the consequent development of agglutinins. For this reason un- 
treated horse serum is a very dangerous substance to use in diflFeren- 
tiating the intestinal bacteria. The great height to which the group 
agglutinins may rise is seen in the following table:* 



Table I. 

Agglutinin in the Serum of a Horse Injected with Paradysentery Bacillus, 

Type, Manila Culture. 

After 18 iDJections. After 21 injections. 



Culture. 



Paradysentery type Manila. 
Colon B. X 



1:3000 

+ -I- 



1:5000 



1:10.000 



1:3000 



1:5000 
+ + 



1:10.000 

+ + 
+ + 



THE SUBSTANCES CONCERNED IN AGGLUTINATION. 167 

The great amount of agglutinins acting upon the colon bacillus X. 
is remarkable. A serum is here seen to be acting in dilutions as high 
as 1:10,000 upon a culture possessing very different characteristics 
from the one used in the injections. 

Although a considerable proportion of the group agglutinins act- 
ing on colon bacillus X. was undoubtedly due to the stimulus of the 
injections of the Flexner paradysentery culture, still a portion of them 
was probably due to the agglutinins developed by the stimulus of the 
absorbed intestinal bacteria. In Table II is seen the marked accumu- 
lation of agglutinins which may occur in a normal horse before injec- 
tions are begim. 

Table II. 

A yoiiQg horee before inoculation. 



Culture 1:100 1:500 1:1000 1:5000 



Dysentery B., Japan 

Paradysentery, Mt. Desert. 



+ - - 

+ - - 

Paradysentery, Manila ++ ++ + + 

Colon B. X ++ + - 



The fact of most importance which appears in this table is the abun- 
dant agglutinins which may be found in the serum of a horse which 
has never received bacterial injections. 

The Ralativa Accumulation of the Group and Specific Agglutinins for 
the Organism Injected and for Allied Varieties. — A test was carried 
out with different types of dysentery bacilli. For the Manila culture 
of Flexner, which is nearest to the colon in its characteristics, the specific 
agglutinins were in the serum of an animal which had received injec- 
tions of the Manilla cultures at the end of the fourth month five times 
as abundant as the group agglutinin acting on the Mt. Desert culture 
of Park, which represents a type Ij^ng between the Flexner and Shiga- 
cultures. For the dysentery bacillus (Shiga) the development of agglu- 
tinins was the least. (Fig. 70). 

Another point of interest is that the proportional amounts of agglu- 
tinins from the different cultures varied at different times. If on tests 
made of a single bleeding we had attempted to draw conclusions as to 
the relative development of specific and group agglutinins between the 
cultures, we would have had an imperfect view. Many conflicting 
statements in literature are undoubtedly due to this lack of appreciation 
of the variability in the relative amount of these two types of agglutinins 
during a long process of immunization. (Fig. 71.) 

The Use of Absorption Methods for Differentiation between 
Specific and Oroup Aigglutinins due to Mixed Lifection and to a 

Sinj^e Infection. — It is now well established that if an infection is due 
to one microorganism there will be specific agglutinins for that organ- 
ism and group agglutinins for that and other more or less allied organ- 
isms. If infection is due to two or more varieties of bacteria, there 
will be specific agglutinins for each of the microorganisms and group 
agglutinins produced because of each of them. 



168 



PATHOGENIC MICRO-ORGANISMS. 



The following expferiments well illustrate these points: A rabbit 
immunized to B, typhi agglutinated B. typhi 1:5000, B, coli (31) 
1 : 600. After saturation with B, typhi all agglutinins were removed 
for both microorganisms. A rabbit immunized to both B. typhi and 
B, coli (31) agglutinated B. typhi 1:4000, B, coli (31) 1: 1000. (After 
saturation with B, typhi the serum did not agglutinate B. typhis but B. 



1st 



2d 



Fig. 70 
3d 



4th 



5th 



6th* 



7th 




The rise and fall of commoQ and specific acglutinins during'^seven months in a rabbit injected 

with the Manila culture. 

Colon bacillus X. 

Paradysentery type (Mt. Desert). 



Paradysentery type (Manila). 

Dysentery type (Japan). 

• Test dates for all four sera. 

♦Injections stopped. 



1st 



Fig. 71 
2d 3d 



4th 



5th 



6th 



7th 



15000 
















I--4500 














/ 


l>*000 












/ 




13500 












/ 




1:3000 












/ 


Am 


12500 










X 


d^ 


r 


12000 








^ 


y^ 


8^ 




1 :| 500 






^ 4 


^^ 




^y 




1:1000 






^-^ 


i^ 


•>*' 






1 : 500 










^/ 


r 




l: 00 


.— -.rrr- 


rr^ . 


— 





Aj 







Similar conditions to those noted in previous chart, except that a young goat has been used for 
the injections of the colon bacillus X. The great accumulation of common agglutinins for the 
paradysentery bacillus in the third month of the injections of the bacillus X is very striking. 

• Tests made. 



coli (31) 1 : 900.) After saturation with B, coli (31) it failed to agglu- 
tinate B. coli (31), but still agglutinated B. typhi 1 : 3500. Some other 
strains of B. coli still agglutinated in 1 : 20 or more because many strains 
included in this group act as differently toward each other in respect to 
agglutinins as they do to the typhoid bacilli. 

The following tables give the outcome of several experiments: 
The great number of varieties of the colon group of bacilli that are 
in the normal intestine and which are absorbed slightly in health and 



THE SUBSTANCES CONCERNED IN AGGLUTINATION, 



169 



more markedly in intestinal diseases make the use of absorption tests 
for diagnostic purposes too complicated except for peculiarly impor- 
tant cases and require trained skill to carry them out. 

Absorption by the Typhoid Bacillus of Group Agglutinins Acting upon a 
Number of Varieties of B. coli which were Produced by 

Another Variety of B. coli. 

Agglutination by Serum of Rabbit Immunized to Colon Bacillus X. 

Before addition of After attempt at absorption 
typhoid bacilli. with typhoid bacilli at 22" C. 

Colon bacillus X 6000 5000 



Colon bacillus 1 500 

Colon bacillus 2 500 

Colon bacillus 3 250 

Colon bacillus 4 250 

Colon bacillus 5 10 

Colon bacillus 6-18 less than 10 

Typhoid bacillus less than 10 



less than 
less than 
leas than 



20 
30 
30 
10 
10 
10 
10 



The absorption tests were carried out by adding the bacilli from 
recent agar cultures to a 10 per cent, solution of the serum in a twenty- 
four-hour bouillon culture. The mixture was allowed to stand for 
twenty-four hours at about 22° C. It was found that the agglutinin 
in a simple dilution of serum when left at 37° C. rapidly deteriorated. 
Thus, in an extreme instance a serum positive at 1 : 1500, when 
diluted with bouillon or salt solution 1 : 25 and left at 37° C. for twenty- 
four hours, lost 30 to 40 per cent, of its strength; at 22° C. it lost at 
times 15 to 20 per cent. Left for three hours only, the loss was only 
5 to 10 per cent. 









Fio. 72 














^2000^ 








m 


c 
•• 




r (d 


CISCO 


E 

a 




o 




U. 


c 


L 


O CP 


l:i600 


• 
<0 




|5 






3 




«;"</) 

?-■= 


|:I400 



c 




?1 




m 


C 




Manila 


l:i200 


en 
'C 

o 




5 " 




Manila 








t 


l:iOOO 


MtOeswt 


Canty 










4> 

9f 


|: 800 


Japan i f*ornai 


■ ¥1 


c 






o— 


1: 600 


1 


1 1 ' 


1 -> 




1 
t 


c 

rt 


"Q 




T 

, "2 


i: 400 


1 


, 1 


1 
1 






a 
d 

-a 


e 




1 a 
1 5 


I: 200 






1 
1 


?s 








•* c E 




1 ? f 


l: «00 


1 


1 > 


-^ 1 










4J Z 






I: 00 




! i T 


1 ; 




Tli 


— w ' T 

T ! i 



Showing the effect of saturating with ba?il!i of type^ of Shiga-Manila and Mt. Desert, a serum 
from a horse which had received combined injections of dysentery bacilli of the three types. Note 
that the Manila type removed almost all the specific and group agglutinins acting upon its own type 
and the group agglutinins acting upon the Coney Island and normal types, leaving the specinc 
agglutinins for types Shiga and Mt. Desert. The same is true for types Shiga and Mt. Desert when 
they were used. 

Manila paradysentery. 

Japan dysentery. 

Mt. Desert paradysentery. 



and Atypical paradysentery. 



The absorption method simply proves, therefore, that when one 
variety of bacteria removes all agglutinins for a second the agglu- 
tinins under question were not produced by that second variety. 



170 



PATHOGENIC MICRO-ORGANISMS. 



Loss of Capacity in Bacteria to be Age^ntinatfld or to Absorb 
Ag^atininB Becaose of Growth in Inunone Sera. — The loss of these 
characteristics by growth in sera has been demonstrated by Marshall 
and Knox. The experiments of Collins and myself are recorded 
because they were undertaken in a slightly different way and also 
because a certain number of confirmatory observations are of value. 

The maltose fermenting paradysentery bacillus of Flexner was 
grown on each of eleven consecutive days in fresh bouillon solutions 
of the serum from a horse immunized through oft-repeated injections 
of the bacillus. The solutions used were 1.5, 4, and 15 per cent. 
The serum agglutinated the culture before its treatment in dilutions 
up to 1 : 800, and was strongly bactericidal in animals. After the 
eleven transfers the culture grown in the 15 per cent, solution ceased 
to be agglutinated by the serum and ceased to absorb its specific agglu- 
tinins. The cultures grown in the 1 .5 and 4 per cent, solutions agglu- 
tinated well in dilutions up to 1 : 60 and 1 : 100 and continued to absorb 
agglutinins. The recovery of the capacity to be a^lutinated was 
very slow when the culture was from time to time transplanted on 
nutrient agar. After growth for sixteen weeks, during which it was 
transplanted forty-three times, it agglutinated in dilutions of 1 : 200. 
The culture grown in 4 per cent, agglutinated 1 : 500, and the one 



1 sad 2 3 B 


Qd * 6 uid a 7 an 


dS Sue 


99«.dl0 11«dl2 13«idl4 16 


udie 


\-MQQ 


Fatal 
















|:IIOO 


doses 






^, 






\ 




1:1000 


£rot«t« 
















1: 900 


Iccwrenv 












/ \ 




1: 800 


40 




/ 




\ 








1: 700 


15 




/ 




\ 




' -1 




I: 600 


W) 




1 








^ 




1: 500 


Z5 




; 






^l 






|: 400 


2i) 








^^ 






\ 


1: 300 


1.5 




y^~ 






\ 1 




\ 


I: 200 


I.D 


y^ 








\ 1 






I: 100 




/ 














i: 00 



















Relatirm of Mglutinal 

*■'' 



ecled with Muiila eullura over a 



1 : 800. This diminution and final cessation of 
utinable substance in bacteria grown in a serum 
id immune bodies is interesting both as showing 
3acteria and as one means of adapting themselves 
iince the bacteria which ceased to produce agglutin- 
labty also produced less substance with affinity 
This inhibition of the production of agglutinable 



THE SUBSTANCES CONCERNED IN AGGLUTINATION. 171 

substance was also very noteworthy in the case of pneumococci grown 
in serum media. 

Relation between Agglutinating and Bactericidal Power. — In 

spite of proof to the contrary good observers hold to the belief that 
there is some relation between the agglutinating and the bactericidal 
strength of a serum. The tests we carried out on the serum of a 
number of horses showed no such relation. In Fig. 73 are recorded 
a number of comparative tests during a period of sixteen months. 
The tests of the bactericidal power of the serum were made by Goodwin. 

Variation in the Agglutinating Strength of a Serum. — ^There is 

usually a continued increase in the amount of agglutinin in the blood 
of an infected person from the fourth day until convalescence and 
then a decrease. At times, however, there is a marked variation from 
day to day, so that it may be abundantly present one day and almost 
absent the next. 

Precipitin. — A substance similar to, but altogether distinct from, 
agglutinin is precipitin. This substance was discovered by Kraus 
in 1897. He found that when a little immune serum was added to 
the bacteria-free filtrate of a culture of the organism used to produce 
the immunization there occurred a precipitate. This same reaction 
took place between the serum of an animal injected with various 
proteid substances, such as white of egg, blood serum, milk, etc. 
Precipitins in their development, their resistance to heat and chemicals 
and in their specific and non-specific forms are similar to agglutinins. 
The precipitins have been used more in relation to blood identification 
than in bacteriology. The specificity of precipitins is, like that of 
the agglutinins, not absolute. Group precipitins act upon similar 
chemical substances derived from cells having very different char*- 
acteristics. The precipitin test is mostly employed in testing sera and 
tissue extracts rather than bacterial filtrates. 

As the action of bacterial precipitins seems to be parallel with the 
action of the agglutinins, it is not possible that where tube reactions 
are depended upon, some confusion may occur as to which substance 
is really aflFected by certain processes or agents, especially those having 
a solvent action upon the bodies of the bacteria. 



CHAPTER XIV. 

OPSONINS^— EXTRACT OF LEUKOCYTES— BACTERIAL 

VACCINES. 

The original theory of Metchnikoff, that the leukocytes were the 
only actual protective bodies which warded oflF disease, and that they 
did this by attacking the bacteria, was founded on the now well-known 
fact that certain of the white cells possess the power of taking up 
into themselves pathogenic bacteria, and that they are there de- 
stroyed. It was later observed that these cells have the property of 
taking from the blood many lifeless foreign elements, thereby keeping 
the blood channels free of foreign particles. 

The question thereby arose as to whether these cells engulfed and 
then killed the bacteria, or whether perhaps other substances killed 
or prepared them before the cells took them up. It became known 
that certain bacteria are killed solely by the bactericidal substances 
in the serum, while others are not killed until taken up by the leuko- 
cytes. The leukocytes and the chemical substances of the blood thus 
both play an important part. The death of the bacteria also liberates 
positive chemotactic substances, and the disintegration of the white 
blood cells gives rise to bactericidal bodies. We find that phagocy- 
tosis is most marked when the disease is on the decline or the infection 
mild, but is usually absent in rapidly increasing infection. This 
would seem to indicate that the course of the infection is often already 
determined before the leukocytes become massed at the point of its 
entrance. The first determining influence is given by the condition of 
the tissues and the amount of bactericidal substances contained in 
them, and then, later, in cases where the bacteria have been checked, 
comes the additional help of the leukocytes. If the tissues are wholly 
free of bactericidal and sensitizing substances, neither they nor the 
leukocytes, nor both combined, can prevent the bacterial increase. 
The simple absorption by the cells of bacteria is not necessarily a 
destructive process. Metchnikoff believes that the polymorphonuclear 
leukocytes are especially antibacterial in relation to acute infections. 
The large phagocytes are conceived to deal chiefly with the resorption 
of tissue cells and with immunitv to certain chronic diseases, such as 
tuberculosis. 

The present great interest in the subject of the opsonins is largely 
due to the investigations and influence of Wright. W^e should, how- 
ever, recognize the important earlier work of others. Denys and 

* Greek "opsone" — I cater for. 

172 



OPSONINS, 



173 



Leclef had previously shown that in the ease of rabbits immunized 
against streptococci, the increased phagocytosis was due to an altera- 
tion in the serum and not to changes induced in the leukocytes. They 
demonstrated that the leukocytes of the immunized animal when 
placed in normal serum showed no greater phagocytic activity than 
normal leukocytes did. Wright added the important fact that the 
substances in the serum favoring phagocytosis united with the bacteria. 
Neufeld and Rimpau discovered the same point independently. 
Wright dealt mostly with normal serum, while Neufeld used serum 
from immunized animals. 

Wright originated the idea of estimating the changes in the opsonic 
power of the blood for the purpose of guiding the use of vaccines in the 
treatment of bacterial infections. Thus he states : 

" I have found that there exists in the serum of the successfully inoculated 
patient an increase of opsonin. This is a substance which lends itself to 
very accurate measurement by a modification of Leishman's method. By the 
aid of this method the patient's progress or regress can be very accurately 
foUowed." 

Where vaccines are injected Wright states there "supervenes a negative 
phase where there is a diminished content in protective substances. This is 
succeeded by a positive phase. This inflowing wave of protective substances 



Fio. 74 



1.7- 
1.6- 

1.5- 

1.4- 

1.8- 

IJt- 

1.1- 

1.0- 

.»- 

.8- 

.7- 

.6 - 

.5- 










— 


— , 


— , 












1 














' 


r 




















t 






— 


U -1 h -H 

1 
























:/ 


\ 


i 






— 


— 




» 








j 


; 












,/ 


\ 








— ' 






1 — ' 


1 


















/ \ 


>■ 








} — ' 


— t 

1 


1 


■--t— 












i 








/ \r 


\ 














-t — *■ — 








t 


r 












i 


V 






















^ 












/ 








V 


1 ' 






^ 1 










r^ 












/ 










\ 


k. 






_^^ 


-^>J 


^^ 


^^ 




-'\ 








r* 










1 




y 


r^^ — ^ — -» — 1 


. 







A-^ 


f^ 


. - — 







— 






z 




\ 


d 


/■ 


— 4 


\-- 


' "1 


^— 


1 






V\ 


- - ^ 


^ — 






V 












J 


~1 
1 




1 










OATE 


s *\ i '* n 10 ii| i« I'ls |i4 ii& 




Ottmh^r, |» M 



An opsonic curve showing the >Usht immediate rise and the later negative and positive phases 
following inoculation. Tne changes here are more regular than generally occuis. 



rapidly flows out again, but leaves behind in the blood a more or less per- 
manently increased content of protective substances. When a small dose of 
vaccine is given the negative phase may hardly appear, but the positive phase 
may be correspondingly diminished. Where an unduly large dose of vaccine 
is inoculated the negative phase is prolonged and much attenuated. The posi- 
tive phase may in such a case make default." 

"It will be obvious that, if we, in the case of a patient who is already the 
subject of a bacterial invasion, produce by the injection of an excessive dose 
of vaccine a prolonged and well-marked negative phase, we may, instead of 



174 PATHOGENIC MICRO-ORGANISMS, 

benefitiag the patient, bring about conditions which will enable the bacteria 
to run riot in his system." 

" Now, consideration will show that we may obtain, according as we choose 
our time and our dose wisely or unwisely, either a cumulative effect in the 
direction of a positive phase or a cumulative effect in the direction of a nega- 
tive phase. We may, in other words, by the agency of two or more successive 
inoculations, raise the patient by successive steps to a higher level of immu- 
nity, or, as the case may be, bring down by successive steps to a lower level. 
We can select the appropriate time and dose with certainty only by examining 
the blood and measuring its content in protective substances in each case 
before reinoculating.'' 

These statements of Wright have exerted a great influence, for, if 
he is correct, it will be desirable for every city to have laboratories 
equipped not only for suppljring vaccines, but also for determining the 
opsonic index in cases suitable for inoculation. Wright claims two 
fundamental points — first, that it is possible to determine the real 
opsonic power of the blood with suflScient accuracy to make it avail- 
able for treatment, and second, that the opsonins are either the most 
important of the protective substances of the blood or that they un- 
dergo a sufficient proportional development with the latter to be a 
safe guide as to their amount. A knowledge of the opsonic content 
of the blood is also believed by him to give information as to the 
presence and gravity of an infection. 

An immense amount of investigation has revealed the fact that the 
index cannot be obtained accurately enough to be a safe guide in single 
tests to be used in diagnosis or treatment unless the variation from the 
normal is exceptionally great and that the opsonic content is not a 
safe guide for the measure of the total antibodies in the blood. 

THE 0P80NI0 INDEX. 

Technique. — Wright's technique of measuring the opsonic power is 
a slight modification of the Leishman^ method and is as follows: An 
emulsion of fresh human leukocytes is made by dropping twenty drops 
of blood from a finger prick into 20 c.c. normal salt solution containing 
1 per cent, sodium citrate. The mixture is centrifuged, the super- 
natant clear fluid i-emoved and the upper layers of the sedimented 
blood cells transferred by means of a fine pipette to 10 c.c. normal 
salt solution. After centrifuging this second mixture the supernatant 
fluid is pipetted oflF and the remaining suspension used for the opsonic 
tests. Such a "leukocyte emulsion," of course, contains a mixture of 
leukocytes and of red blood cells; the proportion of leukocytes, how- 
ever, is much greater than in the original blood. The bacterial emul- 
sion is prepared by gently rubbing a little of the culture to be tested 
in salt solution (0.85 to 1.2 per cent.). When thoroughly mixed the 
fluid is centrifuged for a few minutes so as to remove any clumps. The 
emulsion should be so thick that in a trial test the leukocytes take up 
about five apiece on the average. 

* Leiehman, British Medical Journal, Jan., 1902. 



OPSONINS, 175 

One volume of the leukocytes is mixed with one volume of the bac- 
terial suspension to be tested and with one volume of the serum. This 
is best accomplished by means of a pipette whose end has been drawn 
out into a capillary tube several inches in length. With a mark made 
about three-quarters of an inch from the end it is easy to suck up one 
such volume of each of the fluids, allowing a small air bubble to inter- 
vene between each volume. All three are now expelled on a slide and 
thoroughly mixed by drawing back and forth into the pipette. Then 
the mixture is sucked into the pipette, the end sealed, and the whole 
put into the incubator at 37° C. The identical test is made using a nor- 
mal serum in place of the serum to be tested. Both tubes are allowed 
to incubate fifteen minutes and then the end of the tube is broken off, a 
large drop mounted on a clean sUde the surface of which was previously 
roughened by emery-paper and a spread made with a second slide as in 
ordinary blood work, only a little thicker and using no force whatever. 
After drying in the air the smears are stained without previous fixation 
either with a 1 per cent, aqueous solution of methylene blue or some 
other suitable stain. The degree of phagocytosis is then determined in 
each by counting a consecutive series of fifty or one hundred leukocytes 
and finding the average number of bacteria ingested per leukocyte. 
This number for the serum to be tested is divided by the number ob- 
tained with the normal serum and the result regarded as the opsonic 
index of the serum in question. Thus, if the tubercle bacilli, sensitized 
by a patient's blood, are taken up by the leukocytes to the average 
number of three per leukocytes, and bacilli from the same emulsion 
sensitized b) normal blood are taken up by leukocytes to the average 
of five, then the index will be three-fifths of one, or 0.6. In this case 
the index would indicate a deficiency in opsonins. The presence of 
a high opsonic index Wright regards as indicative of increased resis- 
tance. He further states that the fluctuation of the opsonic index in 
normal health v individuals is not more than from 0.8 to 1.2, and 
that an index below 0.8 is, therefore, almost diagnostic of the presence 
of an infection with the organism tested. 

Simon's Method: Simon has suggested a modification of Wright's 
method. He estimates the percentage of phagocyting cells in the 
mixture containing the serum to be tested and compares this with 
the mixtures containing normal serum. He also suggests that dilu- 
tions of blood be tested. 

The DiltUion or Extinction Method recommended by Dean and by 
Klein. The degree of dilution of the serum necessary for the extinc- 
tion of its opsonic index is determined; that is, the serum to be tested 
is diluted until a dilution is found which shows the same small 
amount of phagocytosis shown in preparations in which no serum is 
used, namely an index below 0.5. Klein claims that results by this 
method are more accurate than by the method of Wright. The 
method is too tedious for practical use in routine work. 

Most workers are now agreed that the use of the opsonic index is 
limited to experimental investigations. 



176 



PATHOGENIC MICRO-ORGANISMS. 



The Accuracy with Which the Opsonic Power of the Blood 
CAN BE Determined by Wright's Method. — ^An examination of 
any slide will show that the different leukocytes vary in their size 
and in their content of bacteria. This is due partly to variation in 
phagocytic activity, and partly to the interference of the red blood 
cells, which are present in great numbers in the emulsion and separate 
the bacteria in different degrees from the white cells. These and 
other reasons bring it about that the different leukocytes vary greatly 
in the number of bacteria they take up and in their distribution on 
the slide. Partly to overcome this, large numbers of leukocytes are 
counted. Beyond one hundred, or at most one hundred and fifty, 
the increase of accuracy hardly compensates for the extra labor. 
The following table shows the difference between counting larger or 
smaller numbers of cells in five opsonic tests as determined by count- 
ing different numbers of cells in one specimen. 

Opsonic Index Estimations in Five Blood Specimens. 



Cells counted 



Average number of bacteria in each leukocyte. 



50, 
100 
150 
200. 
600. 
1,200. 



1.18 
1.22 
1.18 
1.18 
1.28 
1.34 





1.8S 


1.34 


1.42 


1.90 




1.78 


1.24 


1.42 


1.59 




1.62 


1.22 


1.44 


1.50 




1.51 


1.22 


1.46 


1.37 


1.62 


1.23 


1.36 


1.36 


1 1.44 

1 


1.25 


1.30 


1.42 



It is noticed that the variation between the average cell count 
obtained from fifty cells and larger numbers is much greater than 
between that obtained at from one hundred or one hundred and fifty. 

It is necessary to have the counts that are compared all counted by 
the same person, as each individual has a somewhat diflFerent method 
and will average higher or lower for all counts than any other person. 

When two specimens of blood are tested not only the inaccuracy of 
counting due to the diflFerent arrangement of the unequally filled cells 
on the slides to be counted is met, but the fact that in making the test 
the conditions are not similar, for in different mixtures slightly dif- 
ferent proportions of leukocytes, bacteria, and red cells will always be 
mixed together. If smears from a series of tubes of the same blood 
are compared with a series of smears from one of the tubes, the former 
will always show the greater variation. 

This variation is much greater than most examiners believe. North 
has collected a series of tests carried out in nearly all the important 
laboratories in the Eastern United States that are working upon 
opsonins. The results recorded prove absolutely that while an average 
counting error of only about ten per cent, is present, there may be 
an exceptional error of at least 100 per cent., and one of at least 



OPSONINS. 



Ill 



20 per cent, may be expected once in about every ten determinations. 
The following is a fair average of the correctness of routine tests 
by experienced workers. 

Absolute Count of Bacteria in One Hundred Leukocytes. 



Blood specimen A. 


Blood specimen B. 


Blood specimen C. 


Tube 1 

Tube 2 

Tube 3 

Tube 4 


156 
168 
172 
198 


Tube 1 

Tube 2 

Tube 3 


142 

182 

188 


1 

Tube 1 89 

Tube 2 102 

Tube 3 121 

1 



This error, which occurs because of the technique, applies not only 
to the examination of the specimen of blood, but also to the measure 
we employ to estimate the amount of opsonins. As these are not 
stable, we cannot have a standardized solution, as we do with anti- 
toxins. We must, therefore, determine our measure afresh in each 
test, taking for this purpose a supposedly normal blood. Wright, 
from a great many tests, has determined that the opsonic power of 
the blood in non-infected persons for tubercle bacilli does not vary, 
as a rule, more than 10 per cent, above or below the average power 
of healthy blood. For staphylococci there is more variation. It is 
found also that many things besides infection decrease the amount of 
opsonins in the blood. Hemorrhage, fatigue, starvation, and other 
influences which lower the resistance of the body have this effect. 

Wright gets this measure as uniform as possible by determining 
the average opsonic strength of five supposedly healthy persons at 
the time of each test. If any one of these five is considerably below 
or above others it is omitted for that day. The measure so obtained 
will probably vary above 5 per cent, from day to day, though seldom 
getting far away from what we might call the absolute normal. The 
following results were obtained by us from examining at one test a 
number of supposedly normal persons against tubercle bacilli and 
staphylococci. 



Opsonic Counts in Test of Twenty-one Normal Sera with Stock 

Staphylococcus Culture. 



1 : 4.13 

2 2.93 

3 2.78 

4 4.37 

5 3.58 

6 2.90 

7 i 3.56 



8 3.82 

9 3.95 

10 3.98 

11 4.27 

12 * 3.69 

13 3.80 

14 3.59 



15 9.09 

16 5.17 

17 4 04 

18 3.82 

19 4.00 

20 3.79 

21 3 44 



12 



178 



PATHOGENIC MICRO-ORGANISMS. 



Opsonic Counts in Eighteen Consecutive Normal Cases with 

Tubercle Bacilu. 



Case 


Average 

number 

bacilli 


Opsonic 
index 


Case 

I 


Average 

number 

bacilli 


Opsonic 
index 


1 

2 


2.46 

3.20 

2.90 

2.66 

! 2.75 

1 2.30 

1 2.40 

1 1.88 

1 1.73 


1.00 

1.30 

1.14 

1.08 

1.12 

.94 

.98 

.76 

.70 


10 

11 

i 12 

13 

14 

15 

' 16 

17 

18 


2.05 
2.21 
; 2.86 
i 2.81 
2.79 
3.34 
2.96 
2.16 
3.12 


.83 
.90 


3 


1 17 


4 


1 14 


5 

6 

7 

8 

9 


1.14 
1.32 
1.02 

.88 
1.27 



The Influence Upon the Opsonic Test of the Specific Differ- 
ences Between Strains of a Single Species. — The general practice 
in laboratories is to use stock cultures of tubercle bacilli, staphylococci, 
and other bacteria for the opsonic tests. To obtain a culture from a 
case may be at first impossible and, if successful, causes a delay of at least 
one or two days. The culture when obtained may also, as is frequently 
the case with pneumococci and streptococci, fail readily to opsonize. 

These and other reasons tend to establish the use of laboratory 
stock cultures, and yet we must acknowledge that when we test the 
amount of opsonins by both the stock and fresh cultures a marked 
difference sometimes develops. This factor of individual specificity 
must therefore b.e taken into account in our interpretation of the 
accuracy of an opsonic test. 

The Leukocytes to be Employed. — To many it seems a matter 
of indifference whether one person's leukocytes or another's are used, 
but our experience agrees with that of others that the leukocytes from 
diflFerent persons not only vary in their activity, but also in their selective 
action, and that the index is not the same when obtained with one 
person's leukocytes as with another's. 

The Influence of the Strength of the Bacterial Emulsion. 
— The more abundant the bacteria the greater will be the number 
taken up by the leukocytes. It is very important therefore that the 
tests be made with the same strength of emulsion. 

'^Fhe Opsonic Variation During Treatment by Inoculations. 
— Wright lays stress on the considerable uniformity of the degree and 
persistence of development of opsonins after inoculation. We have 
found in a small percentage of cases typical increases and decreases, 
as seen in Fig. 74, but in the majority of those inoculated there has 
been great irregularity. Frequently the negative phase does not occur 
or at least it is not detected. 

The following chart for three staphylococcus cases illustrates this 
(Fig. 75) : 

The Variation in the Amount of Opsonins in Supposedly 
Healthy Persons. — It has already been noted that in getting our 



OPSONINS. 



179 



measure we teat a number of persons and exclude the blood of those 
which varies greatly from the average. We are so in the habit of 
seeing the normal blood placed at unity because it is each (lay the 
measure of comparison and therefore is one that even investigators are 



apt to think of the indices of normal persons as being unchanged from 
day to day. This is not the fact. A glance at the next chart, in which 
three cases of tuberculosis are charted together with two normal 
persons, shows that the variation is only slightly greater in infected 



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180 PATHOGENIC MICRO-ORGANISMS. 

than in normal cases. If one normal person is charted against another 
for several weeks, marked differences will usually appear. The 
indices of the twenty-one normal cases tested against staphylococci 
and the eighteen against tubercle bacilli (pages 177 and 178), illustrate 
this variation in the amount of opsonins in normal blood. . 

The Opsonic Index Cannot be Known at the Time the Treat- 
ment IS Given. — Most of those who have not carried out inoculations 
under the guide of the opsonic tests think that the vaccinator is guided 
at the moment of injection by his knowledge of the opsonic power of 
the blood at the time. A moment's thought reveals that this is an 
absolute impossibility. In fact, except under very unusual conditions, 
it is impossible to have the test of the opsonic power reported within 
twenty-four hours, and in the treatment of the poor in out-patient 
practice longer intervals usually elapse, so that the treatment is given 
on a test made either the day before or, more often, on from three to 
seven days before. As can be seen by the three curves on page 179, 
which are quite as uniform as the average, it is impossible to judge 
what the index is at any moment by looking at the indices of blood 
taken from one to seven days previously. 

Other methods have been devised to get more accurate information 
upon the opsonic contents of the blood. The dilution method and 
that combined with the determining the percentage of phagocytes ab- 
sorbing bacteria are the most valuable^ (p. 175). For experimental 
work they have advantages, but for practical use in governing the dos- 
age of vaccines they have most of the drawbacks of Wright's method. 

The Nature of Opsonins. — Wright and Neufeld, in their orig- 
inal experiments differed as to the effect of heat on opsonins. Further 
investigation has shown that opsonins in those not immunized are 
largely thermo-labile, while opsonins developed after immunization 
are resistant. Muir and Martin believe from their experiments that 
the thermo-labile opsonin of normal serum and the thermo-stable 
opsonin are two entirely distinct classes of substances. The thermo- 
stable substance is of the nature of a true antibody and possesses the 
comparatively specific qualities of antibodies in general. Powerful 
complement absorbers have no effect on the thermo-stable opsonin, 
but do remove almost completely the thermo-labile opsonin. 

Emulsions of other than the organisms used in immunization do 
not absorb a large percentage of the immune opsonin, but do of the 
complement opsonin. 

We have carried out absorption experiments with staphylococci, 
colon, and tubercle bacilli. Our results were similar to those of Muir 
and Martin. 

Opsonin Deficient in Cerebrospinal Fluid and in Exudates. 
— Opie' has shown that exudates produced by injecting microorgan- 
isms usually have little or no opsonin for the variety injected or for 
other varieties. Hektoen has showed that opsonins, like other anti- 

^ Simon, Jour. Exp. Medicine, Vol. 9, No. 5, 1907, page 487. 
^ Opie, Jour. Exp. Med., Vol. 9, No. 5, p. 515. 



OPSONINS. 181 

bodies, are almost absent in the spinal fluid. McKenzie and Martin* 
showed that in a case of cerebrospinal meningitis the spinal fluid 
showed no immune bodies while the blood contained them in 
abundance. 

Comparison Between Opsonins and Bactericidal Substance 
IN the Serum. — We have made comparative tests between the op- 
sonic and bactericidal power of the cell-free serum in typhoid infection 
and found that they did not run parallel. The frequent rapid increase 
in opsonic power within twelve hours of an injection of bacteria is 
striking and verj' different from the development of bactericidal 
strength. 

Rasults Obtained by Vacdne Therapy. — In most cases, the employ- 
ment of vaccine therapy directed to the destruction of a single species 
of microbe leaves the other species quite unaffected. When in cases 
of mixed infection measures are taken to immunize the patient against 
each of the different infections, the task of the immunizator is more 
laborious and more intricate. On the other hand, the organism of 
the patient does not seem to find the task of responding to a series 
of different vaccines (always supposing that each of these is admin- 
istered in appropriate and properly interspaced doses) much more 
diflScult than the task of responding to one variety of vaccine only. 

Although during the past three years many thousands of cases of 
different types of bacterial infection have been treated by vaccines, there 
is at present considerable difference of opinion as to their value. The 
majority of observers agree that it is in subacute and chronic infections 
that vaccines give the best results. Thus a case of acute streptococcus 
septicaemia, which after a week or ten days shows a tendency to abate 
with localization in a joint or in a valve of the heart, offers a much 
better chance for vaccine treatment than such a case during the early 
more acute stage. Pneumonia, which after partial recovery persists, a 
gonorrhceal joint, a persistent pus sinus, a localized inflammation due 
to colon bacilli, are all considered suitable for treatment. The use 
of vaccines in cases of acute inflammation of the mucous membranes 
of the intestines, bladder, throat, etc., have in most hands given rather 
negative results. Most believe that the inflammation is lessened, but 
as a rule the bacterial infection, though it becomes latent, still remains 
and a relapse may occur later. 

The different bacteria seem to differ in the successes with which 
they are used as vaccines. Nearly all observers report the greatest 
success with staphylococcus infections. These not only when they are 
severe as a subacute septicaemia or a very severe carbuncle, but also 
when they are mild, such as ordinary furuncles or acne, seem to be aided 
by the vaccine treatment. Gonorrhceal joints seem to respond well, 
while acute gonorrhoea of the mucous membranes responds less readily. 
Suitable cases of inflammations due to Friedlander's bacillus, to the 
micrococcus catarrhalis, and many other varieties respond quite well 
to inoculations. 

* Jour, of Path, and Bact., Vol. xii, p. 539. 



182 PATHOGENIC MICRO-ORGANISMS. 

The streptococcus infections seem to be more resistant, and, although 
in the something like forty cases of subacute septicaemia due to these 
organisms which have been treated the results indicate some benefit, 
the effects of the vaccines are not well-established. The pneumococcus 
also seems resistant, perhaps even more so than the streptococcus. 
Numerous cases of pneumonia have been treated with somewhat 
doubtful results. Some observers treat these pneumococcus and strep- 
tococcus inflammations with daily doses of 5 million instead of the large 
semi-weekly inoculations. Various sinus infections of the head have 
been treated, but with doubtful results. Streptococcus and other 
inflammations of the gums and teeth have yielded apparently good 
results. The treatment of tuberculosis with the different tuberculins 
has given perhaps the most extended use of vaccines. The majority 
of observers are convinced that in cases of tuberculosis, in which the 
symptoms have become quiescent, whether they are incipient or fairly 
advanced, that tuberculin injections do good. Some believe in giving 
very minute doses, and only slightly increasing their size, others believe 
that different cases will receive with benefit different amounts, and so 
try to detect which will stand larger amounts. Nearly all physicians 
try to prevent definite reactions. The treatment of tuberculosis is 
considered in detail under tuberculosis. 

Preparation of Vaccines. — Bacterial vaccines heated above 60° show 
marked changes in their chemical composition, and do not yield the 
same amount of antibodies as those not so heated. Even lower degrees, 
such as 56*^, are preferable to 60*^. It has lately been suggested that 
the vaccines be killed by ^ per cent, carbolic acid, or i per cent, lysol 
instead of by heating. These vaccines seem to be somewhat more 
capable of giving good response and they preserve their characteristics 
for a longer period. Such vaccines can be kept for a period of six 
months to one year. 

Amount of Vaccine Injected. — Different observers advise that 
for each organism the following number of millions be given : 







Minimum. 


Maximum. 


Average. 


Staphylococcus 




50.0 m. 


1000.0 m. 


250.0 m. 


Streptococcus 




2.5 m. 


100.0 m. 


25.0 m. 


Pneumococcus 




2.5 m. 


100.0 m. 


25.0 m. 


Gonococcus 




2.5 m. 


300.0 m. 


30.0 m. 


B. coli group 




5.0 m. 


1000.0 m. 


100.0 m. 


B. pyocyaneus 




5.0 m. 


1000.0 m. 


100.0 m. 


Tuberculin (B. 


E.) 


.00003 mg. 


.0005 mg. 


.0003 mg. 



The injections are usually given at intervals of five to ten days but 
sometimes daily. 

Sensitised Vaccines. — Besredka* has suggested a plan of injecting 
virus which has been immersed in specific serum; in other words, of 
using virus which has been sensitized. Heated vaccines alone some- 
times give severe symptoms when inoculated, e.g., typhoid, cholera; 
but after exposure to serum, inoculations of the sensitized vaccines are 

* Review of previous work. Bull. Inst. Pasteur, 1910, viii, 241. 



OPSONINS. 183 

found to give practically no negative phase and only slight local and 
general reactions. Immunity, moreover, is produced rapidly by this 
procedure. Such sensitized vaccines can be given in doses thirtyfold 
those of pure virus. Marie* has used the same method in rabies treat- 
ment. We have only used sensitized vaccines in animal experiments 
and have not found as good a response as to the corticated bacteria. 
The method is as follows: (1) For bacteria, — A twenty-four-hour 
agar culture of bacteria is mixed with specific serum at room temperature 
and allowed to stand for three hours. The bacteria are then washed 
free from unattached serum by repeated centrifugalizations with 
sterile normal salt solution. 

Typhoid and cholera bacteria are killed at this stage of the procedure 
by heating at 56® to 60° C. for one hour. Plague bacilli are killed be- 
fore the addition of the serum. By this method the endotoxins are 
neutralized. 

Immunity in plague develops forty-eight hours after injection and 
lasts one and one-half months. 

In tjT)hoid and cholera injections immunity develops in twenty- 
four hours (with non-sensitized vaccine, it does not appear for forty- 
eight hours) and lasts over five months. 

After injections with sensitized dysentery vaccine, immunity develops 
in four days and lasts over four months. 

Autogenous and Stock Vaccines. — Wherever possible it is well to 
make the injections with cultures derived from the patient (autogenous 
culture). The usual practice is to use a stock vaccine for the first 
inoculation and autogenous vaccines later. Sometimes it is impossible 
to obtain a culture from the patient. The staphylococci, gonococci, 
tubercle, and typhoid bacilli from different cases seem much alike, so 
that it is less important with these organisms to get autogenous vaccines 
than when streptococci, pneumococci, or colon bacilli must be used. 

The Diagnostic Value of Opsonins. — The presence of a great 
excess or deficiency of opsonins for a microorganism, or of marked 
variation in the index after massage or exercise, has been thought by 
some to indicate the type of infection. Extreme caution should be 
used in making such an application of the index determinations. 

Leukocyic Extract in Infections. — Hiss^ recommends the treatment of 
certain diseases with a leukocyte extract. He makes this extract as 
follows: 

The leukocytes are obtained by double pleural inoculations with 
aleuronat into the animal (rabbit, dog). The amount of leukocyte- 
filled fluid obtained after twenty-four hours from rabbits has usually 
been from 30 to 60 c.c. This is immediately centrifugalized, the serum 
poured off, and the extracting fluid (distilled water) added in amounts 
about equal to the fluid poured off. The cells are then thoroughly 
emulsified in the distilled water, allowed to stand for a few hours at 
37.5° C, and then at ice-box temperature until used. Varying amounts 

*See Rabies, Sec. III. 

^ Jour. Med. Research, xiv, No. 3. 



184 PATHOGENIC MICRO-ORGANISMS. 

of the entire fluid (after shaking) are inoculated. Hiss's animal ex- 
periments were made on rabbits and guinea-pigs infected with staphyl- 
ococcus, streptococcus, pneumonoccus, tv-phoid bacillus, or menin- 
gococci. Hiss states that animals suffering from severe septicxemias 
and poisonings following intravenous injection of anyone of the above 
oi^anisms have shown the beneficial effect of treatments with extracts 
of leukocytes, and have, in many instances, survived infections fatal to 
the control animals in thirly-six hours, even when treatment has been 
delayed as late as twenty-four hours. Zinsser' has carefully studied 
the nature of the substances extracted from leukocytes. He finds 
they contain no complement, are not destroyed by heating to 56*" C, 
and are no more abundant in cells derived from immunized than 
from normal rabbits. 

The Hiss leukocytic extract has now been used by a number of ob- 
servers for some two years. Cases of pneumonia, erysipelas, septi- 
caemia and some other infections have been treated. It is difficult to 
determine just what the value of the treatment is. No harmful re- 
sults have been noticed. In a certain number of eases the tempera- 
ture and .symptoms have bettered, in a way which seemed clearly to 
indicate that the extract had done good. In other cases no results 
whatever were apparent. It is usually given subcutaneously in 10 c.c. 
doses, every four to six hours. As high as 500 c.c. have been given 
in some cases. 

TacdneB as Immunising Agents. — The injection of vaccines in healthy 
subjects for the prevention of disease have been made so extensively 
that no one doubts the advisability of their use. Typhoid vaccines are 
used extensively in the army and among persons going into special 
danger. The usual injections are either 100 million, 300 million, and 
500 million or two injections of 500 million and 800 million. Tests 
of the blood of such individuals show a large development of antibodies. 
Cholera vaccines and vaccines against bubonic plague also have been 
widely used. 

'Jour. Med. Research, Vol. xxii, No. 3, 



CHAPTER XV. 
THE USE OF ANIMALS FOR DIAGNOSTIC AND TEST PURPOSES. 

Suitable animals are necessarily employed for many bacteriological 
purposes. 1. To obtain a growth of varieties that for any reason 
grow with diflBculty on artificial culture media, as in the case of tubercle 
bacilli: hence material suspected to contain tubercle bacilli is injected 
into guinea-pigs7 with the knowledge that, if present, although in too 
small numbers to be detected by microscopic or culture methods, 
they will develop in the animals' bodies, and thus reveal themselves. 
The same may be true of glanders, tetanus, and anthrax bacilli, of 
pneumococci, of other bacteria, and of protozoa. Certain micro- 
organisms cannot be grown at all on artificial media. This is true of 
few bacteria, of most protozoa, of most of the spirochetes, and of 
certain unknown infectious agents such as produce smallpox and Rocky 
Mountain spotted fever. 2. To cause an increase of one variety of 
organisms in a mixture and thus obtain a pure culture: An injection 
of sputum subcutaneously in rabbits may give rise to a pure pneumo- 
coccus septicaemia or a pure tuberculosis. 3. To test virulence: 
Animals are used to test the virulence or toxin production of organisms, 
where, as in the case of diphtheria, we have very virulent, attenuated, 
and non-virulent bacilli of, so far as we know, identical cultural 
characteristics. Here the injection of a susceptible animal, such as the 
guinea-pig, is the only way that we can differentiate between those 
capable of producing diseases from those that are harmless. Still 
another use of the animals is to differentiate between two virulent 
organisms, which, though entirely different in their specific disease 
poisons, are yet so closely allied morphologically and in culture char- 
acteristics that they cannot always be separated except by studying 
their action in the animal body both with and without the influence 
of specific serums. In this way the typhoid and colon bacilli may be 
separated, or the pneumococcus and streptococcus. 4. To test the 
antitoxic or bactericidal strength of sera: Diphtheria antitoxin is 
added to diphtheria toxin and injected into guinea-pigs, and strepto- 
coccus immunizing serum is mixed with living streptococci and 
injected into the vein of a rabbit. 5. To produce antitoxic, bacteri- 
cidal, or agglutinating sera. 

The Inoculation of Animals. — ^The inoculation of animals may be 
made either through natural channels or through artificial ones : 

1. Cutaneous. Cultures are rubbed into the abraded skin. 

2. Subcutaneous. The bacteria are injected by means of a hypo- 
dermic needle under the skin, or are introduced by a platinum loop 
into a pocket made by an incision. 

185 



186 PATHOGENIC MICRO-ORGANISMS. 

3. Intravenous. The bacteria are injected by means of a hypo- 
dermic needle into the vein. This is usually carried out in the ear 
vein of the rabbit. If rabbits are placed in a holder, so that the animal 
remains quiet and only the head projects, it is usually easy to pass a 
small needle directly into one of the ear veins, especially those running 
along the edge of the ear. If the ear is first moistened with a 3 per 
cent, carbolic acid solution, and then supported between the finger 
inside and the thumb outside, the vein is usually clearly seen and entered 
with ease, if a small, sharp needle is held almost parallel with the ear 
surface and gently pushed into it. When no holder is present, the 
rabbit can be held by an assistant seizing the forelegs in one hand and 
the hind in another and holding the rabbit head downward, or the 
animal may be held between the knees of the operator, its body resting 
on the operator's apron. 

4. Into the anterior chamber of the eye. 

5. Into the body cavities. The peritoneal and less often the pleural 
cavities are used for bacterial injection. The hypodermic needle is 
usually employed, less often a glass tube drawn out to a fine point. 
The needle or the pointed glass tube is gently pushed through the 
abdominal wall, moved about to be certain that the intestines have not 
been perforated and the fluid injected. 

6. By inhalation. This method is carried out by forcing the animal 
to inhale an infected spray or dust. 

7. By the trachea. This method is carried out by making an incision 
in the trachea and then inoculating the mucous membrane or injecting 
substances into the trachea and bronchi. 

8. Through the intestinal tract by swallowing or by the passage of" 
a rubber tube. Morphine may be given to prevent peristalsis. 

9. Into the brain substance or ventricles after trephining, or when 
the parietal bones are thin as in the guinea-pig and the rabbit, after 
making a tiny opening with the point of a small, heavy scalpel. 

In these injections guinea-pigs are held, as a rule, by an assistant 
grasping in one hand the forelegs and in the other the hind legs. 

Rabbits can be held in the same manner or, they may be placed in 
some holder or strung up by their hind legs, or held between the 
knees. 

Mice, which are usually inoculated subcutaneously in the body or 
at the root of the tail, are best placed in a mouse holder, but can be 
inoculated by grasping the tail in a pair of forceps, and then, while 
allowing the mouse to hang head downward in a jar, a glass plate 
is pushed across the top until only space for its tail is left. 

Monkeys and apes are used for certain infections, such as syphilis 
and smallpox, where only man and they are markedly susceptible. 

All these methods must be carried out with the greatest care as to 
cleanliness, the hair being clipped and the skin partially, at least, 
disinfected. The operator must be careful not to infect himself or 
his surroundings. After the inoculations the animals should be 
given the best of care, unless, for special purposes, we want to study 



USE OF ANIMALS FOR DIAGNOSTIC AND TEST PURPOSES. 187 

them under unusual conditions. For food, rabbits and guinea-pigs 
require only carrots and hay. 

When possible, all animals should be anesthetized during painful 
experiments. 

If animals die, autopsy should be made at the earliest moment pos- 
sible, for soon after death some of the species of the bacteria in the 
intestines are able to penetrate through the intestinal walls and infect 
the body tissues. If delay is unavoidable, the animals should be 
placed immediately in a place where the temperature is near the 
freezing point. In making cultures from the dead bodies the greatest 
care should be taken to avoid contamination. The skin should be 
disinfected, and any dust prevented by wetting with a 5 per cent, 
solution of carbolic acid. All instruments are sterilized by boiling in 
3 per cent, washing soda solution for five minutes. Changes of 
knives, scissors, and forceps should be made as frequently as the old 
ones become infected. When organs are examined the portion 
of the surface through which an incision is to be made must be sterilized, 
if there is danger that the surrounding cavity is infected, by sear- 
ing with the flat blade of an iron spatula which has been heated to a 
dull red heat. Tissues if removed should be immediately placed under 
cover so as not to become infected. Sterile deep Petri plates are useful 
for this purpose. 

When it is necessary to transport tissues from a distance they should 
be wrapped in bichloride cloths and sent to the point of destination 
as soon as possible. In warm weather they may be kept cool by sur- 
rounding the vessels which contain them with ice. 

Animals rarely show the same gross lesions as man when both 
suffer from the same infection. The cell changes, however, are similar, 
and, also, so far as we can test them, the curative or immunizing effects 
of protective serums. 

Leukocytes for Testing Phagocytosis.— Inoculate into the pleural 
cavity of a rabbit 5 c.c. of a thick suspension of aleuronat powder in 
a boiled starch solution. The solution should be thick enough to 
hold the aleuronat in suspension. A 20 to 25 per cent, solution of 
peptone gives good results. The fluid is withdrawn eighteen to 
twenty-four hours after the injection. 

For purposes of obtaining the opsonic index the whole blood is 
taken. For description of the method see chapter on opsonins. 

Leukocytes from the horse can be readily obtained by mixing the 
blood with 1 per cent, of sodium citrate and allowing the mixture 
to stand. The red cells rapidly sink and leave the leukocytes in the 
supernatant fluid. 



CHAPTER XVI. 

THE PROCURING AND HANDLING OF MATERIAL FOR BACTE- 
RIOLOGIC EXAMINATION FROM THOSE SUFFERING 

FROM DISEASE. 

A LONG experience has taught us that physicians very frequently 
take a great amount of trouble, and yet, on account of not carrying 
out certain simple but necessary precautions, make worthless cul- 
tures or send material almost useless for bacteriologic study. 

In making cultures from diseased tissues various procedures may 
be carried out, according to the facilities which the physician has and 
the kind of information that he desires to obtain. From the dead 
body culture material should be removed at the first moment pos- 
sible after death. Every hour's delay makes the results less reliable. 
From both dead and living tissues, the less the alteration that occurs 
in any substance between its removal from the body and its examination 
and inoculation upon or in culture media or animals, the more exact will 
be the information obtained. If the material is allowed to dry many 
bacteria will be destroyed in the process, and certain forms which 
were present will be obliterated or, at least, entirely altered in the 
proportion which they bear to others. If possible, therefore, smears 
should be made and culture media should be inoculated directlv 
from the patient or dead body. For the latter purpose a bacteriologist 
should take the most suitable of the culture media to the bedside or 
autopsy table. Such a list of media, if fairly complete, would com- 
prise nutrient bouillon alone and mixed with one-third its quantity 
of ascitic fluid, slanted nutrient agar, slanted agar streaked with rabbit 
or human blood, firmly solidified slanted blood serum and slanted 
ascitic glucose agar. Additional media will be necessary for special 
purposes, such as the isolation of typhoid or tetanus bacilli. If only 
one variety of media is to be used the solidified blood serum is most 
useful for parasitic bacteria, and this can be easily carried by the 
physician and inoculated by him, even if he is not very familiar with 
bacteriologic technique. In the first place some of the infected material 
should always be smeared on a couple of clean slides or cover-glasses 
and allowed to dry. These can be stained and examined later, and 
may give much valuable information. 

The material must be obtained in different ways, according to the 
nature of the infection. 

For the detection of the bacteria causing septioemia we are met 
with the difficulty that there are apt to be very few organisms present 
in the blood until shortly before death. It will, therefore, be almost 
useless to take only a drop of blood for cultures, as even when present 

188 



MATERIAL FOR BACTERIOLOGIC EXAMINATION. 189 

there may not be more than eight or ten organisms in a cubic centi- 
metre. If cultures are to be made at all, it is, therefore, best to make 
them correctly by taking from 5 to 20 c.c. of blood by means of a 
sterile hypodermic needle or a suitable glass tube armed with a hypo- 
dermic needle from the vein of the arm, after proper cleansing of the 
skin and a tiny incision. To each of five different tubes containing 
bouillon we add 1 c.c. of blood, and to a flask containing 100 c.c. we 
add 5 c.c. We have made by this mixture of blood and bouillon a most 
suitable medium for the growth of all bacteria which produce septi- 
caemia, and, at the same time, have added a sufficient quantity of blood 
to insure us the best possible chance of having added some of the 
bacteria producing the disease. We also add to each of several tubes 
of melted nutrient agar, at 40° C, 1 c.c. of blood and pour the mixture 
into Petri plates, so as to indicate roughly the number of organisms 
present if they happen to be in abundance. When blood must be 
carried to a distance, clotting should be prevented by having in the 
test-tube sufficient 10 per cent, solution of sodium citrate, bile, or 
ammonium oxalate to prevent clotting. 

From wounds, abscesses, cellulitis, etc., the substance for bacterio- 
logic examination can, as a rule, best be obtained by means of a 
syringe, or when the lesion is opened, by small rods armed with a little 
absorbent cotton. A number of these swabs can be sterilized in a 
test-tube and so carried. The swab is inserted in the wound, then 
streaked gently over the oblique surface of the nutrient agar in one 
tube, over the blood serum in another, and then inserted in the bouillon. 
Finally, either at the bedside or in the laboratory, material is thinly 
streaked over the surface of nutrient agar contained in several Petri 
dishes. W^e inoculate several varieties of media, with the hope that 
one at least will prove a suitable soil for the growth of the organisms 
present. From surface infections of mucous membranes, as in the 
nose, throat, vagina, etc., the swab, again, is probably the most useful 
instrument for obtaining the material for examination. The greatest 
care, of course, must be used in all cases to remove the material for 
study without contaminating it in any way by other material which 
does not belong to it. Thus, for instance, if we wish to obtain material 
from an abscess of the liver, where the organ lies in a peritoneal cavity 
infected with bacteria, one must first absolutely sterilize the surface 
of the liver by pressing on it the blade of a hot iron spatula before 
cutting into the abscess, so that we may not attribute the infection 
which caused the abscess to the germs which we obtained from the 
infected surface of the liver. From such an organ as the uterus it is 
only with the greatest care that we can avoid outside contamination, 
and only an expert bacteriologist familiar with such material will be 
able to eliminate the vaginal from the uterine bacteria. 

A statement of the conditions under which materials are obtained 
should always accompany them when sent to the laboratory for ex- 
amination, even if the examination is to be made by the one who 
made the cultures. These facts should be noted, or otherwise at some 



190 PATHOGENIC MICRO-ORGANISMS. 

future date they may be forgotten and misleading information sent 
out. The work of obtaining material for examination without con- 
tamination is at times one of extreme difficulty. It simply must 
be remembered that if contamination does take place our results may 
become entirely vitiated, and if the difficulties are so great that we 
cannot avoid it, it may simply mean that under such conditions no 
suitable examination can be made. Where the substance to be studied 
cannot be immediately subjected to cultures or animal inoculations, 
it should be transferred in a sterile bottle as soon as possible to a 
location where the cultures can be made. If for any reason delay 
must take place, the material should at least be put in a refrigerator, 
where cold will both prevent any further growth of some varieties 
of bacteria and lessen the danger of the death of others. 

In obtaining samples of fluid, such as uHne, feces, etc., the bottles 
in which they are placed should always be sterile, and, of course, no 
antiseptic should be added. It is necessary clearly to explain this (o 
the nurse, for she has probably been instructed to add disinfectants 
to all discharges. Disinfected material is, of course, entirely useless 
for complete bacteriologic investigations. It cannot be too much em- 
phasized that materials which are not immediately used should be sent 
to the laboratory as quickly as possible, for in such substances as 
faces, where enormous numbers- of various kinds of bacteria are 
present, those which we seek most, such as the typhoid bacilli, fre- 
quently succumb to the deleterious products of the other bacteria 
present. Even when abundantly present, living typhoid bacilli may 
entirely disappear from the fffices in the course of twelve hours, while 
at other times they may remain for weeks. These differences depend 
on the associated organisms present, the chemical constitution of the 
fasces or urine, and the conditions under which the material is obtained. 
Water and milk rapidly change in their bacterial content if not kept 
under 40° F. 

For obtaining fluid for agglutination and other purposes, blister 
fluid is valuable. A blister can be raised quickly by placing a piece 
of blotting-paper moistened with a little strong ammonia on the .skin 
and covering with a watch-glass, or one may be more slowly formed by 
a cantharides plaster. 

Roatine Technique Oanied Out at Laboratory when Thorough 
Ezaminatioii Required. — As has just been indicated, the bacterio- 
logical examination proceeds somewhat differently according to the 
information needed. When, as is the case with most chnical material, 
definite knowlede^ in regard to the presence or absence of a particular 
ed, the special methods, which have been already 
which are later fully described under each micro- 
1; but when, as is generally the case with autopsy 
times with clinical, a complete examination is needed, 
■ be as follows: 
sv table the routine cultures and smears are made 



MATERIAL FOR BACTERIOLOGIC EXAMINATION. 



191 



2. Material from the different parts is secured under aseptic pre- 
cautions in sterile receptacles and taken to the bacteriological labora- 
tory. The receptacles should be surrounded by ice if the laboratory 
is at a distance. 

3. A smear from each part is stained and examined in order to 
determine in some measure the kind and number of bacteria present, 
so we may more wisely select suitable culture media, if other than 
those already used be needed, and may make the right culture dilu- 
tions if these be necessary. 

Gram's stain (see p. 33) gives more information, especially in regard 
to the first point, than any other one stain, so when possible this stain 
should be used. Other stains, however, may help, if for any reason 
Gram's is not at hand; and smears made from blood or from sus- 
pected syphilitic material should be stainejl by Giemsa's method (see 
Sec. Ill) or an equivalent (see under malarial organisms, treponema 
pallidum, etc.). 

A Gram-stained smear may show all Gram-negative or all Gram- 
positive bacteria or a mixture of the two. ^ / - 

The following points must be remembered in using this stain and in inter- 
preting the results: 

(a) The smears should be thin and evenly spread. 

(6) The staining solutions should be fresh (aniline water, gentian violet, 
lasts about 3 weeks.) 

(c) Controls of fresh cultures (about 24 hrs. old) of a Gram-negative and 
a Gram-positive bacterium should be used on the same slide with the smear 
to be examined. 

(c/) If there is much albumin in the suspected material less heat should 
be used in fixing. 

{e) If the urine is very acid the results may not be good. 

(/) Mix urinary sediment with egg albumen, better to fix it, and wash 
out urinary salts with tap water and stain. 

{g) Too much dependence should not be placed upon the finding of Gram- 
negative bacteria in tissues, because bacteria which in pure young cultures 
may be positive to Gram, may, as they grow older both in tissues and in 
cultures, show forms intermediate between negative forms, as well as a vary- 
ing number of the latter. 



If the 
probably 



Gram- 
Negative 
Bacilli. 



\ 



Most frequently 
from intestinal 
tract. 

Most frequently 
from chest con- 
tents. 



smears show only Gram-negative organisms, the material 
contains one or more of the following: 

B. coli group. 

B. typhosus group. 

B. dysenterijB group. 

B. proteus. 

B. mucosus capsulatus. 

B. pyocyaneus. 

B. influenzse group. 

B. fusiformis. 

B. mallei. 

B. edematis (malignant 

cedema). 
B. of symptomatic anthrax. 
B. pestis. 
B. of Morax-Axenfeld. 



Most frequently 
found, and 
some indication 
of their pres- 
ence in history. 



Less frequently found, and gen- 
erally a marked indication of 
their presence in history. 



PATHOGENIC MICRO-ORGANISMS. 



Negativi 
Cocci. 



Gram- 
Negative 
Spirilla. 



I Micrococcus intracellularis. 
Micrococcus catan-halis. 
Micrococcus goooirhcete. 
Micrococcus melitensis. 



Generally marked indication of 
their presence in history. 



Mouth spirals. 



1 Marked indication of presence of first 
J form in history. 

) Unimportant, unless indication of syph- 
ilis in history when Tr. pallidum 
should be looked for. 



If only Gram-positive organisms are demonstrated, the material 
may coatain one or more of tbe-following: 



dram- 
Positive 
Bacilli. 






B. diphtherite group. 
B. tetani (not often demon- 
strated in smears from lesion). 
B. tuberculosis. 
B. anthracis. 
B. leprae. 

B. welchii and some other in- 
testinal anaerobes. 



Generally marked indication of 
their presence in historj'. 



Staphyli 

Strep to( 

pneumococcus and its 
ety, pneumococcus m 

Micrococcus tetragenut 

None. 



group. 

.^™!IL""l''','*i"'^ [ Some indication of their pres- 
ence in history. 



From the different parts of the body the following more important 
organisms are found in order of their probable frequency. 



Meningeal 

( Cerebrospinal). 



Pericardial a 
pleural. 



Micrococcus intracel- 
lularis. 

Streptococcus (in- 
cluding pneumo- 
coccus group). 

B. influenzK. 

B. tuberculosis 

I Streptococcus ( i n - 
eluding pneumo- 
coccus group) . 
B, mucosus capsu- 
laris. 
B, inHuenzx. 
B. tuberculosis. 

( B. coli group. 

I Streptococcus group. 

( B. tuberculcsis. 



is (including pneumococc 
ca|>sularis. 



Fluid generally 
cloudy with many 
leukocytes. 

Fluid generally clear. 



Fluid may be cloudy. 



} Fluid generally clear. 



MATERIAL FOR BACTERIOLOGIC EXAMINATION. 



193 



Nose and 
Throat. 



Feces. 



Urine. 



Pelvic 
Organs. 



B. diphtherise group. 
B. influenzse group. 
Streptococcus group. 
B. mucosus group. 
B. tuberculosis. 

B. coli group (including B. fcecalis alcaligenes and B. acidi 

lactici). 
B. tjrphosus group. 
B. dysenteric group. 
Gram-i>ositive anaerobes. 
Many forms whose importance has not been worked out. 

B. coli group. 
Streptococcus (kidney). 
M. gonorrhoese. 
B. typhosus. 
B. tuberculosis. 

M. gonorrhoese. 

Streptococcus. 

B. tuberculosis. 

Many other forms probably unimportant. 



The following media should be used for the reasons given below : 

Nutrient broth, for motiUty, morphology, and arrangement (chains, 
groups, etc.). 

Potato for color and abundance of growth. 

Peptone broth for indol. 

Fermentation tube for anaerobes, acidity and gas. 

(a) Poured plates for isolated colonies (dilutions accord- 
ing to the number of organisms seen in smears) . ( Blood 
^ agar if pneimiococcus or streptococcus indicated.) 

(6) Streaked plates for surface colonies. (Blood agar if 
influenza bacilU are indicated.) 

Special media according to the kinds of organisms demonstrated in smears 
or indicated in histories. Such special media are described under the indi- 
vidual organisms. 



Nutrient Agar 
and Gelatin. 



13 



PART II. 

BACTERIA PATHOGENIC TO MAN INDIVIDU 

ALLY CONSIDERED. 



CHAPTER XVII. 
THE BACILLUS AND THE BACTERIOLOGY OF DIPHTHERIA. 

The lesions of diphtheria are caused by toxaemia. The concen- 
trated poison at the seat of the exudate causes intense local inflam- 
mation, while the absorbed poison diffused throughout the body causes 
widespread cellular injury, giving rise to constitutional disturbance 
and definite injury of the cells of the muscle, nerve, and other tissues. 

Historical Notes. — This specific contagious disease can be traced 
back under various names to almost the Homeric period of Grecian 
history. From time to time during the following centuries we hear of 
epidemics both in Italy and in other portions of the civilized world 
which indicate that the disease never absolutely ceased. 

In 1765 Home, a Scotchman, tried to show that "croup "and pharyn- 
geal diphtheria were different diseases. 

In 1771 Bard, an American, supported the opposite theory from 
Home, considering the process the same wherever located. 

In 1821 Bretonneau published his first essay on diphtheria in Paris 
and gave to the disease its present name. His observations were so 
extensive and so correct that little advance in knowledge took place 
until the causal relations of the diphtheria bacilli and their associated 
micoorganisms to the disease began to be recognized. 

Eyidence of Causal RelatLonship. — As early as 1840 observers be- 
gan to notice microorganisms in the pseudomembranes. Gradually 
the observations became more exact. The most importance was attrib- 
uted to micrococci. In the year»1883, however, bacilli which were 
very peculiar and striking in appearance were shown by Klebs to be 
of constant occurrence in the pseudomembranes from the throats of 
those dying of true epidemic diphtheria. He described the peculiar 
staining of the organisms. One year later, LoeflBer separated these 
bacilli from the other bacteria and grew them in pure culture. When 
he inoculated the bacilli upon the abraded mucous membrane of sus- 
ceptible animals more or less characteristic pseudomembranes were 

195 



' 



196 PATHOGENIC MICRO-ORGANISMS. 

produced, and frequently death or paralysis followed with character- 
istic lesions. These animal experiments have been fortified by a 
number of accidental human infections with bacilli in laboratories 
with subsequent development of diphtheria. 

The Diphtheria Bacillus.— This bacillus is one of the most inter- 
esting of bacteria. Grown in the animal body or in suitable culture 
fluid, it produces a powerful toxin. Its morphology and staining 
are peculiar. Outside of the body it grows best on serum media. 

Morphology. — When cover-glass preparations made from the exu- 
date or from the cultures grown on blood serum are examined, the 
diphtheria bacilli are found to possess the following morphological 
characteristics: The diameter of the bacilli varies from 0.3 to 0.8// 
and the length from 1 to 6/£. They occur singly and in pairs (see 
Figs. 77 to 84) and very infrequently in chains of three or four. The 
rods are straight or slightly curved, and usually are not uniformly 
cylindrical throughout their entire length, but are swollen at the end, 
or pointed at the ends and swollen in the middle portion. The aver- 
age length of the bacilli in pure cultures from diflferent sources fre- 
quently varies greatly, and even from the same culture individual bacilli 
diflfer much in their size and shape. This is especially true when the 
bacilli are grown in association with other bacteria. The two bacilli of 
a pair may lie with their long diameter in the same axis or at an obtuse or 
an acute angle. The bacilli possess no spores, but have in them highly 
refractive bodies, some of which are the starting point for new bacilli 
(see p. 16). There are no fiagella. For mode of division, see p. 16. 

Staining. — ^The Klebs-Loeffler bacilli stain readily with ordinary 
aniline dyes, and retain fairly well their color after staining by Gram's 
method. With LoeflSer's alkaline solution of methylene blue, and to 
a less extent with Roux's and dilute Ziehl's solutions, the bacilli from 
blood serum cultures especially, and from other media less constantly, 
stain in an irregular and extremely characteristic way. (See Fig. 77.) 
The bacilli do not stain uniformly. In many cultures round or oval 
bodies, situated at the ends or in the central portions, stain much more 
intensely than the rest of the bacillus, usually showing metachromatism 
(the so-called metachromatic granules. See p. 14). Sometimes 
these highly stained bodies are thicker than the rest of the bacillus; 
again, they are thinner and surrounded by a more slightly stained 
portion. Other bacilli have barred staining. The bacilli stain in this 
peculiar manner at a certain period of their growth, so that only a 
portion of the organisms taken from a culture at any one time will 
show the characteristic staining. The young cultures have the most 
regular forms, an eighteen-hour growth showing more clubbed forms 
than at twelve hours. After twenty-four hours the bacilli do not 
stain quite as well. In still older cultures it is often difficult to stain 
the bacilli, and the staining, when it does occur, is frequently not at 
all characteristic. The same round or oval bodies which take the 
methylene blue more intensely than the remainder of the bacillus are 
brought out still more distinctly by the Neisser stain. 



THE BACILLUS OF DIPHTHERIA. 



197 



The Neisser stain is carried out by placing the cover-slip smear of diph- 
theria or other bacilli in solution No. 1 for from two to three seconds, and then, 
after washing, in No. 2 for from three to five seconds. The bacilli will then 
appear either entirely brown or will show at one or both ends a dark blue, 
round body. With characteristic diphtheria bacilli, taken from a twelve 

Flo. 78 




fomu of diphtheria bi 
>uia. XllOOdi •— "■ 



Tta. 79.— DiphUiena builH chancleristiB m ihapeB, but ihowins even ■(auunc. XIOOO 
diunetcn. Slun. methyknc blue. 

Fla. SO.— Non-virulant diphtbeiu bacilli, ihowinc stain with Neinaer'a aolutioiu. Thii aDIKar- 
ann wu formerly ■uppated lo be cbscBcteHatiii of virulent bacilli. Bodiea of bkcilb in imear, 
yellowiab-biDwn: pcnnis, dark blue. 

to eighteen hours' growth on serum, nearly all will show the blue bodies (Fig. 
80), while with the pseudo type (Fig. 86, page 206), to be described hereafter, 
few will be seen. 

The solutions are as follows: 

No. I. 

Alcohol (96 per cent.) , . , . . , 20 parts. 

Methylene blue (Grilbler) 1 part. 

Distilled water ', 1150 parts. 

Acetic acid (glacial) 50 parts. 

No. 2. 

Bismarck brown 1 part. 

BoiUng distilled water 500 parts. 

The Neisser stain has been advocated in order to separate the viru- 
lent from the non-virulent bacilli, without the delay of inoculating 



198 PATHOGENIC MICRO-ORGANISMS. 

animals; but in our hands, with a very large experience, neither the 
Netsser stain nor other stains, such as the modifications of the Koux 
stain, have given much more information as to the virulence of the 
bacilli than the usual methylene-blue solution of Loeffler. A few 
strains of virulent bacilli fail to show a marked characteristic stain, 
and quite a few pseudodiphtheria bacilli show the dark bodies. There 
are also in many throats bacilli which seem to have all the staining 



, IndiAn-rlubbed r 





Forty-eight houm' wtar cullurr. Mbhv segme 


nts: lol 


Id,. Oney«roi. 


. »tJliiH»1 m«)iB. X 1410 diameWiB. 




.— B, rtiphtherie. 


Twi?nty-roiir houm' nsiir culture. Cocrua farms. 


Se^me 


<im. Only v.riely found; io casta of diphlherin . 


C (Sildi 






and cultural characteristics of the virulent bacilli, and yet have no re- 
lation to the disease diphtheria. They are therefore non-virulent in 
the sen.se that they produce no diphtheria toxin. As will be state<i 
mnn. fiiHv latur nothing but animal inoculations with the suspected 
Ith control injections of diphtheria antitoxin will 
bacilli from those capable of producing diphtheria. 

of the Diphtheria Baeillns on Seram-free Media.— 

ably with different culture media employed. On gly- 
nutrient agar there are two distinct types. One grows 
■ule, more regular forms than when grown on serum cul- 
I. The other type shows many thick, Indian-dubbed 
itc number of segments (Figs. '82-84). Short, spindle, 



THE BACILLUS OF DIPHTHERIA, 199 

lancet, or club-shaped forms, staining uniformly, are all observed. The 
bacilli which have developed in the pseudomembranes or exudate in cases 
of diphtheria resemble in shape young bacilli grown on blood serum, but stain 
more evenly. 

Biology. — The Klebs-Loeffler bacillus is non-motile and non-lique- 
fying. It is aerobic. It grows most readily in the presence of oxy- 
gen, but also without it. It does not form spores. It begins to 
develop, but grows slowly at a temperature of 20° C, or even less. 
It attains its maximum development at 37° C. In old cultures -in 
fluid media, Williams has observed fusion of one bacillus with another. 
The fused forms live the longest (see p. 18). 

Orowth on Culture Media.— Blood Semm. — Blood serum, espe- 
cially coagulated in the form of LoeflBer's mixture, is the most favorable 
medium for the growth of the diphtheria bacillus, and is used partic- 
ularly for diagnostic purposes' in examining cultures from the throats 
of persons suspected of having diphtheria. For its preparation, see 
p. 227. If we examine the growth of diphtheria bacillus in pure culture 
on blood serum we shall find at the end of from eight to twelve hours 
small colonies of bacilli, which appear as pearl-gray, whitish-gray or, 
more rarely, yellowish-gray, slightly raised points. The colonies 
when separated from each other may increase in forty-eight hours 
so that the diameter may be one-eighth of an inch. The borders are 
usually somewhat uneven. The colonies lying together become 
confluent and fuse into one mass when the serum is moist. During 
the first twelve hours the colonies of the diphtheria bacilli are about 
equal in size to those of the other pathogenic bacteria which are often 
present in the throat; but after this time the diphtheria colonies become 
larger than those of the streptococci and smaller than those of the 
staphylococci. The diphtheria bacilli in their growth never liquefy 
blood serum. 

Orowth on Agar. — On 1 per cent, slightly alkaline, nutrient or 
glycerin-agar the growth of the diphtheria bacillus is less certain 
and luxuriant than upon blood serum; but the appearance of the 
colonies when examined under a low-power lens, though very variable, 
is often far more characteristic. (See Fig. 53, page 75, and Fig. 85.) 
For this reason nutrient agar in Petri dishes is used to obtain diph- 
theria bacilli in pure culture. The diphtheria bacillus obtained from 
cultures which have developed for some time on culture media grows 
well, or fairly well, on, suitable nutrient agar, but when fresh from 
pseudomembranes one prevalent type of bacilli grows on these media 
wijji £reat diflSculty, and the colonies develop so slowly as to be fre- 
quently covered up by the more luxuriant growth of other bacteria 
when present, or they may fail to develop at all. 

If the colonies develop deep in the substance of the agar they are 
usually round or oval, and, as a rule, present no extensions; but if 
near the surface, commonly from one, but sometimes from both sides, 
they spread out an apron-like extension, which exceeds in surface area 
the rest of the colony. When the colonies develop entirely on the 



200 PATHOGENIC MICRO-ORGANISMS. 

surface they are more or less coarsely granular, and usually have a 
dark centre and vary markedly in their thickness. The colonies from 
some are almost translucent; from others are thick and almost as lux- 
uriant as the staphylococcus. The edges are sometimes jagged, and 
frequently shade off into a delicate lace-like fringe; at other times the 
margins are more even and the colonies are neariy circular. 

The growth of the diphtheria bacillus upon agar presents certain 
peculiarities which are of practical importance. If a large number 
of the bacilli from a recent culture are implanted upon a properiy 
prepared agar plate a certain and fairiy vigorous growth will always 
Fro. sa take place. If, however, the agar 

is inoculated with an exudate From 
the throat, which contains but a 
few bacilli, no growth whatever 
may occur, while the tubes of 
coagulated blood serum inoculated 
with the same exudate contain the 
bacilli abundantly. Because of 
the uncertainty, therefore, of ob- 
taining a growth by the inocula- 
tion of agar with bacilli unac- 
customed to this medium, agar is 
not a reliable medium for use in 
primary cultures for diagnostic 
purposes. A mixture composed 

Colonia of diphtheria b&cilli. X 200 diunetan. • : _ _, • ,- 

"^ of two parts of a 1.5 per cent, 

nutrient agar and one part of sterile ascitic fluid makes a medium 
upon which the bacillus grows much more luxuriantly, but not so 
characteristi call y , 

Isolation of the Diphiheria Bacillvs from Plate Cultures. — Nutrient 
plain or glycerin-agar should be freshly melted and poured in the Petri 
dish for this purpose. After it has hardened, the medium in a number 
of plates is streaked across with bacteria from colonies on the serum 
culture, which appear in size and color like the diphtheria bacilli. 
Other plates are made from a general mixture of all bacteria, selected, 
as a rule, from the drier portion of the serum. Still others are inocu- 
lated from the pellicles of ascitic broth cultures. The plates are left 
in the incubator for about sixteen hours at 37° C. In the examination 
of the plates one should first seek for typical colonies, then if these are 
not found, for any that look most nearly like the characteristic picture. 
Diphiheria colonics are very apt to be found at the edges of the streaks 
of bacterial growth. The pickings from the colonies are inoculated 
upon Loeffler's blood serum, or into ascitic bouillon. 

Growth in Bouillon. — The diphtheria bacilli from about one-half the 
cultures ^row readily in broth slightly alkaline to litmus; the other cultures 
gtani^icphHy . The characteristic growth in ucutral bouillon ie one showing 
tine graitis. These deposit along the sides and bottom of the tube, leaving 
the broth nearly clear. A few cultures in neutral bouillon and mauy in alka- 



THE BACILLUS OF DIPHTHERIA. 201 

line bouillon produce for twenty-four or forty-eight hours a more or less diffuse 
cloudiness, and frequently a film forms over tlje surface of the broth. On 
shaking the tube this film breaks up and slowly sinks to the bottom. This 
film is apt to develop during the growth of cultures which have long been 
cultivated in bouillon, and, indeed, after a time the entire development may 
appear on the surface in the form of a friable pellicle. The diphtheria bacil- 
lus in its growth causes a fermentation of meat-sugars and glucose, and thus 
if these are present changes the reaction of the bouiUon, rendering it distinctly 
less alkaline within forty-eight hours, and then, after a variable time, when all 
the fermentable sugars have been decomposed, more alkaline again through 
the progressing fermentation of other substances. Among the products 
formed by its growth is the diphtheria toxin. 

Growth in Ascitic or Serum Bouilion. — All varieties of diphtheria 
bacilli grow well in this medium, even when first removed from the 
throat. They almost always form a slight pellicle at the end of 
twenty-four to forty-eight hours. This culture medium is, as pointed 
out by Williams, of the greatest value in attempts to get pure cultures 
of the diphtheria bacillus from solidified serum cultures containing 
few bacilli among many other bacteria. Plate cultures are made 
from the pellicle. The fluid is prepared by adding to the nutrient 
bouillon 25 per cent, ascitic fluid or blood serum. 

Growth on Gelatin. — The growth on this medium is much slower, more 
scanty, and less characteristic than that on the other media mentioned. 
This is partly on account of the lower temperature at which it must be used. 

Growth in Blilk. — The diphtheria bacillus grows readily in milk, 
beginning to develop at a comparatively low temperature (20° C). 
Thus, market milk having become inoculated with the bacillus from 
cases of diphtheria may, under certain conditions, be the means of 
conveying infection to previously healthy persons. The milk re- 
mains unchanged in appearance as lactose is not fermented by the 
diphtheria bacillus. 

Pathogenesis. In Lower Animals. — ^The diphtheria bacillus through 
its toxins is, when injected into their bodies, pathogenic for guinea- 
pigs, rabbits, chickens, pigeons, small birds, and cats; also in a lesser 
degree for dogs, goats, cattle, and horses, but hardly at all for rats and 
mice. In spite of its pathogenic qualities for these animals true 
diphtheria occurs in them with extreme rarity. As a rule, supposed 
diphtheritic inflammations in them are due to other bacteria which 
cannot produce the disease in man. The cat is the only animal that 
we have known to contract true diphtheria from contact with the dis- 
ease. Cobbett reports a case in a colt. At the autopsy of animals 
dying from the poisons produced by the bacilli, the characteristic 
lesions described by Loeffler are found. At the seat of inoculation 
there is a grayish focus surrounded by an area of congestion; the 
subcutaneous tissues for some distance around are oedematous; the 
adjacent lymph nodes are swollen; and the serous cavities, especially 
the pleura and the pericardium, frequently contain an excess of fluid 
usually clear, but at times turbid; the lungs are generally congested, 
the suprarenals are markedly congested. In the organs are found 



202 PATHOGENIC MICRO-ORGANISMS. 

numerous smaller and larger masses of necrotic cells, which are 
permeated by leukocytes. The heart and certain voluntary muscular 
fibres and nervous tissues usually show degenerative changes. Occa- 
sionally there is fatty degeneration of the liver and kidneys. The 
number of leukocytes in the blood is increased. From the area sur- 
rounding the point of inoculation virulent bacilli may be obtained, but 
in the internal organs they are only occasionally found, unless an enor- 
mous number of bacilli have been injected. Paralysis, commencing 
usually in the posterior extremities and then gradually extending to 
the whole body and causing death by paralysis of the heart or respira- 
tion, is also produced in many cases in which the inoculated animab do 
not succumb to a too rapid intoxication. In a number of animals we 
have seen recovery take place three to six weeks after the onset of the 
paralysis. The occurrene of these paralyses, following the introduc- 
tion of the diphtheria bacilli, completes the resemblance of the experi- 
mental disease to the natural malady in man. 

Tissue Changes in Natural (Human) Infection. — The characteristic 
lesions are a pseudomembranous inflammation on some of the mucous 
membranes or occasionally on the surface of wounds and the general 
hyperplasias and parenchymatous inflammations produced by the 
absorbed toxic substances. Pneumonia is apt to occur as a compli- 
cation of laryngeal diphtheria. The membrane may be simply a thin 
pellicle, which is easily removed without causing bleeding or it may 
be thick and firmly attached and leaving when removed a ragged 
bleeding surface. The tissue beneath the pseudomembrane is always 
intensely injected and often hemorrhagic. The cells show marked 
degenerative changes. 

Causes of Deatih. — These are chiefly toxsemia, septicaemia, laryn- 
geal obstruction and broncho-pneumonia. 

Diphtheria Toxin. — This poison was assumed by Loeffler (1884) to 
be produced by the bacilli, but it was first partially isolated by 
Roux and Yersin, who obtained it by filtration through porous porce- 
lain from cultures of the living bacilli. It has not yet been successfully 
analyzed, so that its chemical composition is unknown, but it has 
many of the properties of proteid substances, and can well be desig- 
nated by the term active proteid. It resembles in many ways the 
ferments. After injection into the body there is a latent period be- 
fore its poisonous action appears. The poison produced is probably 
composed of a mixture of several nearly related toxins. Diphtheria 
toxin is totally destroyed by boiling for five minutes, and loses some 
95 per cent, of its strength when exposed to 75° C. for the same time; 
73° C. destroys only about 85 per cent., and 60° very little. Ix>wer 
temperatures only alter it very gradually. Kept cool and from light 
and air, it deteriorates very slowly. Freezing injures it. 



The Production of Toxin in Culture Media. — The artificial production 
of toxin from cultures of the diphtheria bacillus has been found to depend upon 
definite conditions, which are of practical importance in obtaining toxin for 
the inoculation of horses, and also of theoretic interest in explaining why cases 



THE BACILLUS OF DIPHTHERIA. 203 

of apparently equal local severity have such diflferent degrees of toxic absorp- 
tion. The researches of Roux and Yersin laid the foundation of our knowl- 
edge. Their investigations have been continued by Theobald Smith, Spronck, 
ourselves, and others. After an extensive series of investigations we (Park 
and Williams) came to the following conclusions : Toxin is produced by fully 
virulent diphtheria bacilli at all times during their life when the conditions are 
favorable. Under less favorable conditions some bacilli are able to produce 
toxin while others are not. Diphtheria bacilli may find conditions suitable 
for luxuriant growth, but unsuitable for the production of toxin. The 
requisite conditions for good development of toxin, as judged by the behavior 
of a number of cultures, are a temperature from about 32° to 37° C, a suitable 
culture medium, such as a 2 per cent, peptone nutrient bouillon made from 
veal, of an alkalinity which should be about 9 c.c. of normal soda solution per 
litre above the neutral point to litmus, and prepared from a suitable peptone 
(Witte) and meat. The culture fluid should be in comparatively thin layers 
and in large-necked Erlenmeyer flasks, so as to allow of a free access of air. 
The greatest accumulation of toxin in bouillon is after a duration of growth 
of the culture of from five to ten days, according to the peculiarities of the 
culture employed. At a too early period toxin has not sufficiently accumu- 
lated ; at a too late period it has begun to degenerate. In our experience the 
amount of muscle-sugar present in the meat makes no appreciable difference 
in the toxin produced when a vigorously growing bacillus is used, so long as 
the bouillon has been made sufficiently alkaline to prevent the acid produced 
by the fermentation of the sugar from producing in the bouillon an acidity 
sufficient to inhibit the growth of the bacilli. With the meat as we obtain it 
in New York, we get better results with unfermented meat than with fer- 
mented. In Boston, with the same bacillus, Smith gets more toxin from the 
bouillon in which the sugar has been fermented bv the colon bacillus. Instead 
of colon bacilli, yeast may be added to the soalcing meat, which is allowed 
to stand at about 25° C. We have obtained especially good results with veal 
broth made from calves two to four weeks old (bob veal). When strong toxin 
is desirable the muscle is separated from aU fat, tendon and fibrous tissue be- 
fore being chopped. 

Under the best conditions we can devise, toxin begins to be produced by 
baciUi from some cultures when freshly sown in bouillon some time during 
the first twenty-four hours; from other cultures, for reasons not well under- 
stood, not for from two to foiir days. In neutral bouillon the culture fluid 
frequently becomes slightly acid and toxin production may be delayed for 
from one to three weeks. The greatest accumulation of toxin is on the fourth 
day, on the average, after the rapid production of toxin has commenced. 
After that time the number of living baciUi rapidly diminishes in the culture, 
and the conditions for those remaining alive are not suitable for the rapid 
production of toxin. As the toxin is not stable at 35° C, the deterioration 
taking place in the toxin already produced is greater than the amount of new 
toxin still forming. 

Bacilh, when repeatedly transplanted from bouillon to bouillon, gradually 
come to grow on the surface ociy. This characteristic keeps the bacilli in 
contact with the oxygen and seems to aid in the development of toxin. 

Comparative Virulence of Different Cultures.— The virulence of 

diphtheria bacilli from different sources, as measured by their toxin 
prbduction, varies considerably. Thus, as an extreme instance, 
0.002 c.c. of a forty-hour bouillon culture of our most virulent bacillus 
will kill a guinea-pig, which it would require 0.1 c.c. of the culture 
of our least virulent bacillus to kill. This difference frequently de- 
pends on the unequal growth of the bacilli, one culture having fifty 
times as many bacilli as the other. When the different strains are 



204 PATHOGENIC MICRO-ORGANISMS. 

grown on ascitic broth, upon which their growth is usually good, the 
majority of cultures are nearly equal in virulence, but some still 
show marked differences. Moreover, the diphtheria bacilli differ in 
the tenacity with which they retain their virulence when grown outside 
the body. The bacillus that we have used to produce toxin in the 
laboratory of the Board of Health has retained its virulence unaltered 
for fifteen years in bouillon cultures. Other bacilli have apparently 
lessened their capacity for toxin production after being kept six 
months. The passage of diphtheria bacilli through the bodies of sus- 
ceptible animals does not increase their toxin production to any consid- 
erable extent. 
Comparative Vimlence of Bacilli and Severity of Case.^From the 

severity of an isolated case the virulence of the bacilli cannot be de- 
termined. The most virulent bacillus we have ever found was ob- 
tained from a mild case of diphtheria simulating tonsillitis. Another 
case, however, infected by the bacillus proved to be very severe. In 
localized epidemics the average severity of the cases probably in- 
dicates roughly the virulence of the bacillus causing the infection, as 
here the individual susceptibility of the different persons infected 
would, in all likelihood, when taken together, be similar to that of 
other groups; but even in this instance special conditions of climate, 
food, or race may influence certain localities. Moreover, the bacteria 
associated with the diphtheria bacilli, and which are liable to be trans- 
mitted with them, may influence the severity of and the complications 
arising in the cases. It must be remembered that bacilli of like toxic 
power may differ in their liability to infect the mucous membrane. 
Virulence has thus two distinct meanings when used in connection 
with diphtheria bacilli. 

Virulent Bacilli in Healthy Throats.— ^Fully virulent bacilli have 

frequently been found in healthy throats of persons who have been 
brought in direct contact with diphtheria patients or infected cloth- 
ing without contracting the disease. It is, therefore, apparent that 
infection in diphtheria, as in other infectious diseases, requires not 
only the presence of virulent bacilli, but also a susceptibility to the 
disease, which may be local or general. Among the predisposing 
influences which contribute to the production of diphtheritic infec- 
tion may be mentioned the breathing of foul air and living in over- 
crowded and ill-ventilated rooms, impure food, certain diseases, 
more particularly catarrhal inflammations of the mucous membranes, 
and depressing conditions generally. Under these conditions an 
infected mucous membrane may become susceptible to disease. In 
connection with Beebe (1894) we made an examination of the throats 
of 330 healthy persons who had not come in contact, so far as known, 
with diphtheria, and we found virulent bacilli in 8, only 2 of whom 
later developed the disease. In 24 of the 330 healthy throats non- 
virulent bacilli or attenuated forms of the diphtheria bacillus were 
found. Very similar observations have since been made in Boston 
and by others in many widely separated countries. In 1905 Von 



THE BACILLUS OF DIPHTHERIA. 205 

Sholly in our laboratory examined 1000 throats of those who had not 
knowingly been in contact with diphtheria and found true diphtheria 
bacilli in 0.5 per cent, of the cases. 

Persistence of Diphtheria Bacilli in the Throat.— The continued 
presence of virulent diphtheria bacilli in the throats of patients who 
have recovered from the disease has been demonstrated by all inves- 
tigators. In the investigations of 1894 we found that in 304 of 605 
consecutive cases the bacilli disappeared within three days after the 
disappearance of the pseudomembrane; in 176 cases they persisted for 
seven days, in 64 cases for twelve days, in 36 cases for fifteen days, in 
12 cases for three weeks, in 4 cases for four weeks, and in 2 cases for 
nine weeks. Since then we have met with a case in which they 
persisted with full virulence for eight months. It is safe to say 
that in over 5 per cent, of the cases a few bacilli persist two weeks 
after the disappearance of the exudate and in over 1 per cent, four 
weeks. 

Dq>htheria-like Baoilli Not Producing Diphtheria Toxin. — In the 
tests of the bacilli obtained from hundreds of cases of suspected diph- 
theria which have been carried out during the past fiifteen years in 
the laboratories of the Health Department of New York City, in over 
95 per cent, of cases the bacilli derived from exudates or pseudomem- 
branes and possessing the characteristics of the LoeflSer bacilli have 
been found to be virulent, that is, producers of diphtheria toxin. But 
there are, however, in inflamed throats as well as in healthy throats, 
either alone or associated with the virulent bacilli, occasionally bacilli 
which, though morphologically and in their behavior on culture media 
identical with the Klebs-LoefBer bacillus, are yet producers, at least in 
artificial culture media and the usual test animals, of no diphtheria toxin. 
Between bacilli which produce a great deal of toxin and those which 
produce none we find a few minor grades of virulence. We believe, 
therefore, in accordance with Roux and Yersin these non-virulent 
bacilli should be considered as possibly attenuated varieties of the 
diphtheria bacillus which have lost their power to produce diphtheria 
toxin. This supposition is, however, not proven and it may be that 
the ancestors of these bacilli were never toxin producers. These ob- 
servers, and others following them, have shown that the virulent 
bacilli can be artificially attenuated; but the reverse has not been proven 
that bacilli which produce no specific toxin have later been found to 
develop it. In our experience some cultures hold their virulence even 
when grown at 41° C. for a number of months, while others become 
partly attenuated rather quickly. We have never yet been able to 
change a virulent culture into an absolutely non-virulent one. Diph- 
theria-like bacilli are also found which resemble diphtheria bacilli very 
closely except in toxin production, but differ in one or more particu- 
lars. Both these and the characteristic non-virulent bacilli are found 
occasionally upon all the mucous membranes both when inflamed and 
when apparently nonnal. From varieties of this sort having been 
found in a number of cases of the condition known as xerosis con- 



206 PATHOGENIC MICRO-ORGANISMS. 

junctivte, these bacilli are often called xerosis bacilli. Under this name 
different observers have placed bacilli identical with the diphtheria 
bacilli and others differing quite markedly from them. 

Diphtheria-like Bacilli Pathogenic to OiiiBea-pigs Frodociiig no 
Diphtheria Toxin. — These bacilli are obtained frequently from normal 
or slightly inflamed throats and may be slightly pathogenic in guinea- 
pigs, since they may kill, as we have found in a number of instances, 
ID doses of 2 to 5 c.c. of broth culture subcutaneously or intraperi- 
toneally injected. Animals are not protected by diphtheria antitoxin 
from the action of these bacilli. At autopsy the bacilli are usually 
found more or less abundantly in the blood and internal organs. The 
fact that large injections of antitoxic serum hastens the death of 
guinea-pigs injected with these bacilli has given rise to the notion 
that injections of antitoxin might be dangerous in persons in whose 
throats these bacilli were present, either as saprophytes or, possibly, 
as inciters of slight disease. It is not the antitoxin, but the serum, 
which in large doses injures the vitality of the guinea-pigs and so 
slightly hastens death. Any serum has the effect. These bacilli were 
first described by Davis ' from our laboratory and later by Hamilton 
in 1904. In our judgment the possibility of their being present af- 
fords no reason to avoid giving antitoxin in suspected cases. They 
.should in this respect be considered as the streptococci. When 
pathogenic in man they are usually only feebly so. 

Fsendodiphtheiia Bacilli. — Besides the typical bacilli which produce 
diphtheria toxin and those which do not, but which, so far as we can 
determine, are olherwise identical with the Loeffler bacillus, there are 
Fia. S6 other bacilH found in positions similar to 

those in which diphtheria bacilli abound, 
which, though resembling these organisms 
in many particulars, yet differ from them 
as a class in others equally important. The 
variety most prevalent is rather short, 
plump, and more uniform in size and shape 
than the true Ijoeffler bacillus {Fig. 86). 
On blood-serum their colony growth is 
very similar to that of the diphtheria 
bacilli. The great majority of them in 
Pseu.iodiphih6ri» bacilli. *"y youug culturc show uo poUr granules 
(B.hofmanni.) when Stained by the Neisser method, and 

stain evenly throughout with the alkaline methylene-blue solution. 
They do not produce acid by the fermentation of glucose, as do all 
known vinilent and many non-virulent diphtheria bacilli; therefore, 
there is no increase in acidity in the bouillon in which they are grown 
during the first twenty-four hours from the fermentation of the meat- 
sugar regularly present. They are found in varying abundance in 
different localities in New York City, in about 1 per cent, of the normal 
throat and nasal secretions, and seem to have now at least no connection 
' Medical News, April 2fl, 18'(9. 



THE BACILLUS OF DIPHTHERIA. 207 

with diphtheria; whether they were originally derived from diphtheria 
bacillus is doubtful; they certainly seem to have no connection with it 
now. They have been called pseudodiphiheria bacilli, and more prop- 
erly, B, hojmanni} In bouillon they grow, as a rule, less luxuriantly 
than the diphtheria bacilli, and never produce diphtheria toxin. Some 
of the varieties of the pseudodiphtheria bacilli are as long as the shorter 
forms of the virulent bacilli. WTien these are found in cultures from 
cases of suspected diphtheria they may lead to an incorrect diagnosis. 
There are also some varieties which resemble the short pseudobacilli 
in form and staining, but which produce acid in glucose bouillon. 
These bacilli are found occasionally in all countries where search has 
been made for them. It may be added here that no facts have come 
to light which indicate that bacilli which do not produce diphtheria 
toxin in animals ever produce it in man. It must also be borne 
in mind, however, that such proof is necessarily very difficult to 
obtain. 

Persistence of Varieties of the Bacillus Diphtherise and of Diph- 
theria-like Bacilli. — The fact that there are distinct differences between 
strains of bacilli producing specific diphtheria toxins which are as 
great as between these and bacilli producing no specific toxins has, we 
think, been fully established. 

But that such varieties are true sub-species with constant charac- 
teristics, one variety not changing into another of the established 
forms, has not been accepted by all. On the contrary, the opinion 
is held by some investigators that all of the various forms of diph- 
theria-like bacilli are the result of more or less transitory variations 
of the same species, and hence that the virulent forms are the result 
of a rapid adaptation to environment and consequent pathogenesis 
of the non-virulent forms, both typical and atypical. 

This question of the relationship of the specifically virulent diph- 
theria bacillus to non-virulent, diphtheria-like bacilli has been dis- 
cussed since 1887. It is certainly theoretically possible that the non- 
virulent forms have been derived from virulent forms. Whether or 
not this is true is an interesting problem for discussion, but has little 
practical importance. On the other hand, the possibility of 
the non-toxin producing forms readily assuming power to produce 
toxin is of the greatest importance, and if true would cause us 
to change our present methods of trying to prevent the spread of 
diphtheria. 

Until 1896 no one had brought forward evidence which tended to 
show that fully non-virulent forms could be made virulent. In this 
year Trump' states that he converted a non-virulent acid-producing 
bacillus into one capable of killing guinea-pigs with all the symptoms 
of true diphtheria, by successive passages through guinea-pigs plus a 
non-fatal dose of diphtheria toxin. Hewlett and Knight' state (1897) 

»Clark. Jour, of Inf. Dis., 1910, vii., 335. 

* Centralblatt fQr Bakt., etc., 1896, Band xx., p. 721. 

■Trans, of the Brit. Inst, of JPrev. Med., 1897, 1st series. 



208 PATHOGENIC MICRO-ORGANISMS. 

that they changed a non-acid pseudodiphtheria bacillus into a typ- 
ical virulent diphtheria bacillus by culture and passage through 
guinea-pigs. 

Richmond and Salter^ (1898) and Salter' (1899) state that they 
have changed five pseudodiphtheria bacilli into typical diphtheria 
bacilli specifically virulent for guinea-pigs by passage through a number 
of goldfinches. 

In the work of Wesbrook, Wilson, and McDaniel,' on Varieties of 
Bacillus DiphthericB, the study is based upon the morphology of the 
individual bacillus found in smears of throat cultures and pure cul- 
tures. They make a provisional classification based upon the mor- 
phology of the individual bacilli, into three groups, called granular, 
barred, and solid, two of the groups into seven types and the other 
into five, two of the types corresponding with those in the other groups 
not having been seen. In a study of the types found in the smears 
from a series of direct cultures derived from clinical cases of diphtheria 
the authors state that there is generally a sequence of types in the 
variations which appear throughout the course of the disease, the 
granular types, as a rule, predominating at the outset of the 
disease, and these giving place wholly or in part to the barred and 
solid types shortly before the disappearance of diphtheria-like 
organisms. 

The inference drawn from this work is that the diphtheria bacillus 
may be rather easily, especially in the throat, converted into non- 
granular, solidly staining forms of the "pseudodiphtheria" type, and 
that the converse may occur, and that therefore all diphtheria-like 
bacilli must be considered a possible source of danger. The more 
extreme views of Hewlett and Knight are rejected by most investi- 
gators and even the conclusions of Wesbrook are considered too 
extreme. 

Bergey* was not able to give virulence to non-virulent forms, neither 
did he find that these latter gave immunity against the former; for 
these reasons he considers them distinct members of a large group of 
bacilli at the head of which stands the diphtheria bacillus. 

Cobbett* considers the pseudodiphtheria bacillus as perfectly in- 
nocuous to man, but that the relation between the pseudodiphtheria 
and the diphtheria bacillus remains undecided. He did not meet 
with bacilli of low virulence. He found a few non-virulent and 
the others all highly virulent. He thinks that the reason why the 
pseudo-diphtheria bacilli appear so infrequently during the acute 
stage is that they are overlooked then because one discovers the 
virulent bacilli so easily and does not trouble to look any more, 
and they are found more easily later because the diphtheria bacilli 

» Guy's Hospital Reports, 1898. 

' Trans of the Jenner Inst, of Prev. Med., 1899. 

* Transactions of the Association of American Physicians, 1900. 

* Pub. of the Univ. of Penn., 1898, new series. No. 4 (other references). 

* Journal of Hygiene, 1901. 



THE BACILLUS OF DIPHTHERIA. 209 

are disappearing and are hard to find; consequently a long and 
careful search is made, and the pseudodiphtheria bacilli are seen for 
the first time. 

The central idea in the statements of those who believe that 
diphtheria-like bacilli are simply transitory variations of the species 
BcLcillus diphthericB is that both the diphtheria bacillus and those 
bacilli which resemble it have many unstable properties, their form, 
their cultural characteristics, their pathogenicity all varying within 
a wide limit, so that one form may assume readily the properties of 
another form. 

The separatists, on the other hand, have found that certain forms 
possess such stable properties that one is not converted into another, 
and hence they regard them as distinct species. 

In order to make a thorough test of this whole matter Williams^ 
undertook a careful investigation of the subject which covered all 
the tests just described and many others. 

The conclusions reached were as follows: Though some cultures 
change on some of the media, each changes in its own way, and each 
culture still has its distinct individuality. After many culture gen- 
erations, especially when transplanted at short intervals, the different 
varieties of virulent diphtheria bacilli tend to run in lines parallel 
with a common norm, which seems to be a medium-sized, non-seg- 
mented bacillus producing granules in early cultures on serum and 
growing well on all of the ordinary culture media. The non-virulent 
morphologically typical bacilli must be classed with the virulent varieties 
as one species, though there is little doubt that more minute study 
would show that the former constitute a distinct group. The atypical 
pseudo forms, however, which show no tendency to approach the norm 
of the typical forms, must be classed as distinct species. All of the 
pseudo and the non-virulent morphologically typical varieties when 
inoculated into the peritoneum of guinea-pigs in immense doses cause 
death. Attempts have been made to give more virulence to some of 
these varieties by successive peritoneal inoculations, but in no instance 
has any increase of virulence or decided change in morphological 
or cultural characteristics been noted. Two of the non-virulent, 
morphologically typical varieties have also been grown in symbiosis 
with virulent streptococci in broth for ninety culture generations 
transplanted every three to four days, but when separated no change 
in virulence or other characteristics was noted. Two other varieties 
of non-virulent morphologically typical bacilli have been inoculated 
into goldfinches with no result. In large doses they appear to be per- 
fectly innocuous to these birds as well as do four varieties of pseudo- 
bacilli, contrary to the results of Richmond and Salter. 

Since there are so many different forms or varieties of diphtheria- 
like bacilli, it is quite possible that some of them are derived from 
strains of the diphtheria bacillus and that under certain conditions 
they readily regain its characteristics. This seems to be the only way 

' Journal of Med. Research, June, 1902. 



210 PATHOGENIC MICRO-ORGANISMS, 

to explain the apparent discrepancies in the results obtained by dif- 
ferent observers. Such closely related varieties, however, do not 
appear to be common in New York City at the present time. So we 
may safely say that in this region at least, non-virulent diphtheria- 
like organisms retain their characteristics under various artificial and 
natural conditions, and that they may be regarded from a public 
health standpoint as harmless. 

Resistance to Heat, Drying, and Ohemicals. — ^The thermal death point 
with ten minutes' exposure is about 60° C, with five minutes 70° C. 
Boiling kills in one minute. It has about the average resistance of 
non-spore-bearing bacteria to disintectants. In the dry state and ex- 
posed to diffuse light diphtheria bacilli usually die in a few days but 
they may live for months; when in the dark, or protected by a film of 
mucus or albumin, they may live for even longer periods. Thus we 
found scrapings from a dry bit of membrane to contain vigorous and 
virulent living bacilli for a period of four months after removal from 
the throat, and if the membrane had not been at that time completely 
used, living bacilli could probably have been obtained for a much 
longer period. On slate- and lead-pencils, toys, tumblers, as well as on 
paper money, they may live for several weeks, while on coins they die 
in twelve to thirty-six hours. In culture media, when kept at the 
blood heat, they usually die after a few weeks; but under certain con- 
ditions, as when sealed in tubes and protected from heat and light, 
they retain their virulence for years. The bacillus is not very sensi- 
tive to cold, for we found about 10 per cent, of the bacilli to retain their 
vitality and virulence after exposure for two hours to several hundred 
degrees below zero. At temperatures just below freezing they may 
remain alive for a number of weeks. 

Transmission of Diphtheria. — ^The possibility of the transmission 
of diphtheria from animals to man cannot be disputed; we have met 
with two instances where cats had malignant diphtheria, and many 
other animals can be infected, but there are no authentic cases of 
such transmission on record. So-called diphtheritic disease in animals 
and birds is usually, if not always, due to other microorganisms 
than the diphtheria bacilli. 

Let us consider some of the means by which the bacilli may be 
communicated. In actual experiment the bacilli have been observed 
to remain virulent in bits of dried membrane for twenty weeks. Dried 
on silk'threads Abel reports that they may sometimes live one hundred 
and seventy-two days, and upon a child's plaything which had been 
kept in a dark place they lived for five months. The virulent bacilli 
have been found on soiled bedding or clothing of a diphtheria patient, 
or drinking-cups, candy, shoes, hair, slate-pencils, etc. Besides these 
sources of infection by which the disease may be indirectly transmitted, 
virulent bacilli may be directly received from the pseudomembrane, 
exudate, or discharges of diphtheria patients; from the secretions of 
the nose and throat of convalescent cases of diphtheria in which the 
virulent bacilli persist; and from the healthy throats of indi\'iduals 



THE BACILLUS OF DIPHTHERIA. 211 

who acquired the bacilli from being in contact with others having 
virulent germs on their persons or clothing. In such cases the bacilli 
may sometimes live and develop for days or weeks in the throat with- 
out causing any lesion. When we consider that it is only the severe 
types of diphtheria that remain isolated during their actual illness, 
the wonder is not that so many, but that so few, persons contract the 
disease. It indicates that very frequently virulent bacilli are received 
into the mouth, and then either find no condition there suitable for 
their growth or are swept away by food or drink before they can effect 
a lodgment. 

Susceptibility to and Immunity against Diphtheria. — An individ- 
ual susceptibility, both general and local, to diphtheria, as in all 
infectious diseases, is necessary to contract the disease. Age has 
long been recognized to be an important factor in diphtheria. 
Children within the first six months of life are but little susceptible, 
but exceptionally infants of a few weeks are attacked, the greatest 
degree of susceptibility being between the third and tenth year. 
After that age susceptibility decreases. Young animals bom of 
mothers immune to diphtheria possess nearly the same degree of 
immunity as their mothers. They gradually lose this but retain 
traces up to four to six months. 

DiphUieria Antitoxin. — As the result of animal experiments, it is 
now known that an artificial immunity against diphtheria can be 
produced, by the action of toxin on the cells causing the development 
of substances directly antidotal to the diphtheria toxin. Behring, in 
conjunction with others, showed that the blood of immune animals 
contains a substance which neutralizes the diphtheria toxin. The 
blood serum of persons who have recovered from diphtheria has been 
found also to possess this protective property, which it acquires about 
a week after the beginning of the disease, and loses again in a few 
months. Moreover, the blood serum of many individuals, usually 
adults, who have never had diphtheria often' has a slight general 
antitoxic property. 

Neutrauzmg Characteristics of Antitoxin. — Diphtheria antitoxin 
has the power of neutralizing diphtheria toxin, so that when a certain 
amount is injected into an animal before or together with the toxin 
it overcomes its poisonous action. There is a direct action of anti- 
toxins upon their corresponding toxins. 

The various attempts to separate the toxins and antitoxins from 
neutral mixtures have been failures, and it is found that neutralization 
takes place according to the law of multiple proportions, i, e., to save 
an animal from 1000 fatal doses of diphtheria toxin requires little 
more than a hundred times as much antitoxin as is required for 
ten fatal doses, the resistance of the animal itself accounting for the 
difference. 

Nature of Diphtheria Antitoxin. — This has until recently been 
known almost wholly from its physiological properties. Experiments 
have seemed to show that it was either closelv bound to the serum 



212 PATHOGENIC MICRO-ORGANISMS. 

globulins or was itself a substance of proteid nature closely allied to 
serum globulin. A fact developed by Atkinson is that the globulins 
tend to increase markedly in the serum of horses as the antitoxic 
strength increases. It seems possible from the above that diphtheria 
antitoxin has the characteristics of the serum globulins. Antitoxin is 
but little injured by prolonged moderate heat (56*^ C.) but is destroyed 
by short exposure to higher temperatures (95° to 100° C). It is less 
sensitive than diphtheria toxin. Atkinson, when research chemist 
in our laboratory, found that in the case of antitoxic serum the globulin 
precipitate carries with it all of the antitoxic power of the serum, 
leaving the filtrate without any neutralizing power against the diph- 
theria toxin. Independently of Atkinson, Kck obtained similar 
results. These experiments were continued later by Gibson and 
Banzhaf and they proved that the globulins which were insoluble 
in saturated sodium chlorid solution carried with them no antitoxin. 
The soluble globulins which on heating become insoluble also 
contain no antitoxin. With this knowledge a practical method of 
eliminating much of the non-antitoxic portion of the serum was 
perfected. 

Antitoxin Unit — Testing of Antitoxin. — ^This power, possessed by a 
definite quantity of antitoxin to neutraUze a certain amount of toxin, is 
utilized in testing the amount of antitoxin in any serum or solution. 
We measure this amount in units. A unit may be defined as the 
amount of antitoxin which will just neutralize 100 minimal fatal doses 
of toxin for a 250-gram guinea-pig. 

There are certain peculiarities in the composition of toxins which 
require us to use certain precautions in selecting the one to be used 
for testing. This we call a standard toxin. 

In order to facilitate testing we consider that a guinea-pig which 
lives after injection more than four days, is protected. The test is 
carried out as follows: Guinea-pigs of about 250 grams' weight are 
subcutaneously injected with one hundred fatal doses of a carefully 
preserved standardized toxin, which toxin has been previously mixed 
with an amount of antitoxin believed to be sufficient to protect the 
animal. If the guinea-pig lives four days, even if it becomes seri- 
ously ill, the amount of antitoxin added to the one hundred fatal doses 
of toxin is considered to have neutralized it and to measure 1 unit. 
If the guinea-pig dies earlier than four days, less than 1 unit of anti- 
toxin was in the mixture. 

Production of Diphtheria Antitoxin for Therapeutic Purposes.— 
As a result of the work of years in the laboratories of the Health Depart- 
ment of New York City, the following may be laid down as a practical 
method: 

A strong diphtheria toxin should be obtained by taking a very 
virulent culture and growing it in broth under the conditions de- 
scribed on page 203. 

The horses used should be young, vigorous, of fair size, and abso- 
lutely healthy. The horses are severally injected with 10000 units of 



THE BACILLUS OF DIPHTHERIA. 213 

antitoxin and with toxin ^ sufficient to kill five thousand guinea-pigs 
of 250 grams' weight. After from three to five days, so soon as the 
fever reaction has subsided, a second subcutaneous injection of a 
slightly larger dose is given. The following figures give the actual in- 
jections in a horse which produced an unusually high grade of serum. 

Actual Injections in Horse. — Injections of toxin were given every 
three days in the following amounts: 

First day, 12 c.c. toxin (^^ c.c. fatal dose),* together with 10000 
units of diphtheria antitoxin. 

Second and later injections of toxin without antitoxin at three-day 
intervals as follows: 15 c.c, 45 c.c, 55 c.c, 65 c.c, 80 c.c, 95 c.c, 
115 c.c, 140 c.c, (twenty-eighth day), 170 c.c, 205 c.c, 250 c.c, 
300 c.c (fortieth day). The injections were gradually increased 
until on the sixtieth day, 675 c.c were given. 

The antitoxic strength of the serum was on the twenty-eighth day, 
225 units; on the fortieth day, 850 units; and on the sixtieth day, 
1000 units. Regular bleedings were made weekly fqr the next four 
months when the serum had fallen to 600 units in spite of weekly, 
gradually increasing doses of toxin. 

If the antitoxin is not given we begin with .02 c.c of toxin. 

There is absolutely no way of judging which horses will produce 
the highest grades of antitoxin. Very roughly, those horses which 
are extremely sensitive and those which react hardly at all are the 
poorest,^ but even here there are exceptions. The only way, therefore, 
is at the end of six weeks or two months to bleed the horses and test 
their serum. If only high-grade serum is wanted all horses that give 
less than 150 units per c.c are discarded. The retained horses re- 
ceive steadily increasing doses, the rapidity of the increase and the 
interval of time between the doses (three days to one week) depending 
somewhat on the reaction following the injection, an elevation of 
temperature of more than 3° F. being undesirable. At the end of 
three months the antitoxic serum of all the horses should contain 
over 300 units, and in about 10 per cent, as much as 800 units in each 
cubic centimetre. Not more than 1 per cent, give above 1000 units, 
and none so far has given as much as 2000 units per c.c. The very 
best horses if pushed to their limit continue to furnish blood con- 
taining the maximum amount of antitoxin for several months, and 
then, in spite of increasing injections of toxin, begin to furnish blood 
of gradually decreasing strength. If every nine months an interval 
of three months' freedom from inoculations is given, the best horses 
furnish high-grade serum during their periods of treatment for from 
two to four years. 

*The culture, after a week's growth, is removed, and having been tested for 
purity by microscopic and culture tests is rendered sterile by the addition of 10 
per cent, of a 5 per cent, solution of carbolic acid. After forty-eight hours the 
dead bacilli have settled on the bottom of the jar and the clear fluid above is 
siphoned off, filtered, and stored in full bottles in a cold place until needed. Its 
strength is then tested by giving a series of guinea-pigs carefully measured 
amounts. Less than 0.005 c.c, when injected hypodermically, should kill a 250- 
gram guninea-pig. 



214 PATHOGENIC MICRO-ORGANISMS. 

In order to obtain the serum the blood is withdrawn from the jugular 
vein by means of a sharp-pointed cannula, which is plunged through 
the vein wall, a slit having been made in the skin. Tlie blood is 
carried by a sterile rubber tube into large Erienmeyer flasks, held 
slanted or into cylindrical jars, and allowed to clot. The serum is 
drawn off after four days by means of sterile glass and rubber tubing, 
and is stored in large flasks. Instead of this process when the globulins 
are to be separated the blood may be added directly to one-tenth of its 
volume of a 10 per cent, solution of sodium citrate. This prevents 
clotting of the blood. With the serum or globulin solution, small 
phials are filled. The phials and their stoppers, as indeed all the 
utensils used for holding the serum, must be absolutely sterile, and 
every possible precaution must be taken to avoid contamination. An 
antiseptic may be added to the serum as a preservative, but it is not 
necessary except when the serum is to be sent to great distances, 
where it cannot be kept under supervision. 

Kept from access of air and light and in a cold place it is fairly 
stable, deteriorating not more than 30 per cent., and often much less, 
within a year. Diphtheria antitoxin, when stored in phials and kept 
under the above conditions, contains within 10 per cent, of its original 
strength for at least two months; after that it can be used by allowing 
for a maximum deterioration of 2 per cent, for each month. The 
antitoxin in old serum is just as effective as in that freshly bottled, 
only there is less of it. The serum itself is less apt to produce rashes. 
All producers put more units in the phials than the label calls for, so 
as to allow for gradual loss of strength. 

Technical Points upon the Testing of Diphtheria Antitoxin and 
the Relations between the Toxicity and Neutralizing Value of 
Diphtheria Toxin. — During the earlier investigations the filtered or 
sterilized bouillon, in which the diphtheria bacillus had grown and 
produced its "toxin," was supposed to require for its neutralization an 
amount of antitoxin directly proportional to its toxicity as tested in 
guinea-pigs. Thus, if from one bouillon culture ten fatal doses of 
''toxin" were required to neutralize a certain quantity of antitoxin, 
it was believed that ten fatal doses from every culture, without regard 
to the way in which it had been produced or preserved, would also 
neutralize the same amount of antitoxin. Upon this belief was 
founded the original Behring-Ehrlich definition of an antitoxin unit 
that it was ten times the amount of antitoxin which neutralized ten 
fatal doses of toxin. 

The results of tests by different experimenters with the same anti- 
toxic serum, but with different diphtheria toxins, proved this opinion 
to be incorrect. Ehrlich^ deserves the credit for first clearly perceiving 
and publishing this. He obtained from various sources twelve toxins 
and compared their neutralizing value upon antitoxin; these tests 
gave most interesting and important information. The results in four 

* Die Wertberaessung des Diphtherieheilserums und deren theoretische Grund- 
lagen. Klinisches Jahrbuch, 1897. 



THE BACILLUS OF DIPHTHERIA, 



215 



toxins, which are representative of the twelve, are as shown in the 
following table: 



Toxin ' 
specimen 
number of, 
Ehrlich. I 



12 



Estimated 
"minimal" 

fatal dose 
for 250-^m. 
guinea-pigs. 



Smallest number \ 

of fatal doses of i Fatal doses required: 
toxic bouillon re- I to "completely ■ 
quired to kill a neutralise one anti- 
250-gm. gumea- toxin unit" as de- 
pig within 6 days, termined by the 
when mixed with health of the guinea- 
one antitoxin j pig remaining unaf- 
umt. "L^.Ehr- fected "Lo 'Ehrlich 
Uch." 



4 0.009 

7 0.0165 

9 0.039 



0.0025 



39.4 

76.3 
123 

100 



33.4 

54.4 

108 

50 



L4. — Lo| Data upon 
-fatal 



toxin 



t» 



doses. 



6 

22 
15 

50 



specimen ^ven 
by Ehruch. 



Old, deteriorated 
from 0.003 to 
0.009. 
Fresh toxin, preserv- 
ed with tricresol. 

A number of fresh 
cultures grown at 
37° C. 4 and 8 
days. 

Tested immediately 
after its with- 
drawal. 



From the facts set forth in the table, Ehrlich believed that the 
diphtheria bacilli in their growth produce a toxin which, so long as 
it remains chemically unaltered, has a definite poisonous strength 
with a definite value in neutralizing antitoxin. The toxin is, however, 
an unstable compound, and begins to change almost immediately 
into substances which are not, at least acutely, poisonous, but which 
retain their power to neutralize antitoxin. 



The results of some experiments of Atkinson and Park* were fully in 
accord with those published by Ehrlich as to the varying neutralizing value 
of a minimal fatal dose of ^' toxin"; they, however, also indicate roughly a 
general law in accordance with which these changes occur. 

The neutralizing value of a fatal dose of toxin is at its lowest in the culture 
fluid when the first considerable amounts of toxin have been produced. 
After a short period, during which the quantity of toxin in the fluid is increas- 
ing, the neutralizing value of the fatal dose begins to increase, at first rapidly, 
then more slowly. 

While the culture is still in vigorous growth and new toxin is being pro- 
duced, the neutralizing value of the fatal dose fluctuates somewhat, but 
with a generally upward tendency. After the cessation of toxin produc- 
tion the neutralizing value of the fatal dose increases steadily until it becomes 
five to ten times its original amount. 

In our experiments the greatest value for L+ was 126, the least 27. As 
at six hours L4. was only 72 and at twenty-eight hours only 91, we doubt 
whether L-i- ever reaches above 150.* When we seek to analyze the above- 

Lo —fatal doses of toxin required to fully neutralize one unit of antitoxin. 
described process we find certain facts which seem partly to explain it. 

In the fluid holding the living bacilli we have, after the first few hours of 
toxin formation, a double process going on — one of deterioration in the toxin 
already accumulated, which tends to increase the neutralizing value of the 

* Journal of Experimental Medicine, Vol. iii, No. 4. 

' L+ —fatal doses of toxin required to kill a guinea-pig in four days after having 
been mixed with one unit of antitoxin. 



216 PATHOGENIC MICRO-ORGANISMS. 

fatal dose ; the other of new toxin formation, which probably tends to dimin- 
ish the neutralizing value. The chemical changes produced by the growth 
of the baciUi in the bouillon tend to aid one or the other of these processes, and 
so to make, from hour to hour, slight changes in the value of the fatal dose. 
Later, with the period of cessation of toxin production, the gradual deteriora- 
tion of the toxicity alone continues, and the fatal dose gradually and steadily 
increases in its neutraUzing value. We believed that two types of toxin were 
produced by the bacilU. 

With greater information Ehrlich has had to modify greatly the details 
of his earUest explanation of the reason of the variation in the ratio between 
toxicity and neutraUzing value of toxin. He now accepts the fact that diph- 
theria culture fluid contains at least two toxins which differ in their charac- 
teristics. 

To summarize Ehrlich's present views as to the nature of diphtheria 
toxin: The diphtheria bacillus secretes two toxins, one of which, the toxin, 
causes the acute phenomena of diphtheria intoxication, while the other, the 
toxon, causes cachexia and paralysis after a rather long period of incubation. 
The non-toxic toxin, or toxoid, appears as the result of the degeneration of 
the toxophore group of the toxin, the haptophore group remaining intact. 
The toxin may be separated into three divisions, which vary in their affinity 
for antitoxin — prototoxin, deuterotoxin, and tritotoxin. On the same basis 
there are three toxoids — prototoxoids, syntoxoids, and epitoxoid (the toxon) 
— the first having the greatest affinity for antitoxin, while the epitoxoid has 
the least. The toxins are divided into an alpha and a beta portion, depend- 
ing on the ease with which they are changed into toxoids. All of these sub- 
stances unite with tissue cells and with antitoxin through the agency of a 
haptophore group, while the toxicity depends on the presence of a toxophore 
group in the toxin or toxon molecule. 

Bordet and others refuse to accept these complicated conceptions of 
Ehrlich and the whole matter is at the present time under active discussion. 
Thus the existence or non-existence of toxons has excited a great deal of discus- 
sion among investigators. The Swedish chemist, Arrhenius, has quite recently 
given much attention to toxons and is appl3ring the principles of physical 
chemistry to the study of toxins and antitoxins. It is a well-known fact that 
some chemical substances when in solution have the power of breaking up 
into their constituent parts; thus sodium chloride breaks up in part into sodium 
and chlorine, as sodium or chlorine ions or electrolytes. The dissociated 
sodium and chlorine may then enter into combination with any other suitable 
substance which may be present. Arrhenius holds that this is the case with 
the toxin-antitoxin molecule, that it may to a certain extent again break up 
into separate toxin and antitoxin. He believes that this dissociated toxin 
is the substance which EhrUch has been calling toxon. Madsen, who formerly 
had done much work with toxons, has now joined with Arrhenius in support 
of the dissociation theory. In spite of their reasoning Ehrlich and his fol- 
lowers continue to uphold the toxon as an independent toxic substance. 
Recent investigations throw doubt on both explanations as being at all final. 

Standardizing of Antitoxin Testing.— Ehrlich has contributed 
greatly to uniformity of results in testing antitoxin by calling atten- 
tion to the necessity of selecting a suitable toxin and by employing 
and distributing an antitoxin as a standard to test toxins by. In this 
way smaller testing stations can make their results correspond with 
those of the central station. The United States Marine Hospital 
laboratory has also distributed to laboratories in the United States 
an equally carefully standardized serum. 

The old definition of Behring and Ehrlich, that an antitoxin unit 



THE BACILLUS OF DIPHTHERIA. 217 

contains the amount of antitoxin which will protect the life of a guinea- 
pig from one hundred fatal doses of toxin, is true only for a toxin 
similar to that adopted as the standard — namely, one having approxi- 
mately the characteristics of toxins in cultures at the height of their 
toxicity. 

The actual test of an antitoxin serum is, therefore, carried out as 
follows: Six guinea-pigs are injected with mixtures of toxin and 
antitoxin. In each of the mixtures there is the amount of toxin suffi- 
cient to just neutralize 1 unit of the standard antitoxin supplied by 
the central laboratory. In each of the mixtures the amount of anti- 
toxin varies; for instance, No. 1 would contain 0.002 c.c. serum; 
No. 2, 0.003 c.c; No. 3, 0.004 c.c; No. 4, 0.005 cc, etc. If at 
the end of the fourth day Nos. 1, 2, and 3 were dead and Nos. 4, 5, 
and 6 were alive we would consider the serum to contain 200 units 
units of antitoxin for each cubic centimetre. When we test for ex- 
perimental purposes sera with very little antitoxin, we often use only 
one-tenth the above amount of toxin. In this case the resistance of 
the guinea-pig must be considered so that the guinea-pig must not 
only not die but must remain well. The mixed toxin and antitoxin 
must remain together for fifteen minutes before injecting so that com- 
plete union may occur. 

Use of Antitoxin in Treatment and Immnnization.— The antitoxin 
in the higher grades of globulin solution or serum is identical with that 
in the lower grades; there is simply more of it in each drop. In 
treatment, however, for the same amount of antitoxin we have to inject 
less foreign proteids with the higher grades, and, therefore, have some- 
what less danger of rashes and other deleterious results. The amount 
of antitoxin required for immunization is 300 to 500 units for an infant, 
500 to 1000 for an adult, and proportionately for those between these 
extremes. The larger doses are advised when the danger of infection 
is very great. After the observation of the use of antitoxin in the 
immunization of several thousand cases, I have absolute belief in its 
power to prevent an outbreak of diphtheria for at least two weeks, 
and also of its almost complete harmlessness in the small doses required. 
If it is desired to prolong the immunity the antitoxin injection is re- 
j>eated every two weeks. For treatment, mild cases should be given 
1500 units, moderate cases 2000 to 4000 units, and severe cases 10,000 
to 20,000 units. Where no improvement follows in twelve hours the 
dose should be repeated. Intravenous injections give most rapid 
effect, and should be used in all malignant cases. It takes twelve 
to eighteen hours for the absorption into the blood of the greater 
part of the antitoxin from the subcutaneous tissues. This in bad 
cases may be a fatal delay. Antitoxin is only to a very slight extent 
absorbed when given by the mouth. 

Sesnlts of the Antitoxin Treatment of Diphtheria.— The conclu- 
sions arrived at by Biggs and Guerard, after a review of all the sta- 
tbtics and opinions published since the beginning of the antitoxin 
treatment in 1892, were as follows: 



218 PATHOGENIC MICRO-ORGANISMS, 

"It matters not from what point of view the subject is regarded, 
if the evidence now at hand is properly weighed, but one conclusion is 
or can be reached — whether we consider the percentage of mortality 
from diphtheria and croup in cities as a whole, or in hospitals, or in 
private practice; or whether we take the absolute mortality for all the 
cities of Germany whose population is over 15,000, and all the cities 
of France whose population is over 20,000; or the absolute mortality 
for New York City, or for the great hospitals in France, Germany, 
and Austria; or whether we consider only the most fatal cases of 
diphtheria, the laryngeal and operative cases; or whether we study 
the question with relation to the day of the disease on which treatment 
is commenced, or the age of the patient treated; it matters not how 
the subject is regarded or how it is turned for the purpose of compari- 
son with previous results, the conclusion reached is always the same 
— namely, there has been an average reduction of mortality from the 
use of antitoxin in the treatment of diphtheria of not less than 50 
per cent., and under the most favorable conditions a reduction to one- 
quarter, or even less, of the previous death rate. This has occurred 
not in one city at one particular time, but in many cities, in different 
countries, at different seasons of the year, and always in conjunction 
with the introduction of antitoxin serum and proportionate to the 
extent of its use." The combined statistics of deaths of 19 of the 
chief cities of the world show there were in the last four years not 
over 30 per cent, as many deaths as in the four years preceding the 
introduction of antitoxin. Except where immunization has been 
practiced on a large scale, no marked reduction in the number of cases 
of diphtheria has been evident. 

Deleterious Effects Following Injections of Antitoxic Senun. — About 
1 in 10,000 persons develop, within a few minutes after an injection of 
serum, alarming symptoms. About twenty deaths in all have been 
reported. The persons suffering severe symptoms have usually been 
subject to asthma while the fatal cases usually have the pathological 
changes known as status lymphaticus. A few of these rare cases die 
almost instantly. As a rule, when death occurs it takes place 
within a few minutes after the development of symptoms. Usually 
the respiratory rather than the circulatory center seems to be affected. 
It is over three years since a death from this cause has occurred in 
New York City. 

Serum Sickness. — Besides these rare accidents there are the disa- 
greeable after-effects which we group under the name serum sickness. 
Under this name we now include the various clinical manifestations 
following the injection of horse serum into man. The principal 
symptoms of this disease are a period of incubation varying from 
eight to thirteen days, fever, skin eruptions, swelling of the lymph 
glands, leukonemia, joint symptoms, oedema, and albuminuria. The 
term ** serum sickness*' was first used by von Pirquet and Schick,^ 
from whose excellent monograph the following data are chiefly taken. 

' V. Pirquet and Schick, Die Serum Krankheit, Wien, 1905. 



THE BACILLUS OF DIPHTHERIA. 219 

In 1874 Dallera reported that urticarial eruptions may follow the 
transfusion of blood. In the year 1894 the use of diphtheria antitoxin 
introduced the widespread practice of injecting horse serum. In the 
same year several cases were reported in which these injections were 
followed by various skin manifestations, mostly of an urticarial charac- 
ter. Following these came a great mass of evidence which made it 
clear that following the injection of antidiphtheric serum these sequelae 
were usually comparatively harmless. 

Due to Serum as Such. — Heubner in 1894 and von Bokay some- 
what later expressed the opinion that these manifestations were due 
to other properties than the antitoxin in the serum, and this has 
proven to be the case. It has also been shown that the skin eruptions 
and other symptoms follow in a considerable degree according to the 
amount of serum injected, and this has led to attempts to eliminate 
the non-antitoxic portion of the serum as much as possible.^ The 
serum reaction has been studied by many investigators, but is not yet 
fully understood. 

VON PiRQUET AND ScHICK's SENSITIZATION ThEORY (ANAPHYLAXIS). 

— The underlying idea is that the injection of serum into animals 
causes the development of specific reaction products which are able 
to act upon the antigens introduced. These antibodies encounter the 
antigens, i.e., the serum later introduced in the body, and so give rise 
to a deleterious reaction. This accounts for the cases of ''immediate 
reaction " in man described by von Pirquet and Schick, in which second 
injection of a serum produces an attack of serum sickness with a short 
or no period of incubation. It has been known for years that a large 
injection of horse serum is poisonous to guinea-pigs, that have been 
previously injected with small amounts of horse serum.' The time 
necessary to elapse between the first and second injections is ten days 
or more. The greatest effect is present between four and six weeks. 
The symptoms are respiratory embarrassment, paralysis and convul- 
sions, and come on usually within ten minutes after the injection. 
When death results it usually occurs within one hour, frequently in 
less than thirty minutes. The poisonous principle in horse serum in 
these cases appears to act on the respiratory centers. The heart 
continues to beat long after respiration ceases. 

The first injection of horse serum renders the guinea-pig susceptible; 
the quantity required for this purpose is extremely small. Rosenau 
and Anderson find that from ^ttt ^^ Tinnr ^'^' ordinarily suflBces. 
Guinea-pigs may be sensitized to the toxic action of horse serum 
by feeding them with horse serum or horse meat. 

It is probable that man cannot be sensitized in the same way as 
guinea-pigs, the most susceptible of the laboratory animals. Children 
have, in numerous instances, been injected with antidiphtheric horse 

* See Gibson, The Concentration of Diphtheria Antitoxin. Jour, of Biological 
Chemistry, Vol. i, 1906. 

'The Germans usually speak of this as '* Theobald Smith's phenomenon of 
hypersusceptibility " (seep. 162). 



220 PATHOGENIC MICRO-ORGANISMS, 

serum at short and long intervals without, so far as we are aware, 
causing severe symptoms. Certain serums, for example the antitu- 
bercle serum of Maragliano, are habitually uSed by giving injections 
at intervals of days or weeks. The rare fatal cases so far reported 
have all followed primary injections. 

While it may be true that the sensitizing of guinea-pigs by a previous 
injection of serum is analogous to the condition present in man which 
gives rise to the sudden symptoms following an injection of antitoxic 
serum, there is, in our experience, no reason to avoid a second im- 
munizing injection of serum when it is really indicated. A subcu- 
taneous injection in man comparable to the amount required to produce 
sickness in a guinea-pig would be over 200 c.c. We should hesitate, 
however, to give a large intravenous injection in a sensitized child. 
Banzhaf and Famuleuer have recently shown that chloral in large 
doses will prevent sickness in sensitized guinea-pigs. 

Ibe Separation of Antitoxin from Semm. — ^There have already 
been many attempts to accomplish this in the case of the antitoxins. 
Those interested in the chemical side of these investigations are re- 
ferred to the recent article by Gibson, as already stated. In 1900, 
Atkinson, working in the Research Laboratory of the Health Depart- 
ment, eliminated all but the globulin from the antitoxic serum, and we 
tried this partially refined serum in 36 cases. The results were so 
nearly identical with an equal number of cases treated with the whole 
serum from the same horse that it did not seem to be worth while to go 
to the expense of preparing such an antitoxic solution. The idea that 
a practical separation of the antitoxin from much of the proteid non- 
antitoxic portion of the serum was possible was not given up. In 
August of 1905 we began trials with an antitoxic preparation which 
offered grounds for hoping for better success. 'Dr. R. B. Gibson, 
chemist in the Research Laboratory, placed the ammonium sulphate 
precipitate from the antitoxic serum in saturated sodium chloride so- 
lution and found that the portion of the globuUn soluble in this con- 
tained all the antitoxin. In this way the nucleoproteids and the 
insoluble globulins present in the Atkinson preparation were elimi- 
nated, as in the following summary shows. 

Ordinary antitoxic serum contains serum globulins (antitoxic), 
serum globulins (non-antitoxic), serum albumins (non-antitoxic), 
serum nucleoproteids (non-antitoxic), cholesterin, lecithin, traces of 
bile coloring matter, traces of bile salts and acids, traces of inorganic 
blood salts and other non-proteid compounds. Refined serum con- 
tains serum globulins (antitoxic), traces of serum globuUns (non- 
antitoxic), dissolved in dilute saline solution. Later Dr. E. A. Banz- 
haf,^ who had succeeded Gibson, discovered that if the antitoxic serum 
or plasma was heated to 57® for 18 hours there was a change of a con- 
siderable portion of the soluble globulins into insoluble globulins. The 
antitoxin remained unchanged. This permitted a greater elimination 
of the non-antitoxic proteids. 

' Journal of Biological Chemistry. 



THE BACILLUS OF DIPHTHERIA. 221 

Method of Concentration. — ^The material we use is blood plasma 
instead of blood serum* This is obtained by allowing the blood to 
flow directly from the jugular vein of the immunized horse into 10 
per cent, sodium citrate solution, which prevents it from clotting and 
allows the red corpuscles to settle out. This plasma is used, in place 
of serum, merely as a matter of convenience and economy. 

Isolating the Antitoxin Globulins. — ^The globulins of the 
plasma are removed from the other constituents by precipitating them 
from solution by means of ammonium sulphate and filtering off on 
paper. This allows the serum albumins and other soluble, non- 
proteid constituents of the blood, to pass through and thus become 
immediately eliminated in the filtrate. Now this precipitate, formed 
with ammonium sulphate, contains the globulins of the blood which 
are antitoxic in character; those which are non-antitoxic in character; 
and nucleoproteids. The antitoxic globulins are extracted from this 
mass of precipitate by treating with saturated solution of sodium 
chloride, in which this compound is soluble. The problem then re- 
mains to separate this antitoxic substance from the solution and wash 
out of it the salts of ammonium and sodium. 

The antitoxic globulin is next isolated by precipitation with dilute 
acetic acid. 

The ammonium salts are thoroughly washed out by repeated treat- 
ment with saturated sodium chloride solution and filtered each time. 
Finally, the sodium chloride is removed by dialysis, which process is 
accomplished by placing the antitoxic globulins in bags of vegetable 
parchment and immersing in running water so long as salts continue 
to diffuse out. After dialysis, the antitoxic globulins are dissolved 
in dilute saline solution, filtered through paper pulp, to remove the 
traces of undissolved matter, filtered through a Berkefeld clay filter to 
remove bacteria, and then put in sterile syringes. 

This antitoxic solution of globulin and a portion of the other solu- 
ble serum globulins was then tested on a number of children. The 
results were from the start' favorable, except that in the beginning 
more local pain was produced than with the whole serum. Stricter 
attention to the neutralization soon overcame this, so that when the 
serum was injected on one side and the globulin solution on the other 
the patient was unable to tell one from the other. In October, 
1905, the antitoxic globulin solution was administered not only in the 
hospitals, but also in private homes by medical inspectors. Since then 
it has been the only form of antitoxin supplied by the Health Depart- 
ment. Private manufacturers have also recently begun to furnish it. 

Besnlts from the Use of Antitoxic Globulin Solution. — The curative 
effect proved to be identical with that of the whole serum. Our tests 
showed clearly that not only the toxin, but also the poisons produced 
in the animal by injections with virulent bacilli are neutralized as com- 
pletely by the globulin solution as by the antitoxic serum from which 
they are separated. Not only we ourselves, but the resident and attend- 
ing physicians of the contagious disease hospitals noted that following 



222 PATHOGENIC MICRO-ORGANISMS. 

the injectioQs of the globulin solution there seemed to be deddediy less 
severe rashes than formerly fallowed the whole serum, and it was 
especially noted that there were very few who had any constitutional 
disturbances even when the development of the rashes did occur. As 
the serum supplied by di£Ferent horses or from the same horse at differ- 
ent times is known to vary, and as it is therefore difficult accurately to 
compare different bleedings, it was decided to make a test by collecting 
a quantity of serum from four different horses, mixing it thoroughly, and 
then, after precipitating one-half, to treat an equal number ^multane- 
ously with the two preparations. These tests were chiefly carried 
out in the Willard Parker Hospital, but a few of the cases were treated 
at Riverside Hospital. It soon became evident that the serum that 
we had chosen for the test was one of such character that eruptions 
and constitutional disturbances usually appeared in the children in- 
jected. In those over ten years of age almost no rashes occurred. 
The rashes in those given the globulin preparation were much less 
severe. The cases treated with both the whole serum and the anti- 
toxic globulins were most carefully watched, and the course of the 
disease as well as after-affects noted. 

After all the tested cases had become fully convalescent or had left 
the hospital, the histories were finally gone over and compared. It 
was found that fifty children under ten years of age treated with the 
whole serum had lived at least nine days or long enough for the devel- 
opment of serum effects. The first fifty consecutive cases in children 
under ten years treated with the antitoxic globulins precipitated from 
the same lot of serum were taken to compare with these. 

The comparative table giving a summary of the constitutional and 
local reactions obtained in the treatment of fifty cases of diphtheria 
in young children, with a lot of antitoxic serum received from three 
horses and of an equal number of similar cases treated with a solution 
of the antitoxic globulins derived from a portion of the same lot of 
serum is as follows: 

Children who were 



Marked constitutional symptoms 


Children treated « 
the whole serun^ 

28 per cent, 

18 per cent. 

20 per cent. 

4 per cent. 

30 per cent. 

12^4 

,5 7 .1 2 


th 

5 
3 


treated with the 
antitoxic gobulina 


Moderate constitutional symptomK 

erythema or urticaria 

Very alight constitutional disturb- 
ance accompanied by a more or 


4 per cent. 

8 per cent. 

34 per cent. 
54 per cent. 

6 7 8 Totals 
1 -23 


!ir„ ;„i.i. — nalitutional dis- 

re or less general 


•ieterious after- 


Dt-H.T,, 



THE BACILLUS OF DIPHTHERIA. 223 

The concentration of antitoxin made possible by the eUmination 
of the non-antitoxic substances is not only a convenience but is of a 
distinct importance, as it tends to encourage large doses. Some pro- 
ducers supply a product which is too rich in proteid. This is prob- 
ably not so well absorbed as the less concentrated product. The 
total solids in the globulin solution should not be much greater than 
those in the serum. 

The antitoxic globulin solution tends to become slightly cloudy 
when kept at moderate or high temperatures and substances such as 
solutions of carbolic acid and tricresol precipitate it. 

Development of Agglatiiiins for Diphtlieria Bacilli.— By the in- 
jections of the bodies of diphtheria bacilli into animals agglutinins 
have been developed in sufficient amount to act in 1 : 5000 dilutions 
of the serum. The serum produced from diphtheria bacilli does not 
agglutinate pseudodiphtheria bacilli in high dilutions. The serum 
of patients convalescent from diphtheria has, as a rule, little agglu- 
tinating power. This test is not used in diagnosis. 

Persistence of Antitoxin in the Blood — When injections of toxin 
are stopped in a horse the antitoxin is slowly eliminated, so that there 
is a loss of about 20 per cent, a week. In from five to eight months 
all appreciable antitoxin has been eliminated. 

The Persistence in the Man's Blood of Injected Antitoxin 
Produced in the Horse. — All observers from Ransom on, except 
Madsen and Roemer, have noted that antitoxins and other anti- 
bodies produced in an animal disappear more rapidly when 
introduced into the blood of another species than into one of the 
same species. 

Madsen and Roemer claim that the antibodies from each species 
must be tested in other species and that, in some cases, the foreign 
antibody will persist as long as that obtained from one of the same 
species. 

When their experiments are examined it is seen that Madsen's re- 
sults were obtained in four animals only, and that one of the goats 
receiving its own type of antitoxin died on the seventeenth day. 
Roemer's recorded observations really substantiate the claims made 
by other investigators, for when he injected lambs with heterologous 
antitoxin it disappeared just as rapidly as in the animals tested by 
others. He tested for such very slight amounts of antitoxin, how- 
ever, that it appeared to last longer than in the animals of others, 
who did not test for such small amounts. 

In our experiments in guinea-pigs we have found that the homol- 
ogous antitoxin was retained in appreciable amounts for at least six 
months, while the heterologous antibodies were noticeable to the same 
extent for only four weeks. There is a very rapid loss of both types 
of antitoxins during the first two weeks and then a slow loss becom- 
ing more and more gradual until final eUmination. The larger the 
amount of antibodies injected the longer will be the time before the 
elimination of effective amount. 



224 PATHOGENIC MICRO-ORGANISMS. 

Active Immanization. — Theobald Smith has recommended that 
mixtures of toxin and antitoxin be given so as to produce active im- 
munity. It is a well-known fact that when 60% of the L + dose of 
toxin is added to one unit of antitoxin, that this mixture will cause 
the production in the animal of antitoxin, and as a rule cause no 
toxic symptoms. The immunity produced from a single injection is 
slight, but will last for from nine to twelve months. Some guinea- 
pigs, however, some weeks after the injection, show a late paralysis 
and it is questionable whether we would dare to give such mixtures 
to children. Another practical objection is that the immunity dur- 
ing the first two weeks after the injection is almost negligible. Usu- 
ally this is the period during which we desire the greatest immunity, 
because it is then that the danger of infection is greatest. 

Mixed Infection in Diphtheria.— Virulent diphtheria bacilli are 
not the only bacteria present in human diphtheria. Various cocci, 
more particularly streptococci, staphylococci, and pneumococci, are 
also found actively associated with LoeflSer's bacilli in diphtheria, 
playing an important part in the disease and leading often to serious 
complications (sepsis and bronchopneumonia). Though the results 
of these investigations so far have been somewhat indefinite, they 
would seem to indicate that when other bacteria are associated with 
the diphtheria bacilli they mutually assist one another in their attacks 
upon the mucous membrane, the streptococcus being particulariy 
active in this respect, often opening the way for the invasion of the 
Loeffler bacillus into the deeper tissues or supplying needed conditions 
for the development of its toxin. In most fatal cases of broncho- 
pneumonia following laryngeal diphtheria we find not only abundant 
pneumococci or streptococci in the inflamed lung areas, but also in 
the blood and tissues of the organs. As these septic infections due to 
the pyogenic cocci are in no way influenced by the diphtheria antitoxin, 
they frequently are the cause of the fatal termination. Other bacteria 
cause putrefactive changes in the exudate, producing alterations in 
color and offensive odors. 

Pseadomembranous Exadative Inflammations Due to Baeteria 
other than the Diphtheria Bacilli — The diphtheria bacillus, though 
the most usual, is not the only microorganism that is capable of pro- 
ducing pseudomembranous inflammations. There are numerous 
bacteria present almost constantly in the throat secretions, which, under 
certain conditions, can cause local lesions very similar to those in the 
less-marked cases of true diphtheria. The streptococcus and pneu- 
mococcus are the two forms most frequently found in these cases, 
but there are also others, such as the Vincent's bacillus, which, under 
suitable conditions, excite this form of inflammation, but without con- 
stitutional symptoms. 

The pseudomembranous angina accompanying scarlet fever, and 
to a less extent other diseases, may not show the presence of diph- 
theria bacilli, but only the pyogenic cocci, especially streptococci, or, 
more rarely, some varieties of little-known bacilli. The deposit cover- 



THE BACILLUS OF DIPHTHERIA. 225 

ing the inflamed tissues in these non-specific cases is, it is true, usually 
but not always, rather an exudate than a true pseudomembrane. 

Relation of Bacteriology to Diagnosis.— We believe that all expe- 
rienced clinicians will agree that, when left to judge solely by the 
appearance and symptoms of a case, there are certain mild exudative 
inflammations of the throat which are at times excited by diphtheria 
bacilli and at times by other bacteria. 

It is not meant to imply that a case is one of true diphtheria simply 
l>ecause the diphtheria bacilli are present, but rather that the doubtful 
cases not only have the diphtheria bacilli in the exudate, but are 
capable of giving true characteristic diphtheria to others, or later 
<levelop it characteristically themselves; and that those in whose 
throats no diphtheria bacilli exist can under no condition give true 
characteristic diphtheria to others, or develop it themselves unless 
they receive a new infection. It is, indeed, true, as a rule, that cases 
presenting the appearance of ordinary follicular tonsillitis in adults 
are not due to the diphtheria bacillus. On the other hand, in small 
children mild diphtheria very frequently occurs with the semblance of 
rather severe ordinary follicular tonsillitis, due to the pyogenic cocci, 
and in large cities where diphtheria is prevalent all such cases must be 
watched as being more or less suspicious. As showing doubt in our 
judgment, I think most would feel that if in any case exposure to 
diphtheria is known to have occurred, even a slightly suspicious sore 
throat would be regarded as probably due to the* diphtheria bacilli. 
If, on the other hand, no cases of diphtheria have been known to exist 
in the neighborhood, even cases of a more suspicious nature would 
probably not be regarded as diphtheria. 

Appearances Gluuracteristic of Diphtheria.— The presence of irreg- 
ular-shaped patches of adherent grayish or yellowish-gray pseudo- 
membrane on some other portions than the tonsils is, as a rule, an 
indication of the activity of the diphtheria bacilli. Restricted to the 
tonsils alone, their presence is less certain. 

Occasionally, in scarlatinal angina or in severe phlegmonous sore 
throats, patches of exudate may appear on the uvula or borders of the 
faucial pillars, and still the case may not be due to the diphtheria 
bacilli; these are, however, exceptional. Thick, grayish pseudomem- 
branes which cover large portions of the tonsils, soft palate, and nostrils 
are almost invariably the lesions produced by diphtheria bacilli. 

The very great majority of cases of pseudomembranous or exuda- 
tive laryngitis, in the coast cities at least, whether an exudate is present 
in the pharynx or not, are due to the diphtheria bacilli. Nearly all 
membranous affections of the nose are true diphtheria. When the 
membrane is limited to the nose the symptoms are, as a nile, very slight; 
but when the nasopharynx is involved the symptoms are usually grave. 

Most cases of pseudomembranes and exudates, entirely confined 
to portions of the tonsils in adults, are not due to the diphtheria bacilli. 
Cases presenting the appearances found in scarlet fever, in which a 
thin, grayish membrane lines the borders of the uvula and faucial 



226 PATHOGENIC MICRO-ORGANISMS. 

pillars, are rarely diphtheritic. As a rule, pseudomembranous 
inflammations complicating scarlet fever, syphilis, and other infectious 
diseases are due to the activity of the pathogenic cocci and other 
bacteria, induced by the inflamed condition of the mucous membranes 
due to the scarlatinal or other poison. 

Location of Diphtheritic inflammation. — Diphtheria attacks not 
only the fauces, larynx, and na^al cavities, but also occasionally the 
skin, vagina, rectum, conjunctiva, nose, and ear. 

Exudate Due to the Diphtheria Bacilli Contrasted with that Due 

to Other Bacteria. — As a rule, the exudate in diphtheria is firmly 
incorporated with the underlying mucous membrane, and cannot be 
removed without leaving a bleeding surface, at least until convales- 
cence. The tissues surrounding the exudate are more or less inflameii 
and swollen. Where other bacteria produce the irritant the exudate, 
except in cases due to the bacillus described by Vincent, is usually 
loosely attached, collected in small masses, and easily removable. 
Exceptions, however, occur in both these diseases, so that in true diph- 
theria the exudate may be easily removed, and in lesions due to other 
bacteria the exudate may be firmly adherent. 

Paralysis following a pseudomembranous inflammation is an al- 
most positive indication that the case was one of diphtheria, although 
slight paralysis has followed in a very few cases in which careful cul- 
tures revealed no diphtheria bacilli. These, if not true diphtheria, 
must be considered very exceptional cases. 

Bacteriologic Diagnosis. — ^From the above it is apparent that 
fully developed characteristic cases of diphtheria are readily diag- 
nosticated, but that many of the less marked, or at an early period 
undeveloped, cases are diflScult to differentiate the one from the other. 
In these cases cultures are of the utmost value, since they enable us 
to isolate those in which diphtheria-like bacilli are found, and to give 
preventive injections of antitoxin to both the sick and those in contact 
with them, if this has not already been done. As a rule, cultures do 
not give us as much information as to the gravity of the case as the 
clinical appearances, for by the end of twenty-four to forty-eight hours 
the extent of the disease is usually possible of determination. The re- 
ported absence of bacilli in a culture must be given weight in propor- 
tion to the skill with which the culture was made, the suitableness of 
the media, the location of the disease, and the knowledge and experi- 
ence of the one who examined it. 

Diphtheria does not occur without the presence of the diphtheria 
bacilli; but there have been many cases of diphtheria in which, for 
one or another reason, no bacilli were found in the cultures bv the 
examiner. In many of these cases later cultures revealed them. The 
reverse is also true, the presence of diphtheria bacilli in throats without 
the clinical signs of diphtheria in no sense makes it a case of diphtheria. 
In a convalescent case the absence of bacilli in any one culture indicates 
that there are certainly not many bacilli left in the throat. Only 
repeated cultures can prove their total absence. 



THE BACILLUS OF DIPHTHERIA. 227 

Techniqae of the Bacteriologic Diagnosis. — Collection of the Blood Serum 
and its Preparation for Use in Cultures. — A covered glass jar, which has been 
thoroughly cleansed with hot water, is taken to the slaughter-house and filled 
with freshly shed blood from a calf or sheep. The blood is received directly 
in the jar as it spurts from the cut in the throat of the animal. After the edge 
of the jar has been wiped it is covered with the lid and set aside, where it 
may stand quietly until the blood has thoroughly clotted. The jar is then 
carried to the laboratory and placed in an ice-chest. If the jar containing the 
blood is carried about before the latter has clotted, very imperfect separation 
of the serum will take place. It is well to inspect the blood in the jar after 
it has been standing a few hours, and, if the clot is found adhering to the sides, 
to separate it by a rod. The blood is allowed to remain twenty-four hours on 
the ice, and then the serum which surrounds the clot is siphoned off by a 
rubber tube and mixed with one-third its quantity of nutrient beef-broth, to 
which 1 per cent, glucose has been added. This constitutes the Loeffler 
blood-serum mixture. This is poured into tubes, which should be about four 
inches in length and one-half of an inch in diameter, having been previously 
plugged with cotton and sterilized by dry heat at 150° C. for one hour. Care 
should be taken in filling the tubes to avoid the formation of air bubbles, as 
they leave a permanently uneven surface when the serum has been coagulated 
by heat. To prevent this the end of the pipette or funnel which contains 
the serum should be inserted well into the test-tube. About 3 c.c. are suffi- 
cient for each tube if the small size is employed; if not, 5 c.c. are required. 
The tubes, having been filled to the required height, are now to be coagu- 
lated and sterilized. They are placed slanted at the proper angle and then 
kept for two hours at a temperature just below 95° C. For this purpose 
a Koch serum coagulator or a double boiler serves best, though a steam 
sterilizer will suffice. If the latter is used a wire frame must be arranged 
to hold the tubes at the proper inch nation, and the degree of heat must be 
carefully watched, as otherwise the temperature may go too high, and if 
the serum is actually boiled the culture medium will be spoiled. After 
sterilization by this process the tubes containing the sterile, solidified blood 
serum can be placed in covered tin boxes, or stopped with sterile paraffined 
corks and kept for months. The serum thus prepared is quite opaque and 
firm. 

Swab far Inoculating Culture Tubes. — The swab we use to inoculate the 
serum is made as follows: A stiff, thin, iron rod, six inches in length, is 
roughened at one end by a few blows of a hammer, and about this end a little 
absorbent cotton is firmly wound. Each swab is then placed in a separate 
glass tube, and the mouths of the tubes are plugged with cotton. The tubes 
and rods are then sterilized by dry heat at about 150° C. for one hour, and 
stored for future use. These cotton swabs have proved much more ser- 
viceable for making inoculations than platinum-wire needles or wooden sticks, 
especially in young children and in laryngeal cases. It is easier to use the 
cotton swab in such cases, and it gathers up so much more material for the 
inoculation that it has seemed more reliable. 

For convenience and safety in transportation "culture outfits" have been 
devised, which consist usually of a small wooden box containing a tube of 
blood serum, a tube holding a swab, and a record blank. These "culture 
outfits " may be carried or sent by messenger or express to any place desired. 

Directions for Inoculating CuUure Tubes with the Exudate. — The 
patient is placed in a good light, and, if a child, properly held. The 
swab is removed from its tube, and, while the tongue is depressed with 
a spoon, is passed into the pharynx (if possible, without touching the 
tongue or other parts of the mouth), and is rubbed gently but firmly 
against any visible membrane on the tonsils or in the pharynx, and 



228 PATHOGENIC MICRO-ORGANISMS, 

then, without being laid down, the swab is immediately inserted in 
the blood-serum tube, and the portion which has previously been in 
contact with the exudate is rubbed a number of times back and forth 
over the whole surface of the serum. This should be done thoroughly, 
but it is to be gently done, so as not to break the surface of the serum. 
The swab should then be placed in its tube, and both tubes, thin cotton 
plugs having been inserted, are reserved for examination or sent to 
the laboratory or collecting station (as in New York City). If sent 
to the health department laboratories for examination the blank forms 
of report which usually accompany each ''outfit" should be filled out 
and forwarded with the tubes. 

Where there is no visible membrane (it may be present in the nose 
or larynx) the swab should be thoroughly rubbed over the mucous 
membrane of the pharynx and tonsils, and in the nasal cavities, and a 
culture made from these. In very young children care should be 
taken not to use the swab when the throat contains food or vomited 
matter, as then the bacteriological examination is rendered more 
difficult. Under no conditions should any attempt be made to collect 
the material shortly after the application of strong disinfectants 
(especially solutions of corrosive sublimate) to the throat. Cultures 
from the nostrils are often more successful if the nostrils are first 
cleansed with a spray of sterile normal salt solution. 

Examination of Cultures, — The culture tubes which have been 
inoculated, as described above, are kept in an incubator at 37° C. for 
twelve hours, and are then ready for examination. When great haste 
is required, even five hours will often suffice for a sufficient growth of 
bacteria for a skilled examiner to decide as to the presence or absence 
of the bacilli. On inspection it will be seen that the surface of the 
blood serum is dotted with numerous colonies, which are just visible. 
No diagnosis can be made from simple inspection; if, however, the 
serum is found to be liquefied or shows other evidences of contamina- 
tion the examination will probably be unsatisfactory. 

In order to make a microscopic preparation a clean platinum 
needle is inserted in the tube and quite a large number of colonies are 
swept with it from the surface of the culture medium, a part being 
selected where small colonies only are found. A sufficient amount of 
the bacteria adherent to the needle is washed off in the drop of water 
previously placed on the cover-glass and smeared over its surface. 
The bacteria on the glass are then allowed to dry in the air. The 
cover-glass is then passed quickly through the flame of a Bunsen 
burner or alcohol lamp, three times in the usual way, covered with a 
few drops of Loeffler's solution of alkaline methylene blue, and left 
without heating for five to ten minutes. It is then rinsed off in clear 
water, dried, and mounted in balsam. When other methods of stain- 
ing are desired they are carried out in the proper way. 

In the great majority of cases one of two pictures will be seen with 
the y^ oil-immersion lens — either an enormous number of character- 
istic Loeffler bacilli, with a moderate number of cocci, or a pure cul- 



THE BACILLUS OF DIPHTHERIA. 229 

ture of cocci, mostly in pairs or short chains. (See Streptococcus.) 
In a few cases there will be an approximately even mixture of LoeflBer 
bacilli and cocci, and in others a great excess of cocci. Besides these, 
there will be occasionally met preparations in which, with the cocci, 
there are mingled bacilli more or less resembling the LoeflBer bacilli. 
These bacilli, which are usually of the pseudodiphtheria type of 
bacilli (see Fig. 86), are especially frequent in cultures from the nose. 

In not more than one case in twenty will there be any serious diflS- 
culty in making the diagnosis, if the serum in the tube was moist and 
had been properly inoculated. In such a case another culture must 
be made or the bacilli plated out and tested in pure culture. 

Direct Microscopic Examination of the Exudate. — An immediate 
diagnosis without the use of cultures is often possible from a micro- 
scopic examination of the exudate. This is made by smearing a 
slide or cover-glass with a little of the exudate from the swab, drying, 
heating, staining, and examining it microscopically. This examina- 
tion, however, is much more diflScult, and the results are more uncer- 
tain than when the covers are prepared from cultures. The bacilli 
from the membrane are usually less jtypical in appearance than those 
found in cultures, and they are mixed with fibrin, pus, and epithelial 
cells. They may also be very few in number in the parts reached by 
the swab, or bacilli may be met with which closely resemble the 
LoeflBer bacilli in appearance, but which diflFer greatly in growth and 
in other characteristics, and have absolutely no connection with them. 
^Vhen in a smear containing mostly cocci a few of these doubtful bacilli 
are present, it is impossible either to exclude or to make the diagnosis 
of diphtheria with certainty. Although in some cases this immediate 
examination may be of the greatest value, it is not a method suitable 
for general use, and should always be controlled by cultures. 

WTien carried out in the best manner an experienced bacteriologist 
may obtain remarkably accurate results. Higley in New York in a 
series of consecutive throat cases made the same diagnosis from the 
direct examination of smears as the Health Department laboratory 
made from the culture. To get the exudate he used a probe armed 
with a loop of heavy copper wire which has been so flattened as to 
act as a blunt curette. He makes thus thin smears from the exudate. 
After drying and fixing by heat the smears are stained for five seconds 
in a solution made by adding five drops of Kiihne's carbolic methylene 
blue to 7 c.c. of tap water. After washing and drying stain for one 
minute in a solution of 10 drops of carbol-fuchsin in 7 c.c. of water. 
The dilute solution should be freshly prepared. The diphtheria 
bacilli will appear as dark-red or violet rods, and their contour, mode 
of division, and arrangement are manifest. 

Animal Inoculation cw a Test of Virulence. — If the determination 
of the virulence of the bacilli found is of importance, animal inocu- 
lations must be made. Experiments on animals form the only method 
of determining with certainty the virulence of the diphtheria bacillus. 
For this purpose, alkaline broth cultures of forty-eight hours' growth 



230 PATHOGENIC MICRO-ORGANISMS. 

should be used for the subcutaneous inoculation of guinea-pigs. 
The amount injected should not be more than one-fifth per cent, 
of the body-weight of the animal inoculated, unless controls with 
antitoxin are made. In the large majority of cases, when the bacilli 
are virulent, this amount causes death within seventy-two hours. If 
a good growth is not obtained in nutrient bouillon, ascitic broth 
should be used. At the autopsy the characteristic lesions already 
described are found. Bacilli which in cultures and in animal ex- 
periments have shown themselves to be characteristic may be regarded 
as true diphtheria bacilli, and as capable of producing diphtheria in 
man under favorable conditions. 

For an absolute test of Sftecific virulence antitoxin must be used. 
A guinea-pig is injected with antito.\in, and then this and a control 
animal, with 2 c.c. of a broth culture of the bacilli to be tested; if 



the guinea-pig which received the antitoxin hves, while the control 
dies, it was surely a diphtheria bacillus which killed by means of 
diphtheria toxin — or, in other words, not simply a virulent bacillus, 
but a virulent diphtheria bacillus. When the bacilli to be tested 
grow pooriy in a simple nutrient bouillon they should be grown in 
bouillon to which one-third its quantity of ascitic fluid has been added. 
Quite a number of bacilli have been met with which killed 250-grm. 
guinea-pigs in doses of 2 to 15 c.c, and yet were unaffected by anti- 
* — '— ''^' bacilli, though slightly virulent to guinea-pigs, pro- 
pria toxin, and so cannot, to the best of our belief, 
ria in man (see p. 206). 

gina. — The local symptoms are similar to a slight 

a. Exudate or pseu do membrane forms on the tonsils 

ecome necrotic leaving a superficial ulcer, which is 

The general disturbance, outside a little fever, is 



THE BACILLUS OF DIPHTHERIA, 231 

usually slight. The disease runs its course in from one to two weeks. 
It has been frequently noticed that the disease begins with an erup- 
tion of vesicles as in aphthous stomatitis. Paralysis never follows 
from this infection. The bacilli found by Vincent in the lesions are 
6/£ to 12/£ long by . 6/£ to . 8// broad. Their ends are tapering. They 
are frequently bent like the letter S and resemble spirillte. 

The bacilli stain with methyl blue irregularly so that light and 
dark bands alternate (see Fig. 87). 

Stained by the method of Romanowsky there appear sharply 
defined chromatin bodies in the blue stained protoplasm. 

The bacilli are not motile. 

These spindle-shaped bacilli have not been grown in pure culture, 
indeed there is doubt as to their nature, some considering them as 
being spirochaetes. When direct smears are made from the exudate 
tiny spirochaetes are usually found mixed with the bacilli. 

Certain necrotic conditions of the mucous membrane of the cheek 
and about the teeth are accompanied by microorganisms very similar 
to those described by Vincent. 



CHAPTER XVIII. 
THE BACILLUS AND THE BACTERIOLOGY OF TETANUS. 

Tetanus is a disease which is characterized by a gradual onset of 
general spasm of the voluntary muscles, commencing in man most 
often in those of the jaw and neck, and extending in severe cases to 
all the muscles of the body. The disease is usually associated with 
a wound received from four to fourteen days previously. 

In 1884 Nicolaier, under Fliigge's direction, produced tetanus in 
mice and rabbits by the subcutaneous inoculation of particles of gar- 
den earth. The Italians, Carle and Rattone, had just before demon- 
strated that the pus of an infected wound from a person attacked with 
tetanus could produce the same disease in rabbits, and showed that 
the disease was transmissible by inoculation from these animals to 
others. Finally, Kitasato, in 1889, obtained the bacillus of tetanus 
in pure culture and described his method of obtaining it and its bio- 
logical characters. 

Occurrence in Soil, etc. — The tetanus bacillus occurs in nature as 
a common inhabitant of the soil, at least in places where manure has 
been thrown, being abundant in many localities, not only in the super- 
ficial layers, but also at the depth of several feet. It has been found 
in many different substances and places — ^in hay-dust, in horse and 
cow manure (its normal habitat is the intestine of the herbivora), in 
the mortar of old masonry, in the dust from horses' hair; in the 
dust in rooms of houses, barracks, and hospitals; in the air, and in 
the arrow poison of certain savages in the New Hebrides, who obtained 
it by smearing the arrow-heads with dirt from crab holes in the swamps. 

The tetanus bacilli are apparently more numerous in certain locali- 
ties than in others — for example, some parts of Long Island and New 
Jersey have become notorious for the number of cases of tetanus caused 
by small wounds — but they are very generally distributed, as the experi- 
ments on animals inoculated with garden earth have shown, and are 
fairly common in New York City. In some islands and countries in 
the tropics cases of puerperal tetanus and tetanus in the newborn are 
very frequent. Tetanus bacilli are found in the intestines of about 
15 per cent, of horses and calves living in the vicinity of New York 
(^ity. They are also present to a somewhat less extent in the intestines 
of other animals and of man. 

Morphology. — From young gelatin cultures the bacilli appear as 
motile, slender rods, with rounded ends, 0.5/i to 0.8/i in diameter by 
2// to 4/( in length, usually occurring singly, but, especially in old 
cultures, often growing in long threads. They form round spores, 
thicker than the cell (from l/« to l.o/i in diameter), occupying one of 

232 



THE BACILLUS OF TETANUS. 233 

its extremities and giving to the rods the appearance of small pins 
(Fig. 88). 

Staining. ^It is stained with the onlinar^- aniline dyes, and is not 
decolorized by Gram's method^ The spores are readily stained and 
may be demonstrated by double-staining with Ziehl's method. The 
Sagella are fairly easily stmned in very young cultures. 

Biology. — An anaerobic, liquefying, moderately motile bacillus. 
It has abundant peritrichic flagella. Forms spores, and in the spore 
stage it is not motile. It grows slowly at temperatures from 20° to 
24° C, and best at 38° C, when, within twenty-four hours, it forms 
spores. It will not in pure culture grow in the presence of oxygen, 
but grows well in an atmosphere of hydrogen gas. With certain other 
bacteria the tetanus bacillus grows luxuriantly in the presence of 
oxygen. 

Orowth in Hodia. — The bacillus of tetanus grows in ordinary nutrient gela- 
tin and agar of a slightly alkaline reaction. The addition to the media of 
1.5 per cent, of glucose causes the development to be more rapid and abun- 
dant. It also grows abundantly in alkaline _ _ 
bouillon in an atmosphere of hydrogen. On 
gelatin plates the colonies develop slowly; 
they resemble siomewhat the colonies of the 
Bacillus »ublili», and have a dense, opaque 
centre surrounded by fine, diverging rays. 
Liquefaction takes place more slowly, how- 
ever, than with Bacillus subtiiis, and the re- 
»embtance to these colonies is soon lost. 

The colonies on agar are quite character- 
istic. To the naked eye they present the 
appearance of light, fleecy clouds; under the 
microscope, a tangle of fine threads. 

The slab cultures in gelatin exhibit the 
appearance of a cloudy, linear mass, with 
prolongations radiating into the gelatin from 
alt sides (arborescent growth). Liquefaction 
takes place slowly, generally with the production of gas. In slab cultures in 
agar a growth occurs not unlike in structure that of a miniature pine-tree. 
Alkaline bouillon is rendered somewhat turbid by the growth of the tetanus 
bacillus. In all cases a production of gas results, accompanied by a character- 
istic and very disagreeable odor. It develops in milk without coagulating it. 

Kesistance of Spores to Deleterious Influences.— The spore.s of the 
tetanus bacillus are very resistant to outside influences; in a desic- 
cated condition they may retain their vitahty for several years, and 
are not destroyed in two and a half months when present in putrefy- 
ing material. They withstand an exposure of one hour to 80° C, 
but are killed by an exposure of ten minutes at 105° C. to live steam. 
They resist the action of 5 per cent, carbolic acid for ten hours. A 
a per cent, solution of carbolic acid, however, to which 0.5 per cent, 
of hydrochloric acid has been added, destroys them in two hours. 
They are killed when acted upon for three hours by bichloriile of 
mercury (1:1000), and in thirty minutes when 0.5 per cent. HCl is 
atlded to the solution. Silver nitrate solutions destroy the spores of 



Tetutu bsoiUi with gpons ii 



234 PATHOGENIC MICRO-ORGANISMS. 

average resistance in one minute in 1 per cent, solution and in about 
five minutes in 1 : 1000 solution. 

With regard to the persistence of tetanus spores upon objects where 
they have found a resting place, Henrijean reports that by means 
of a splinter of wood which had once caused tetanus he was able 
after eleven years again to cause the disease by inoculating an animal 
with the infective material. 

Isolation of Pure Ctdtures.— The growth of the tetanus bacillus in 
the animal body is comparatively scanty, and is usually associated 
with that of other bacteria; hence, the organism is diflScult to obtain 
in pure culture. The method of procedure proposed by Kitasato, 
which, however, is not always successful, consists in inoculating 
slightly alkaline nutrient agar or glucose bouillon with the tetanus- 
bearing material (pus or tissue from the inoculation wound), keep- 
ing the culture under anaerobic conditions for twenty-four to forty- 
eight hours at a temperature of 37® C, and, after the tetanus spores 
have formed, heating it for one-half an hour at 80*^ C, to destroy the 
associated bacteria. The spores of the tetanus bacillus are able to 
survive this exposure, so that when anaerobic cultures are then made 
in the usual way the tetanus colonies develop. When the tetanus 
bacilli are the only spore-bearing bacteria present, pure cultures are 
readily obtained; when other spore-bearing anaerobic bacteria are 
present, the isolation of a pure culture may be a matter of difficulty, 
but even then the presence of tetanus toxin in the culture fluid will 
indicate the presence of tetanus bacilli. The tetanus cultures can be 
kept for years. 

Pathogenesis. — In mice, guinea-pigs, rabbits, horses, goats, and a 
number of other animals inoculations of pure cultures of the tetanus 
bacillus cause typical tetanus after an incubation of from one to three 
days. A mere trace of an old culture — only as much as remains 
clinging to a platinum needle — is often sufficient to kill very suscep- 
tible animals like mice and guinea-pigs. Other animals require a 
larger amount. Rats and birds are but little susceptible, and fowls 
scarcely at all. Man is more susceptible than any of the animals so 
far tested. A horse is about six times as sensitive as a guinea-pig 
and three hundred thousand times as sensitive as a hen. It is a 
remarkable fact that an amount of toxin sufficient to kill a hen would 
suffice to kill 500 horses. It is estimated that if 1 gram of horse 
requires 1 part of toxin to kill, then 1 gram of guinea-pig requires 
6 parts, 1 of mouse 12, of goat 24, of dog 500, of rabbit 1500, of 
cat 6000, of hen 360,000. Cultures from different cases vary greatly 
in their toxicity. On the inoculation of less than a fatal dose in test 
animals a local tetanus may be produced, which lasts for days and 
weeks and then ends in recovery. On killing the animal there is 
found at autopsy, just at the point of inoculation, a hemorrhagic spot, 
and no changes other than these here or in the internal organs. A 
few tetanus bacilli may be detected locally with great difficulty, often 
none at all; possibly a few may be found in the region of the neigh- 



THE BACILLUS OF TETANUS. 235 

boring lymphatic glands. From this scanty occurrence of bacilli the 
conclusion has been reached that the bacilli of tetanus, when inocu- 
lated in pure culture, do not multiply to any great extent in the living 
body, but only produce lesions through the absorption of the poison 
which they^develop at the point of infection. It has been found that 
pure cultures of tetanus, after the germs have sporulated and the tox- 
ins been destroyed by heat, can be injected into animals without pro- 
ducing tetanus. But if a culture of non-pathogenic organisms is 
injected simultaneously with the spores, or if there is an effusion of 
blood at the point of injection, or if there was a previous bruising 
of the tissues, the animals surely die of tetanus. Even irritating 
foreign bodies have been introduced along with the spores deprived 
of their toxins, and tetanus did not develop; but if the wounds con- 
taining the foreign bodies became infected with other bacteria, tetanus 
developed and the animal died. From such experiments it seems 
that a mixed infection aids greatly in the development of tetanus 
when the infection is produced by spores not accompanied by tetanus 
toxin. 

Natoral Infection. — Here the infection may be considered as prob- 
ably produced by the bacilli in their spore state, and the conditions 
favoring infection are almost always present. A wound of some kind 
has occurred, penetrating at least through the skin, though perhaps 
of a most trivial character, such as might be caused by a dirty splinter 
of wood, and the bacilli or their spores are thus introduced from the 
soil in which they are so widely distributed. If in any given case, 
the tissues being healthy, the ordinary saprophytic germs are killed 
by proper disinfection at once, a mixed infection does not take place, 
and tetanus will not develop. If, however, the tissues infected be 
badly bruised or lacerated, the spores may develop and produce 
the disease. Gelatin is occasionally found to contain tetanus spores. 

Tetanus in Man. — Man and almost all domestic animals are sub- 
ject to tetanus. It is a comparatively rare disease except after the 
Fourth of July celebration, when throughout the United States a 
considerable number of cases develop. In some years more than one 
hundred persons develop tetanus after blank cartridge wounds. On 
examination of an infected individual very little local evidence of 
the disease can be discovered. Generally at the point of infection, 
if there is an external wound, some pus is to be seen, in which, along 
with numerous other bacteria, tetanus bacilli or their spores may be 
found. Although rather deep wounds are usually the seat of infec- 
tion, at times such superficial wounds as an acne pustule or a vaccina- 
tion may give the occasion for infection. Not only undoubted trau- 
matic tetanus, but also all the other forms of tetanus, are now conceded 
to be produced by the tetanus bacillus — puerperal tetanus, tetanus 
neonatorum, and idiopathic tetanus. In tetanus neonatorum in- 
fection is introduced through the navel, in puerperal tetanus through 
the inner surface of the uterus. It should be borne in mind that 
when there is no external and visible wound there may be an internal 



236 PATHOGENIC MICRO-ORGANISMS, 

one. The lesions in the nervous system are still obscure. Congestion, 
cellular exudate into the perivascular spaces, and chromatolysis of the 
ganglion cells are common. This is a pure toxflemic disease. 

Toxins of the Tetanus Bacillus. — It is evident from the localization 
of the tetanus bacilli at the point of inoculation and their slight mul- 
tiplication at this point that they exert their action through the pro- 
duction of powerful toxins. These toxins are named, according to 
their action, the tetanospasmin and the tetanolysin. One one-thou- 
sandths of a cubic centimeter of the filtrate of an eight-day glucose 
bouillon culture of a fully virulent bacillus is sufficient to kill a mouse. 
The purified and dried tetanus toxin prepared by Brieger and Cohn 
was surely fatal to a 15-gram mouse in a dose of 0.000005 gram. 
The appalling strength of tetanus toxin may readily be appreciated 
when it is stated that it is twenty times as poisonous as dried cobra 
venom. 

The quantity of the toxin produced in nutrient media varies accord- 
ing to the age of the culture, the composition of the culture fluid, 
reaction, completeness of the exclusion of oxygen, etc. For some 
reason more toxin develops in broth inoculated with masses of 
tetanus spores than with bacilli. The variation in strength is partly 
due to the extreme sensitiveness of the toxin, which deteriorates on 
keeping or on exposure to light, being also sensibly affected by most 
chemical reagents and destroyed by heating to 55° to 60° C. for any 
length of time. It retains its strength best when protected from heat, 
light, oxygen, and moisture. Under the best conditions the amount 
of toxin produced in cultures by the fifth day is such that 0.000005 c.c. 
is the fatal dose for a 15-gram mouse. 

The tetanus cultures retain their ability to produce toxins unaltered 
when kept under suitable conditions; but when subjected to deleterious 
influences they may entirely lose it. The usual medium for the 
development of the toxin is a slightly alkaline bouillon containing 1 
per cent, of peptone and 0.5 per cent. salt. In addition 1 per cent, 
of dextrose is sometimes added but is not advised. 

Action of Tetanus Toxin in the Body. — After the absorption of the 
poison there is a lapse of time before any effects are noticed. With 
an enormous amount, such as 30,000 fatal doses, this is about twelve 
hours; with ten fatal doses, thirty-six to forty-eight hours; ^ith two 
fatal doses, two to three days. Less than a fatal dose will produce 
local symptoms. The parts first to be affected with tetanus are, in 
about one-third of the cases in man, and usually in animals, the muscles 
lying in the vicinity of the inoculation — for instance, the hind foot of a 
mouse inoculated on that leg is first affected, then the tail, the other 
foot, the back and chest muscles on both sides, and the forelegs, until 
finally there is a general tetanus of the entire body. In mild cases, 
or when a dose too small to be fatal has been received, the tetanic 
spasm may remain confined to the muscles adjacent to the point of 
inoculation or infection. The symptoms following a fatal dose of toxin 
vary greatly with the method of injection. Intraperitoneal injection is 



THE BACILLUS OF TETANUS. 237 

followed by symptoms which can hardly be distinguished from those 
due to many other poisons. Injection into the brain is followed by 
restlessness and epileptiform convulsions. The tetanus toxins un- 
doubtedly combine readily with the cells of the central nervous system. 
They also combine with other tissue cells with less apparent effects. 
The symptoms in tetanus depend upon an increased reflex excitability 
of the motor cells of the spinal cord, the medulla, and pons. 

Presence of* Tetanus Toxin in the Blood of Infected Animals.— 

The blood usually contains the poison, as has been proved experi- 
mentallv on animals. Neisser showed that the blood of a tetanic 
patient was capable of inducing tetanus in animals when injected 
subcutaneously. In St. Louis the serum of a horse dying of tetanus 
was given by accident in doses of 5 to 10 c.c. to a number of children, 
with the development of fatal tetanus. In this connection Bolton 
and Fisch showed by a series of experiments that much toxin might 
accumulate in the serum before symptoms became marked. Ehrlich 
has shown that besides the predominant poison which gives rise to 
spasm (tetanospasmin) there exists a poison capable of producing 
solution of red blood corpuscles. This he calls tetanolysin. It was 
not found in all culture fluids. Whether in actual disease this poison 
is ever in suflScient amount to cause appreciable harm is not known. 
After one or two weeks the blood becomes antitoxic even though the 
symptoms persist. 

Tetanus Antitoxin. — Behring and Kitasato were the first to show 
the possibiKty of immunizing animals against tetanus infection. The 
treatment of tetanus is directed against the action of the toxin and 
this is accomplished by the neutralization of the toxin by antitoxin in 
the body. 

The immunizing experiments in tetanus have borne practical fruit, 
for it was through them that the principle of serum therapeutics first 
became known — the protective and curative effects of the blood serum 
of immunized animals. It was found that animals could be pro- 
tected from tetanus infection by the previous or simultaneous injection 
of tetanus antitoxin, provided that such antitoxic serum was obtained 
from a thoroughly immunized animal. From this it was assumed 
that the same result could be produced in natural tetanus in man. 
Unfortunately, however, the conditions in the natural disease are 
very much less favorable, inasmuch as treatment is usually commenced 
not shortly after the infection has taken place, but only many hours 
after the appearance of tetanic symptoms, when the poison has 
already attacked the cells of the central nervous system. 

The tetanus antitoxin is developed in the same manner as the diph- 
theria antitoxin — by inoculating the tetanus toxin in increasing doses 
into horses. Th e toxin is produc ed in boullloulcu.lt ures .grown, an- f 
aerobically for six jo ^en da \s. Abundant spores should be used to 
inoculate the broth, rhe culture fluid is filtprpfl thr^'?^h porrHp^", 
and the germ^fcee-^filtliateji^ us ed f or the ino culations. The horses re- 
ceive 5 c.c. as the initial dose of a toxin of which 1 c.c. kills 250,000 



238 PATHOGENIC MICRO-ORGANISMS. 

grams of guinea-pig, and along w ith this twice the amount of anj i- 
toxin req uired to neutralize it. In'Tivej Iays this dose is don hied ^ an d 
then every five to seven days, larger amounts ju:e giveii* A|ter the 
tllirdjnjfiition-the Antitoxin h omitterj The dose is increased atTTrsT 
slowly until appreciable amounts of antitoxin are found to be present 
and then as rapidly as the horses can stand it, until they support 700 
to 800 c.c. or more at a time. This amount should not be injected in a 
single place, or severe local and perhaps fatal local tetanus may de- 
velop. After gj^me m^nt^is of this treatment the blood of the horse 
cont ains th e anjito xin in s uMcient__amount for thfi xapeutic use. Some 
horses have produced as high as 600 unlts'per c.c. 

Antitoxin Unit and Technique of Testing Antitoxin Serum. — 

Tetanus antitoxin is tested exactly in the same manner as diphtheria 
antitoxin, except that the unit is different. In April, 1907, the pro- 
ducers of serum in the United States agreed to a unit of antitoxin 
which is approximately ten times the size of the unit of diphtheria anti- 
toxin. A unit is defined as the amount of antitoxin required to just 
neutralize 1000 fatal doses of tetanus toxin for a 350-gram guinea-pig. 
If the test guinea-pig, receiving the mixture of antitoxin, and 1000 
times the amount, is protected from death for four days, neutrali- 
zation is considered to have taken place. The United States govern- 
ment has adopted this unit and supplies the different producers with 
standardized toxin. 

The amount of antitoxic serum which neutralizes an amount of test toxin 
which would destroy 40,000,000 grams of mouse contains 1 unit of antitoxin 
by the German standard. In the French method the amount of antitoxin 
which is required to protect a mouse from a dose of toxin sufficient to kill in 
four days is determined, and the strength of the antitoxin is stated by deter- 
mining the amount of serum required to protect 1 gram of animal. If 0.001 
c.c. protected a 10-gram mouse the strength of that serum would be 1 : 10,000. 
The toxin used for testing is preserved by precipitating it with saturated 
ammonium sulphate and drying and preserving the precipitate in sealed 
tubes. As required, it is dissolved in 10 per cent, salt solution as above 
stated. For small testing stations the best way is to obtain some freshly 
standardized antitoxin and compare serums with this. 

Persistence of Antitoxin in the Blood. — Ransom has clearly shown 
that the tetanus antitoxin, whether directly injected or whether pro- 
duced in the body, is eliminated equally rapidly from the blood of an 
animal, provided that the serum was from an animal of the same species. 
If from a different species it is much more quickly eliminated. From 
this we see a probable explanation of the fact that immunity in man, 
due to an injection of the antitoxic serum of the horse, is less persistent 
than immunitv conferred bv an attack of the disease. 

The same author found some interesting facts in testing the anti- 
toxic values of the serum of an immunized mare, of its foal, and of 
the milk. The foal's serum was one-third the strength of the mare's 
and one hundred and fiftv times that of the mare's milk. In two 
months the mare's serum lost two-thirds in antitoxic strength, the 



THE BACILLUS OF TETANUS, 239 

foal's five-sixth, and the milk one-half. Injections of toxin were 
then given the mare, so that it doubled its original strength in one 
month. The milk increased eightfold, but the foal's continued to 
lose in antitoxin, although it was feeding on antitoxic milk. Under 
diphtheria it was noted that homologous antitoxin remained much 
longer than heterologous. 

Toxin and Antitoxin in the Living Organism. Animal Experi- 
ments. — The experiments of Meyer and Ransom and of Marie and 
Morax have proved to them that the poison is transported to the cen- 
tral nervous system by the way of the motor nerves — and by no other 
channel. These authors thought that they had shown that the essen- 
tial element for the absorption and transportation of the toxin is not 
the nerve sheath or the lymph channels, but the axis-cylinder, the 
intramuscular endings of which the toxin penetrates. The poison 
is taken up quite rapidly. Marie and Morax were able to demonstrate 
the poison in the corresponding nerve trunk (sciatic) one and a half 
hours after the injection. Absorption, however, and conduction 
are dependent to a large extent on the nerves being intact. A nerve 
cut across takes very much longer to take up the poison (about twenty- 
four hours), and a degenerated nerve takes up no poison whatever. 
In other words, we see that section of the nerve prevents the absorption 
of the poison by way of the nerve channels. Similarly section of the 
spinal cord prevents the poison from ascending to the brain. 

According to Meyer and Ransom, the reason why the sensory nerves 
do not play any r6le in the conduction of the poison lies in the pres- 
ence of the spinal ganglion, which places a bar to the advance of the 
poison. Injections of toxin into the posterior root leads to a tetanus 
dolorosus, which is characterized by strictly localized sensitiveness to 
pain. 

Ascending centripetally along the motor paths, the poison reaches 
the motor spinal ganglia on the side of inoculation ; then it affects the 
ganglia of the opposite side, making them hypersensitive. The vis- 
ible result of this is the highly increased muscle tonus — i, e., rigidity. 
If the supply continues, the toxin next affects the nearest sensory 
apparatus; there is an increase in the reflexes, but only when the af- 
fected portion is irritated. In the further course of the poisoning the 
toxin as it ascends. continues to affect more and more motor centres, 
and also the neighboring sensory apparatus. This leads to spasm of 
all the striated muscles and general reflex tetanus. 

A different explanation of the passage of the toxin up the nerve 
trunks has recently been discovered. It is well known that the lymph 1 
flow i^n ni^rves is from the periphery to the center, and Field in our ' 
laboratory has shown that not only tetanus toxin, but diphtheria toxin • 
and inert colloids can be demonstrated in the sciatic nerves after they 
have been injected subcutaneously or intramuscularly, and after vary- 
ing periods may be found in the spinal cord. He believes that the toxins 
are absorbed by way of the lymphatics of the nerves, and not by way of 
the axis-cvlinder. 



240 PATHOGENIC MICRO-ORGANISMS, 

A recent experiment of Cemovodeanu and Henni almost proves 
this contention. They ligated all the muscles and blood vessels in 
a guinea-pig's leg, leaving intact only the sciatic nerve, skin and bone, 
and then injected a large amount of tetanus toxin below the point of 
ligation. The animals in which this was done never developed 
tetanus. 

In this case there was only a very slight flow of lymph into the ligated 
area, and so there could be only a slight flow up the nerve. 

If the toxin gets into the blood the only path of absorption to the 
central nervous system is still by way of the motor-nerve tracts. There 
I seems to be no other direct path, as, for example, by means of the 
i blood vessels supplying the central nervous system. Even after intro- 
i ducing the poison into the subarachnoid space, owing to the passage 
, of the poison into the blood, there is a general poisoning and not a 
cerebral tetanus. This at least is the case if care has been taken dur- 
ing the operation to avoid injuring the brain mechanically. 

Rapidity of Absorption of Tetanus Antitoxin from Tissues. — 

The complete absorption of a given quantity of antitoxin adminis- 
tered subcutaneously takes place rather slowly. In his animal ex- 
periments Knorr found the maximum quantity in the blood only 
after twenty-four hours. From that time on the amount again steadily 
decreased, so that by the sixth day only one-third the optimum quantity 
was present. By the twelfth day only one-fiftieth and at the end of 
three weeks no antitoxin whatever could be demonstrate<l. These 
facts emphasize the necessity of giving the first dose in a case of 
tetanus intravenously. 

Naturally the time during which these changes take place varies 
with the application, the conditions of absorption, and the concentra- 
tion and amount of the preparation injected. When injected intra- 
venously the antitoxin very quickly passes into the lymph. Ransom 
was able to demonstrate it in the thoracic duct of a dog a few minutes 
after intravenous injection. Neither the central nervous system nor 
the peripheral nervous tissti£ take up any antitoxin from the blood. Only 
after very massive intravenous doses are small traces found in the 
cerebrospinal fluid. From this it is at once clear that passively and 
actively immunized animals become tetanic if the poison is injected 
directly into the central nervous system or into a peripheral nerve. 
Antitoxin injected subdurally also passes almost entirely over into 
the blood. 

A rapid and plentiful appearance of antitoxin in the blood is de- 
pendent on the content of serum in antitoxin units. The more units, 
the more rapidly will the blood develop a high content of antitoxin; 
and the higher this is the more thoroughly will the tissue fluids be 
saturated with the antitoxin. 

From the foregoing it is not difficult to formulate the conditions 
under which an antitoxin introduced into the organism can exert its 
neutralizing power on the toxin. We see that the poison deposited 
at any given place takes either of two paths to the central nervous 



THE BACILLUS OF TETANUS, 241 

system, one a direct path by way of the local peripheral nerves and 
the other an indirect path through the lymph channels and blood to 
the end plates of all other motor nerves. Only that portion can be 
neutralized which (a) still lies unabsorbed at the site of inoculation, 
or (b) which, though it has paSwSed into the blood, has not yet been 
taken up by the motor-nerve endings. A curative effect can therefore 
result from antitoxin introduced subcutaneously or intravenously only 
so long as a fatal dose of poison has not been taken up by the nerves. 

So long as the toxin circulates in the blood it is neutralized by anti- 
toxin in about the same proportion as in test-tube experiments. By 
means of intravenous injections of antitoxin Ransom was able to 
render the blood free from toxin in a very few minutes. According 
to Marie and Morax, toxin injected into the muscles is already demon- 
strable in the nerve tissue at the end of one and a half hours — i. e,, 
it has already entered the channel, where it is no longer reached by 
the antitoxin. Donitz injected various rabbits intravenously ea^h 
with 1 c.c. of a toxin solution containing twelve fatal doses. There- 
upon he determined the dose of antitoxin which, when intravenously 
given, would neutralize this poison after various intervals of time. 
The antitoxin was of such a strength that in test-tube experiments 
1 c.c. of a 1 : 2000 solution just neutralized the amount of toxin em- 
ployed. He found that at the end of two minutes double the dose 
required in vitro would still neutralize the poison; at the end of 
four minutes about four times the dose was required, and at the 
end of eight minutes ten times. When one hour had been allowed 
to elapse forty times the original dose just suflSced to protect the 
animal from death, but not from sickness. In order to explain these 
results, the correctness of which has been confirmed by many anal- 
ogous observations, the conception ** loose union of toxin" has been 
introduced. By this is meant a state of union between toxin and 
susceptible cell constituent which can still be disrupted by means of 
large doses of antitoxin. In this particular instance we do not need 
to make use of this conception, for the reason that the tetanus toxin is 
not at all combined during the first hour. Personally, we should 
regard it as more probable that the interval during which the toxin 
can still be neutralized, though with difficulty, corresponds to that 
time during the passage of the toxin in which after leaving the 
capillaries the poison is held up in the fine interstices of the con- 
nective tissue which it must penetrate before it can be taken up by 
the nerves. 

Kesults of the Antitoxin Treatment in Tetanus. — ^The course of 

tetanus varies so much with the individual that it is difficult to judge 
by statistics or personal experience as to the value of the antitoxic 
treatment of the developed form. It is interesting to note that the 
two latest authoritative reviews by American writers differ greatly 
in their conclusions. McFarland, who has had an extensive experi- 
ence, states, "It would seem, therefore, that we have in tetanus anti- 
toxin not a specific, because it fails too often to have merited that 
i6 



242 PATHOGENIC MICRO-ORGANISMS, 

name, but a valuable remedy in the treatment of the disease, and one 
that ought not to be neglected until a better one is supplied." Our 
own opinion, founded on reading and a considerable personal experi- 
ence is even more favorable. We have seen numerous cases of gener- 
alized tetanus that after a large intravenous injection have markedly 
improved and finally recovered, and these cases have certainly done 
better on the average than apparently similar ones receiving palliative 
treatment alone. Lambert, who some years ago made an exhaustive 
study of tetanus, states that in a total of 114 cases of this disease 
treated with antitoxin, according to published and unpublished reports, 
there was a mortality of 40.35 per cent. Of these, 47 were acute 
cases — ^that is, cases with an incubation period of eight days or less and 
with rapid onset, or cases with a longer period of incubation, but in- 
tensely rapid onset of symptoms; of these the mortality was 74.46 per 
cent. Of the chronic type — those with an incubation period of nine 
days or more, or those with shorter incubation with slow onset — there 
were 61 cases, with a mortality of 16.39 per cent. With a still larger 
number of cases the results indicate that with tetanus antitoxin about 
20 per cent, better results are obtained than without. In our own 
diphtheria antitoxin horses we used to lose several almost every year 
from tetanus infection until we immunized all the animals every three 
months with about 5000 units of tetanus antitoxin. 

Methods of Admimstering Tetanus Antitoxin. — For immunization, 
about 1500 units of a serum of medium strength will suffice, unless 
the danger seems great, when the injection is repeated at the end of 
a week. For treatment, begin with an intravenous injection of ^0,000 
units, and repeat every eight to twelve hours until the symptoms 
abate. It is well to continue decreasing daily injections until recovery 
is certain. In some of the gravest cases no curative effect will be 
noticed from the serum. The first injections should be made intra- 
venously, or partly intravenously and partly into the spinal canal 
through lumbar puncture. Later, injections should be made subcu- 
taneously or intravenously. Besides these, injections are advised by 
some to be made into all the nerve trunks leading from the infected 
region. These injections are directed to be made as near the trunk 
as possible and distend the nerve so as partly to neutralize and partly 
mechanically interrupt the passage of toxin to the cord or brain. In 
New York City Rogers believes he has had good results by following 
these methods. The method of injecting from 3 to 15 c.c. of antitoxic 
serum into the lateral ventricles has not, in the writer's opinion, shown 
itself to be advisable. No bad results have followed the injections 
when the serum was sterile and the operation was performed asep- 
tically; but several brain abscesses have already followed the intra- 
cerebral injections. 

The striking results which have been obtained, both in human and 
in veterinary practice, with the prophylactic injection of tetanus anti- 
toxin, would seem to warrant the treating of patients with immunizing 
doses of serum — at least, in neighborhoods where tetanus is not 



THE BACILLUS OF TETANUS. 243 

uncommon — when the lacerated and dirty condition of their wounds 
may indicate the possibility of a tetanus infection. 

Splendid results have followed this practice in many places. It is 
the custom at many dispensaries in New York City and elsewhere to 
immunize all Fourth of July wounds by injecting 1000 units. None 
of these have ever developed tetanus. Even the eleven cases of human 
tetanus reported as occurring in Europe after single injections of anti- 
toxin prove the value of immunizing injections, for the mortality was 
only 27 per cent. They teach also that where tetanus infection is sus- 
pected the antitoxic serum should be given a second and even a third 
time at intervals of seven days. 

In cooperation with Dr. Cyrus W. Field, we have recently tried a 
number of experiments upon guinea-pigs to test the importance of 
intravenous and of intraneural injections of antitoxin in animals in 
which tetanus had already developed. Forty guinea-pigs have been 
experimented upon. These were injected in the lower part of the 
hind leg with ten to twenty times the fatal dose of a mixture of tetanus 
toxin and bacilli. Within from one to two hours after the development 
of the first definite symptoms of tetanus the animals were operated 
upon and given antitoxin. The experiments show clearly that moder- 
ate doses of antitoxin given after the development of tetanus did not 
save the animals from death or even prolong life, while very large doses 
usually did both. Seventy-five per cent, of those receiving 500 units 
recovered. The surprising result developed that amputation of the 
infected leg at the hip-joint hastened the death of the animals in every 
case. • Control animals which had not been infected ^tood the ampu- 
tation perfectly well, and made good recoveries. Without antitoxin, 
excision of a piece of the nerve did not materially prolong life, nor did 
ligation of the nerve. In the guinea-pigs receiving antitoxin the 
ligation of the nerve seemed to be of benefit. The results of the ex- 
periments showed that large doses of antitoxin given shortly after the 
development of tetanus usually saved the animals, and that most of the 
toxin was absorbed by the blood and not by the nerves of the infected 
part. Every minute of delay after the appearance of tetanus was of 
importance. We feel convinced that in human tetanus the most impor- 
tant thing is to give at the earliest possible moment after diagnosis a 
very large intravenous injection of antitoxin. From 50 to 75 c.c. 
of the most potent serum obtainable should be given. During succeed- 
ing days injections can be given either intravenously or subcutaneously 
until marked improvement or death has taken place. If a surgeon is 
at hand intraneural injections into the nerves supplying the infected 
portion of the body may also be given, but these, we believe, are not 
usually necessary if the large intravenous injections have been given. 

Differential Diagnosis. — ^The differential diagnosis of the bacillus 
of tetanus is, generally speaking, not difficult, inasmuch as animal 
inoculation affords a sure test of the specific organism. No other 
microorganism known produces similar effects to the tetanus bacillus, 
nor is any other neutralized by tetanus antitoxin. The other charac- 



244 PATHOGENIC MICRO-ORGANISMS. 

teristics also of this bacillus are usually distinctive, though micro- 
scopic examination alone cannot be depended on to make a differen- 
tial diagnosis. DifBculty arises when other anaerobic or aerobic 
bacilli, almost morphologically identical with the tetanus bacillus, are 
encountered which are non-pathogenic, such as the BaciUiis pseudo- 
teianicus anaerobius, already mentioned, and the Bacillus pseudo- 
telanicus a'erohms. It is possible, however, that both these bacilli, when 
characteristic in cultures, are only varieties of the tetanus bacillus, 
which, under unfavorable conditions of growth, have lost their viru- 
lence. These non-virulent types do not, as a rule, have spores abso- 
lutely at their ends, and the spores themselves are usually more ovoid 
than those in the true tetanus bacilli. 

Methods of Examination in a Oase of Tetanus. — (a) Microscopic. — 
From every wound or point of suppuration film preparations should 
be made and stained with the usual dyes. The typical sporeJbearing 
forms are looked for, but are usually not found. At the same time 
other bacteria are noted if present. 

(6) Ctdtures. — Bits of tissue, pus, cartridge wads, etc., are collected 
and dropped into glucose bouillon contained in small flasks or tubes. 
This bouillon should be slightly alkaline, be free from oxygen, and 
protected from oxygen. A simple way is to cover the bouillon with 
liquid or semi-solid paraffin, and thus boil and afterward cool it. 
Cultures placed in such protected bouillon grow readily. 

(c) Inoculation. — ^A salt solution emulsion of material from the 
wound is inoculated into mice or guinea-pigs subcutaneously. 



CHAPTER XIX. 

INTESTINAL BACTERIA. 

of Bacteria in Intestines. — The constant presence of 
great numbers of bacteria in the intestinal tract has been the subject 
of much investigation which has given somewhat conflicting results. 
On the one hand, certain experiments seem to show that the presence 
of intestinal bacteria is not essential to life. For example, Nuttall and 
Thierfelder experimenting with guinea-pigs succeeded in keeping 
the intestines free from bacteria for a limited time, during which the 
young pigs remained well. Furthermore, Levin makes the interesting 
statement that the intestinal tract of polar animals is for the most part 
sterile. 

On the other hand, the supporters of the opposite theory, namely, 
that certain intestinal bacteria are necessary for perfect physiologic 
action, state that in their experiments on feeding animals with sterile 
food they found that development was retarded; thus Schottelius 
claims this for chickens, and Mme. MetchnikofiF obtained similar results 
with young frogs. However, whether or not the presence of bacteria 
in thi intestinal canal is essential to the animal economy, it is, never- 
theless, evident that microorganisms play a certain r6le in aiding or 
inhibiting some of the alimentary processes dependent upon biological 
activity. Recently new interest has been added to the subject by the 
work of MetchnikofiF who claims that old age is hastened by the in- 
creased growth and action of certain putrefactive bacteria normally 
found in small numbers in the intestines; and he, Herter, and others 
consider that the development of these harmful varieties may be 
checked by the growth of the obligate intestinal bacteria or by some 
substituted variety which has no harmful action upon the host. 

Conditions Influencing Development of Bacteria. — ^The intestinal 
canal presents such varying conditions dependent upon so many dif- 
ferent factors that of necessity its flora will reflect great diversity. As 
the organisms gain access to the tract chiefly through the air, food, and 
drink ingested, the character of these will influence the nature of the 
flora. The condition of the oral cavity and that of the respiratory 
passages on account of swallowing bacteria will also have an influence 
on the kind of bacteria found. Some few microorganisms, such as 
the colon group and the obligate anaerobes have become established 
as regular inhabitants of the intestines and find in the difiFerent locali- 
ties of the canal their best environment. Together with these may be 
found those bacteria which having been ingested with various sub- 
stances have survived the action of the gastric and intestinal fluids. 

245 



246 PATHOGENIC MICRO-ORGANISMS, 

The length of time which the intestinal contents are retained at any 
one point of the tract will cause an increase or decrease of certain 
types, as well as the total number, since all portions of the canal are 
not equally adapted to the development of any one species nor to 
bacteria as a class. 

Under absolutely normal conditions organisms, which are not de- 
stroyed, pass through the intestinal tract without entering the body 
of the host, but if injury occurs to the intestinal wall or the normal 
resistance of the body's tissues is lowered for any reason they may pass 
into the circulation. Makl^zow claims that after twenty-two hours of 
fecal impaction, intestinal organisms were found in the circulation. 
MacFayden has also demonstrated the same. In chronic constipation 
intestinal bacteria are found in the urine. 

Escherich found that in viero and immediately after birth the 
meconium is sterile, unless, in exceptional cases where the mother 
has suffered from a severe bacterial infection and the invading organ- 
isms are found in the foetus. From three to seven hours after birth 
a few bacilli, cocci, and yeasts may be found, having presumably entered 
by the anus; after eighteen hours the number and kinds of bacteria 
increase, being taken in by the food or the swallowing of saliva. The 
stools of drtificially-fed infants show a greater variety of organisms than 
those of the breast-fed child. 

Anaerobic Conditions in the Intestines.— Virchow first questioned 
the presence of free oxygen in the entire intestinal canal and con- 
cluded it was essentially anaerobic, the oxygen which is taken in 
being quickly absorbed or combined with hydrogen. 

The character of the flora also indicates an anaerobic condition 
of the small intestines with more or less aerobic conditions in the 
lower part of the colon and rectum. 

The anaerobes play the chief part in intestinal putrefaction, and 
certain varieties are thought to be at least the predisposing if not the 
chief cause of many cases of appendicitis. 

Regional Distribution of Bacteria in Digestive Tract. — Many 
difiFerent organisms may be found at times in all parts of the tract, 
but each species finds its best environment in some one location and 
is here found with greater frequency. In the stomach, very few 
bacteria develop, the sarcinee, B, gaMricus, and cloacae group are 
rather constant, the larger number and variety taken in being destroyed 
to a great extent by the gastric juice. 

The fact that great numbers of bacteria are destroyed by the diges- 
tive juices, together with a rapid passage of the partly digested food 
and the strict anaerobic condition, account^ for the very few bacteria 
that are usually found in the upper part of the small intestines. It 
is in this location that the obligate anaerobes, which are usually 
spore bearers and often Gram-positive organisms, such as the putrificus 
of Bienstock, capsulatus aerogenes and B. bifidus are usually found. 
The chief bacteria of the lower part of the small and the upper part of 
the large intestines are members of the B. coli group which reach their 



INTESTINAL BACTERIA, 247 

highest development in the caecum and upper colon. Here, too, other 
organisms which have been held in check by the above chemical and 
mechanical causes, finding a more suitable soil, develop, and a marked 
increase is found in many Gram-positive bacilli and cocci of various 
types. In the lower colon and rectum with the accumulation of specific 
antagonistic substances formed from the abundant growth higher up, 
many forms, especially of B. coli group, are more or less destroyed. 

The flora can be materially changed in dogs })y the diet, as has been 
shown by Herter. Lemke and MacFayden demonstrated the same 
in man. 

The range of variation of the bacteria that appear normally from 
time ta time in the intestinal tract is so very great that no one grouping, 
except in the most general sense, such as fermenters or non-fermenters 
of glucose, anaerobes, or aerobes, seems to apply to all cases. Ford 
isolated 50 distinct species from the human faeces. 

Methods XJsed in Examination of Normal FsDces. — The material should 
be taken from a perfectly fresh stool, preferably after a dose of castor oil 
has been given. This induces a quick and thorough emptying of the intes- 
tinal tract, with the least alteration of the chemical nature of the fseces. The 
use of blind tubes or flushing are apt to give only the contents of the lower 
part of the colon and rectum and are useful only when the examination is 
to be limited to this area. 

To 1 gram of the material 100 c.c. of normal salt solution is gradually 
added, first nibbing up the fseces in a small quantity of the diluent and then 
shaking thoroughly as more is added. Definite amounts of this emulsion can 
be used for plating. Two per cent, glucose agar with defibrinated blood 
added to it is very satisfactory for the isolation of anaerobes, while beenvort 
agar (10 per cent, sterile beerwort added to the usual stock agar) is used for 
the acidophile group. A very convenient method, according to Zinsser, of 
growing anaerobic cultures is to take crjrstallizing dishes of different sizes 
so that one dish fits within the other, leaving a space of three-fourths of an 
inch all around. The larger dish is placed over the smaller dish as a cover, 
they are then wrapped in filter-paper and in this way can be easily sterilized . 
When reirdy for plating 1 c.c. of defibrinated blood is placed in the bottom 
of the smaller dish and into this is poured 10 c.c. of glucose agar which has 
been inoculated with 1-10 c.c. of the above emulsion. By gently tipping the 
dish back and forth the blood and agar become very well mixed. This is 
covered with a petri dish or the companion crystallizing dish and allowed to 
stand until it is perfectly cold. In the larger dish are placed two pieces, about 
1^ inches to 2 inches long, of caustic soda and the dish is filled to about i 
its capacity with pyrogallic acid. The smaller dish is carefully inverted over 
this and sufficient sterile water poured into the larger dish to cover the acid. 
The whole is then sealed with paraffin or oil poured over the water that is 
collected outside the smaller dish; this prevents continual absorption of the 
oxygen from the air. Plates are made from beerwort agar and grown both 
ana^robically and aerobically. Fishings are made from these plates upon cor- 
responding tube media and the cultures are further tested on such media as 
may be b^ adapted to each special organism. 

Sabstdtation of one Variety of Bacteria for Others. — It is possible 
for the usual flora of the intestines to be almost entirely replaced 
temporarily by an invading organism. This occurs in disease when 
the microorganism producing a specific disease of the intestines is 



248 PATHOGENIC MICRO-ORGANISMS. 

found in almost pure cultures, as in dysentery or cholera, or where 
a large number of organisms are swallowed, as in the case of a child 
that had dysentery, and the dysentery bacillus was found to be abun- 
dant; but few days after the onset of the attack, tonsillitis developed 
and the stools contained streptococci, greatly in excess of the other 
organisms. 

As already stated, Metchnikoff claims that this possibility of substitu- 
tion can be used in cases where intestinal putrefaction is excessive and 
thus check the process by the introduction of a lactic acid bacterium. 

This work of Metchnikoff and his followers has led to an extensive 
study of organisms causing lactic acid fermentation. Our present 
knowledge of the lactic acid milks is summed up in the following 
paragraphs : 

Lactic Acid Milks. — For many years the people of western Asia and 
eastern Europe have looked upon sour milk as an essential part of daily 



B.bul«iiricus:7lh day (14°) colony. Whey agar plat*. X 50 diameters. (Wlule and Aveiy.) 

diet. In western Europe and America buttermilk has been a favorite 
drink with many, but it never a.ssumed as much importance as in the 
east. The term sour milk covers all milks or parts of milks in which 
lactic acid fermentation is pronounced. The ordinary buttermilk 
sours because of the growth of lactic acid bacteria in the raw milk 
which have been derived from the local surroundings. Sour milk 



INTESTINAL BACTERIA. 249 

from the dealers may be such milk, but it is more usually heated milk 
to which some special culture of bacteria (starter) has been added. 
Sour milk is usually nearly fat-free, but more or less of the cream may 
remain in it. Hansen, of Copenhagen, has for some time supplied a 
lactic acid bacillus which has been much used. Another starter now 
popular is one supplied by Metchnikoif which he obtained from the 
east. There are a number of preparations of sour milk used at present, 
among these are: 

Kumyti, in which the fermentation is due to lactic acid bacteria and 
yeasts, and thus contains not only lactic acid, but carbon dioxide and 
about 1 per cent, of alcohol. 



B.bulgBrickU. X lOOOdianw. (Rflnrd.) ' 

Maadzoun and Yoghurt, the common sour milk of southeastern 
Europe, containing chiefly the B. bulgaricua and streptococci and 
diplococci, all producing lactic acid. 

Zoolak (matzoon) made by adding to heated milk the same bac- 
teria as occur in maadzoun. 

Yohourd, blabberade, aod other sour milks are made by the use of 
much the same organisms. 

The bacilli at present of most interest are those resembling the B. 
bulgaricua (B. of Massol) which are present in the eastern milks 
and are now through the advocacy of Metchnikoff used alone or in 
connection with a lactic acid streptococcus to produce much of the 
souring of milk of Europe and America. In 1906, Cohendy studied 
the action of this bacillus and found that it produced a large amount 
of lactic acid, 3 . 23 per cent, being found after 10 days at 36° C. From 
other preparations slightly different bacilli were found which produce 
a firm clot, while the B. inUgaricus produces a soft curd. Some 



250 PATHOGENIC MICRO-ORGANISMS. 

bacilli which resemble the B. bulgaricug in many respects produce gas 
as well as acid. The bacilli in all strains of B. bulgaricus show wide 
variations in length from 2/t to 50/i. Chains of bacilli occur in some 



i( B. bulgsriciu and a ImIoh fetQwnlinc ■tnpUKOccus. 
Fio. B3 



■t celb tuiil UcU>« fe 



strains to a more marke<l degree than in others. The bacilli are 
non-motile, non-sporulating. Gram-positive, except when in involution 
forms, when they are said to be Gram-negative. Difficult to cultivate 



INTESTINAL BACTERIA. 251 

in most media. When freshly isolated, growth obtfuned only on media 
containing whey or malt or milk. Grow equally well in aerobic and 
anaerobic conditions. Optimum temperature for growth is 44°, 
fair growth at 30°, slight at 25°, none at 20°. Gelatin is not liquefied. 
Colonies on whey agar are round, grayish- white, and measure 0.5 to 
1.5 mm. Periphery of colonies mostly filamentous. The growth in 
whey produces clouding, but this disappears in 5 to 14 days, leaving a 
sediment. Coagulates milk in 8 to IS hours at 44°, and after longer 
time at lower temperatures. The lactic acid formed is either inactive 
or bevorotatory. A small quantity of volatile acid is also produced. 
No appreciable peptonization of the curd. 



id whey « 
id HonetB. 



It 42° C. Small colony 



The bacilli are non-pathogenic. These bacilli are probably widely 
distributed in nature, being frequently present in the intestines of man 
and animals. White and Avery, who have made sn exhaustive study 
of this group of bacilli, consider that they all belong to one group 
which is identical with the group Bacterium caucasicum (Kern). There 
are apparently at least two distinct types which differ in the amount 
and kind of lactic acid formed.' 

Prevalent Intestinal Bacteria. — B. Bifidoi.— Tissier found the 
B. bifidus in the stools of breast-fed infants which at times forms 
nearly the entire flora. He found it, though less frequently, in arti- 
ficially fed infants. He also isolated it in the superficial ducts of the 

' Helen Baldwin has just shown in the case of one man that when milk plus a 
lactic acid bacillus was added to a mixed diet, the ethereal sulphates in the urine 
were increased. 



252 PATHOGENIC MICRO-ORGANISMS. 

mammary gland of the mother. It is a strict anaerobe. In the fteces 
and fresh cultures it presents the form of a slender bacillus with one end 
tapering and the other club-shaped. It varies in length from 2/i to 
3/1 and even 4/t. 

It occurs mostly as a diplobacillua (see Fig. 94) with the pointed ends adja- 
cent and the swollen ends free, but at times this order is reversed and a fusi- 
form appearance results. As the line of separation is often obscure and as 
the two organisms come together at different angles these various arrange- 
ments give the impression of many different forms. 

The bacilli lie sometimes in parallel groups, but are seldom entangled. In 
old cultures, the swollen ends seen in the young cultures become bifurcated, 
others take the stain irregularly and Tissier designates this the vesicular form. 
In some instances several bacilli become grouped together at different angles, 
giving the appearance of multiple branching forms. As the medium becomes 
more acid, the bifurcated forms become more numerous. Vesicular forms 
bear a relation to vitality and bifurcated forms a relation to media. It is 




^. 



^ 



I. BiGdus. repnaenting lh« varioiu fomu deacribed; the irreauUrly-aUined or vrsicrular fon 
lieiai Irom old cultures. X about ISOO diam. 



non-motile, stains by Gram's method, old cultures staining irregularly. Does 
not seem to possess spores. Killed at 60° for 15 minutes. Does not die out 
quickly. Can be transplanted after three weeks. Grows best at 37° C, but 
also grows at 20° C. 

On glucose agar, after three days, fine regular colonies, oval In shape, 

appear. It is innocuous to guinea-pigs. It can be cultivated on beerwort 

agar and on glucose agar. In stab cultures made from the feces, in either 

medium the bacilli may be found in almost pure cultures at the bottom of 

the stab after two to fifteen days; the other organisms dying out unless the 

is a facultati\'e anaerobe, is present, then B. bifidvs, being 

ergrown. Fermentation tubes of glucose broth inoculated 

A-ill show an abundant growth of bifidus in the sediment. 

urpjuweit claim that in infants the B. bijidua plays 
ihibiting the growth of the more harmful organisms 
es in the adults. Cahn states that the B. bipdua is 



INTESTINAL BACTERIA. 253 

not found so constantly in artificially fed infants, but that the B. 
addopkilvs of Mora takes its place. 

B. Addophilos. — This organism belongs to the acidophile group and 
differs from the bifidus in several respects. It never shows the bifid 
forms. It is only found in the artificially fed infants and in milk 
from cows. Colonies are irregular and send out filaments. It is a 
facultative anaerobe. Some consider it to be the same as the B. 
bifidus. 

Kntorococcua. — Thiercelin in 1903 described the Enterococeua 
proteiformis (Fig. 95) as occurring as a coccus, diplocoecus, strepto- 
coccus, staphylococcus, tetrad filaments and rods. It has a capsule 
barely visible, sometimes forming a halo. 



ReprcHota the (rBttstioD of the EutcroHKcua (Thier»liD) (mm Ihe ftppanot baciUary [onus 
lo Ihc focciB without a capgule. X lOOO diara. 

The arrangement depends upon the mode of divisions. When the 
organism assumes the form of a bacillus the division takes place in line 
of the short axis, the capsule being tough does not rupture but encloses 
2 or 4 more organisms. This form is observed in culture media con- 
taining alcohol, quinine, chromic acid, permanganate, and especially 
bichromate of potash. This bacillary form, before division takes place, 
is confusing as it is difficult to tell whether or not the culture is pure 
until transferred to a medium in which alcohol is present when the 
forms all become coccal. In strongly alkaline broth it grows in 
tetrads, on agar it resembles the staphylococcus, on gelatin the same. 
In broth containing a httle methylene blue, picric or acetic acid and in 
bay infusions it is a distinct streptococcus. It is present in normal stools 
but may become pathogenic. It is found in the upper respiratory 
tract, skin, vagina, and is obtained in pure cultures from purulent 
discharges, being easily isolated by ordinary methods. 

It is sensitive to heat and direct sunlight, rather resistant to dis- 
infectants, does not grow in distilled water and sparingly in broth 
containing 2 per cent, sodium carbonate or nitrate. Grows in sterilized 
tap water, does not ferment sugars, does not constantly coagulate 



254 PATHOGENIC MICRO-ORGANISMS. 

milk, does not liquefy gelatin, grows well in the digestive fluids, no 
gas, indol, or odor in tlie presence of sugar. 

It produces a toxin that kills mice in 24 to 48 hours. The organisms 
are found in all the organs after death. 

B. Pntiificas. — Bienstock found in putrid mixtures an anaerobic, 
spore-bearing bacillus resembling tetanus in its morphology, which 
is capable of decomposing fibrin in the absence of oxygen, in this 
case the end products of putrefaction, such as indol, are not formed. 
When, however, B. pulrifums is associated with some aerobes it acts 
upon fibrin in the presence of oxygen, forming the characteristic 
putrefactive products, which are further split up by the aerobes, 
forming indol. This action is not observed with all aerobes, for ex- 
ample, with B. coli and B. lactis a^ogenes inoculated on fibrin with 
B. puirifiau. 

B. putrificua is found commonly in the small intestines where it 
enters through the respiratory and digestive tract; that putrefaction 
does not occur, normally is supposed to be due to the presence of in- 
hibiting bacteria. It is isolated with difficulty from the fteces. Is an 
obligate anaerobe with drum-stick spores. It is a slender rod with 
blunt ends, and sometimes forms threads, especially on liquid gelatin, 
is actively motile with flagella arranged on either side. Liquefies blood 
serum with the production of a foul odor. Is Gram-positive. la not 
pathogenic for animals. 

B. AingvntB Oapsnlatas (5. welekti, B. perfringens). — Found 
usually in small numbers in healthy adults. Increased in old age. 
It is considered by Herter to be the chief cause of intestinal putre- 
faction. For a full description of its pathogenicity and other chai^ 
acterisfics, sec p. 440. 

Klein found in the fseces of patients during an outbreak of diarrhoea 
at St. Bartholomew's Hospital, London, an organism which he named 
the B. aerogenes sporogettes, which may be a variety of B. vrelchii. {See 
also p. 442.) 

BlDLIOORAPHV. 

Bienstock, Untersuchungen Uber die Aetiologie des Encisofftulniss, -Archiv. f . 
Hygiene, 1X99, xxxvi. 

Central, f. Bakt., Zweite .\bt., Bd. xxv. No. 5/9. 

Escherich, Darin bakterien dea S&uglinga und ihre Besiehungen xur Fhysiologie 
iler Verdauung. Stuttgart, 18S6. 

Herter, Bacterial Infections of the IntestiDal Tract, New York, 1907. 

Kleia. Ucl>er einen pathocenen anaeroben Darmbacillen B. enteritidis sporo- 
((ene?, Centralbl. t. Bakt., Bd. xviii., 1895. 

MacNeal, Latser and Kerr, Fsecal Bacteria of Healthy Men, J. Inf. Dis., vi.. 

ur lea microbea de la putrification inteatinale, C. R. Acad. 
. Etude aur la flore inteatinale, Am. Inat. Past., 1908, xxii. 
I d'involution de enterocoque enterobacteria. Comptea 
de Biologic, 1902-1903. 
I sur La Klora Intcstinale, Normale et Pathologique du 



CHAPTER XX. 

THE COLON-TYPHOID GROUP OF BACILLI. 
THE COLON BACILLUS GROUP. 

There are a number of varieties of bacilli occupying the intestines 
of man and animals which, because they have similar characteristics 
and live in the colon, are generally grouped together as colon bacilli. 
These bacilli are only pathogenic under unusual conditions. The 
specific pathogenic typhoid, paratyphoid, dysentery, and paradysentery 
bacilli, and those responsible for meat poisoning also have among 
themselves and between them and the colon bacilli resemblances and 
are often classed together in the group of the colon-typhoid bacilli. 

The chief common characteristics of this whole group are: (1) a 
similar morphology, i,e,, short, rather plump rods with a tendency to 
thread formation; (2) a Gram-negative staining reaction; (3) similar 
growths on agar and gelatin; (4) non-liquefaction of gelatin (a few 
organisms which might be placed in the colon group, such as B. 
cloaccB, liquefy gelatin very slowly). 

In order to see more clearly the main points of difference between 
the subdivisions of this great group the tabulation on page 256 may 
be studied. 

The chief differential points between the individual species may be 
seen in the table on page 257. 

The Bacillus Coli Group. — The first description of an organism of 
the colon type was by Emmerich (1885), who obtained it from the 
intestinal discharges of cholera patients. A similar organism was 
found by Escherich (1886) in the faeces of healthy infants. He gave 
it the name of Bacterium coli commune. It has since been demon- 
strated that closely allied types of bacilli are normal inhabitants of 
the intestines of most of the lower animals. They are transferred 
through the ffleces as manure and sewage to cultivated land, surface 
waters, etc. During warm weather they may increase outside of the 
animal body. Those strains having the chief cultural characteristics 
of the original strain are classed as colon bacilli, while those differing 
considerably from it are, while considered in the general group, given 
different names, such as paracolon, etc. 

The group of the B. coli has interest not only because it excites 
disease at times in man and animals, but also because it is an index 
of faecal pollution from man or animals. If from man it indicates 
the possibility of infection with the typhoid or dysentery bacilli. 

Morphology. — Bacillus coli varies considerably in its morphology, 
according to the sources and the culture media from which it is ob- 

255 



PATHOGENIC MICROORGANISMS. 






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THE COLON TYPHOID BACILLUS GROUP. 



257 



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258 PATHOGENIC MICRO-ORGANISMS. 

tained. The typical form (Fig, 96) is that of short rods with rounded 
ends, from 0.4/i to 0.7/i in diameter by l/i to 3/i in length; sometimes, 
especially where the culture media are not suitable for their growth 
and in tissues, the rods are so short as to be almost spherical, resembling 
micrococci in appearance, and, again, they are somewhat oval in 
form or are seen as threads of 6/1 or more in length. The various 
forms may often be associated in the same culture. The bacilli occur 
as single cells or as pairs joined end-to-end, rarely as short chains. 
There is nothing in the morphology of this bacillus sufficiently char- 
acteristic for its identification. 

Flagella. — Upon some varieties seven or eight peritnchic flagella 
have been demonstrated, upon others none. The flagella are shorter 
and more delicate than those characteristic of the typhoid bacilli. 

Staining. — -The colon bacillus stains readily with the ordinary aniline 
colors; it is always decolorized by Gram's method. Under certain 
conditions the stained bacilli exhibit bipolar granules. 

. Biology. — It is an aerobic, facultaiive anaerobic, non-tiqueftfing 
bacillus. It develops best at 37° C, but grows well at 20" C, and 
slowly at 10° C. It is usually motile, but the movemenU in some 
of the cultures are so sluggish that a positive opinion is often difficult. 
In fresh cultures, frequently, only 
one or two individuals out of 
many show motility. MacConkey 
recommends examining under 
dark ground illumination a drop 
of a 6-hour broth culture placed 
on an ordinary glass slide without 
a coverslip, using a J-inch objec- 
tive and a X 8 eye-piece. Thi.s 
gives a good idea of the power of 
movement of an organism. 
TheB.co/idoes not form .spores. 
Cultivation.— The colon bacillus 
develops well on all the usual cul- 
ture media. Its growth on them 
is usually more abundant than 

Colon bacilli. Twsnty-four-hour mar culture. .1 . » .l » L -J L -11 

X iioudiaoieten. that of the typhoid bacillus or 

the dysentery bacillus, but the 
difference is not sufficient for a differential diagnosis. 

OelatiB. — In gelatin plates, colonies are developed in from eighteen 
to thirty-six hours. They resemble greatly the colonies of the typhoid 
bacillus, except that many of them are somewhat larger and more 
opaque. {See Figs. 42-44, page 73.) When located in the depths 
of the gelatin and examined by a low-power lens they are at first 
.seen to be finely granular, almost homogeneous, and of a pale 
yellowish to brownish color; later they become larger, denser, darker, 
and more coarsely granular. In shape they may be round, oval, or 
whetstone-like. When the gelatin is not firm the mai^ns of many 




THE COLON BACILLUS GROUP, 259 

colonies are broken by outgrowths, which are rather characteristic of 
colon bacilli. 

In stab cultures on gelatin the growth usually takes the form of a 
nail with a flattened head, the surface extension generally reaching 
out rapidly to the sides of the tube. 

Nutrient Agar. — In plate cultures: Surface colonies mostly circu- 
lar, finely granular, and rather opaque. The deep colonies are apt 
to have protuberances. In streak cultures an abundant, soft, white 
layer is quickly developed, but the growth is not characteristic. 

Bouillon. — In bouillon the Bacillus coli produces diffuse clouding 
with sedimentation; in some cultures a tendency to pellicle formation 
on the surface is occasionally seen. 

Potato. — On potato the growth is rapid and abundant, appearing 
after twenty-four to thirty-six hours in the incubator as a yellowish- 
brown to dark cream-colored deposit covering the greater part of the 
surface. But there are considerable variations from the typical 
growth; there may be no visible growth at all, or it may be scanty and 
of a white color. These variations are due often to variations in the 
potato. 

Milk. — Milk coagulates in from four to ten days at 20° C, and in 
from one to four days at 37° C. The acids formed are lactic, acetic, 
formic, and succinic acids. Coagulation is due principally but not 
altogether to acids. A ferment is produced which is capable of causing 
coagulation in the presence of lime salts, especially in acid solutions. 

Chemical Activities. — Behavior Toward Carbohydrates. — In cultures 
of colon bacilli many carbohydrates, especially sugars, become fer- 
mented with production of acid and gas. 

There are differences, however, among the organisms thus included 
under the name colon bacilli; thus, some ferment cane-sugar, others 
do not. The great majority ferment glucose and lactose with the 
production of gas. 

The important fermentation products, both qualitatively and 
quantitatively, are produced from grape-sugar, probably, according to 
the following reaction: 

2CtHi20« + HsO - 2C«H«Oi + CHiCOOH + CtH»COOH + 2C0» + 2Hj 
Grape-sugar. Water. Lactic acid. Acetic acid. Ethyl alcohoL Carbonic Hydrogen. 

acid. 

There are reasons to think that lactic acid is first produced and that 
from this other acids and products develop. Under aerobic conditions 
lactic is produced in excess of acetic acid, while in the absence of oxygen 
the reverse is apt to be true. 

Gas Production. — When colon bacilli are grown in a solution of 
glucose (dextrose), CO2 and H, are produced, iCO, to IH, up to 
iCOj to 3Hj. Anaerobic conditions aid gas formation. A very few 
intestinal varieties of Gram negative bacilli produce gas from no 
sugars and some from a few only. Nearly all produce gas from glu- 
cose, and about 80 per cent, of all varieties produce gas from milk- 
sugar (lactose). Very slight traces of gases other than H and CO, are 



260 PATHOGENIC MICRO-ORGANISMS, 

produced. The amount of gas varies in different varieties; the closed 
arm of the tube half-filled, and the H and CO, in the proportion 
2 to 1, is the characteristic type. It is also true of Gartner's B. en- 
teritidis. In another type the whole of the closed arm is filled, — 
H, : C03= 1 : 2 or 3. This type is usually called Bacillus cloacce. In 
a third type the arm is nearly filled, — H, : CO, =1:1. This type is 
the 5. a'&rogenes. 

The fermentation is not a simple hydrolytic action, but one in which 
combinations between the C and O atoms are sundered and formed. 
This is not an oxidation process, but a change through breaking 
down — that is, a true decomposition. What oxidation takes place 
is chiefly due to the oxygen liberated from splitting the sugar molecules. 

Use of Neutral Sugar Litmus Agars to Differentiate between Colon 

AND Typhoid Bacilli. 

Color of media after 24 hours* growth of culture. 

List of sugars. ■ . 

Colon bacillus. Typhoid bacillus. 

Grape-sugar (Dextrose). . .Red. Red. 

Saccharose Red or blue. Blue. 

Mannite Red. Red. 

Maltose Red or moderately red. Red or pink. 

Milk-sugar (Lactose) Red. Blue. 

Dextrin Blue Violet blue. 

To bouillon used to detect acid and gas formation no sodium hydrate or car- 
bonate should be added. 



« 



Effect of Colon Bacilli in Nitrogenous Oompounds. — Indol Formation. — 
None of the Colon bacilli liquefy gelatin nor peptonize any albu- 
mins. They do, however, break down some of the higher nitrog- 
enous compounds into smaller atom groups. The first noted of 

these compounds was indol, C^H^v puyCH. This is one of the 

most important products of colon activity, although a few varieties lack 
it. (Witte's peptone solution is used to develop indol.) Sugars interfere 
with its production, as also does the absence of oxygen. The maximum 
amount of indol is present about the tenth day. In the intestinal 
canal in health very little indol appears to be produced by colon bacilli. 
Sulphurated hydrogen is liberated from sugar-free fermentable proteid 
substances. Mercaptan and sometimes skatol have been noted in 
peptone solution cultures. The colon bacillus liquefies, according to 
some minute quantities of gelatin, but so little as to be inappreciable. 

In media containing fermentable sugars and proteid substances 
simultaneous action takes place on both with the production of both 
alkalies and acids. 

Test for Indol. — The test is carried out by adding little by little 
i to 2 c.c. of 0.005 per cent, potassium nitrite to lOc.c. of fluid cul- 
ture, to which has been added 5 c.c. of 10 per cent, sulphuric acid. 
To prevent confusion with other colors a little amyl alcohol is added 
and shaken to dissolve and concentrate the color. 

Effect on Fats. — No action has been noted. 



THE COLON BACILLUS GROUP. 261 

Bedaction Processes. — Reduction of Pigments. — The action on cer- 
tain pigments which are reduced to colorless products and interme- 
diate colors is more vigorous than that of typhoid bacilli. This eflFect 
occurs in litmus bouillon in the closed arm of the tube, in bouillon 
and agar (not in gelatin), indigo-sodium-sulphate-methyl blue, in 
sugar media, etc. 

Bedaction of Inorganic Salts. — ^From nitrates the bacilli produce 
nitrites and from them ammonia and free nitrogen as follows: 

2NaN0, +H2O =2NaOH +N, + 0.. 

Toxins. — ^The bodies of dead colon bacilli contain pyogenic sub- 
stances (and others), which, injected into the circulation, produce 
paralysis of the striped muscle fibres, convulsions, coma, and death. 
Extracts from some cultures produce irritation of the mucous mem- 
branes of the large intestines with dysenteric symptoms. 

Orowth with Other Bacteria. — ^The colon baciUi act antagonist- 
ically to many of the proteolytic bacteria in the intestinal tract, and 
so inhibit alkaline putrefaction otherwise caused by the latter. In milk 
the same antagonism exists, probably because of the acidity caused by 
the colon growth. 

Beaction to High and Low Temperatures. — Colon bacilli are killed 
at 60° C. in from five to fifteen minutes. Frozen in ice a large proportion 
die, but some resist for six months. Frozen in liquid air 95 per cent, 
are killed in two hours. 

Besistance to Jiiymg and Antiseptics. — Simple drying destroys 
the majority of organisms dried at any one time, but some bacilli of 
the number dried may remain alive, especially when held in the 
texture of threads, for five or six months, or all may die in forty-eight 
hours. 

To most antiseptics they are moderately resistant. They are killed 
in 5 to 15 minutes in a 1 per cent, solution of carbolic acid. 

Effect of Acids. — ^The colon bacilli grow in a wider range of acids 
and alkalies than most other bacteria. They develop in from 0.2 to 
. 4 per cent, of mineral acids, in from . 3 to . 45 per cent, of vetegable 
acids, and in from 0.1 to . 2 per cent, of alkalies. 

Effect of Animal Fluids and Juices. — Gastric juice kills colon 

bacilli when not protected or too greatly diluted by food. All the 
members of the typhoid-colon group are more resistant to the gastric 
juices than most non-spore-bearing bacteria. With the food they 
readily pass from the stomach into the intestines.^T'hey grow in bile 
and in the intestinal juices. ^*"^^-^<--i*-^ ta^y ^.d/Vt;,^: 

Occurrence in Man and Animals during Health. — The Badllus 

coli is a common inhabitant of the intestinal canal in man and in almost 
all domestic animals. Many of the varieties found in animals seem 
to be identical with those found in man. It is also found occasion- 
ally in wild animals and appears at times even to occur in fishes. 

Occurrence Outside of the Intestines. — Colon bacilli are found 

wherever human or animal faeces are carried. They are therefore 




^ 



262 PATHOGENIC MICRO-ORGANISMS. 

present in all cultivated soil and inhabited country. In surface water 
a few bacilli, less than one to each cubic centimeter, are not suflScient 
to give rise to the opinion that it is contaminated. Even the presence 
of ten colon bacilli in 1 c.c. does not necessarily show dangerous 
pollution, since such a number could equally well come from the 
rain-water from streets or fields. Inspection alone can hope to reveal 
the source of the bacilli and therefore their significance. Colon bacilli 
are apt to be found in everything which comes in contact with man 
or animals. 

Association with Other Bacteria in the Intestines. — In the breast- 
fed infant within a few hours or days after birth one or two varieties 
of typical colon bacilli are found in the colon, and these bacilli form 
the great majority of all the bacteria present which grow in media. 
The bacilli in one infant's intestines usually all agglutinate with the same 
serum, but those from different infants vary. The bacilli find their 
way through the food or from the anus upward. In the small intes- 
tines the Bacilltis aerogenes is most prevalent, while in the caecum and 
below the characteristic colon types predominate. 
^ Only about 10 per cent, of the bacteria from stools seen under the 

^ microscope appear as colonies, and whereas in infant stools the ma- 

' jority of the bacteria in spreads are frequently Gram-positive, the 

I larger number of the colonies are composed of Gram-negative bacteria. 

N^ Some of the Gram-positive bacteria are anaerobic; others fail to grow 

^^ at all on ordinary culture media. These conditions, the normal pres- 

ence of colon bacilli and the tendency of other bacteria not to grow in 
culture media, make the greatest care necessary in weighing conclu- 
sions as to the pathogenic significance of colon bacilli in disease. 

Pathogenesis. — In Lower Animals. — Intraperitoneal and intravenous 

inoculation of guinea-pigs and rabbits may produce death, which, 

when it follows, usually takes place within the first forty-eight hours, 

. r I accompanied by a decided fall of temperature, the symptoms of enter- 

K \ itis, diarrhoea, etc., and finally fibropurulent peritonitis. 

^ I Subcutaneous inoculation in rabbits is followed usually by abscess 

^ I formation at the point of inoculation. Dogs and cats are similarly 

^ ' affected. 

Albarran and Hall^ have caused cystitis and pyelonephritis by 

^ direct injections into the bladder and ureters, the urine being artificially 

\ suppressed; Chassin and Roger produced angiocholitis and abscess 

of the liver in the same way. Akermann produced osteomyelitis in 

young rabbits by intravenous injections of cultures. 

From experiments on animals it would, therefore, appear that the 
true explanation of the pathogenesis of the colon bacillus is undoubt- 
edly to be found in the toxic effects of the chemical substance and 
roducts of the cells. 
In Man. — In normal intestines with intact mucous membranes the 
toxic products formed by the colon bacilli are absorbed but little or 
not at all, and the bacilli themselves are prevented from invading the 
tissues by the epithelial layer and the bactericidal properties of the 



;i 




r 



THE COLON BACILLUS GROUP. 263 

body fluids. Possibly there is an acquired immunity to the colon 
varieties which have long inhabited the intestines. 

The colon bacillus was at first regarded purely as a saprophyte. 
Later, through not realizing the post-mortem invasion and the great 
ease of growth of the colon bacillus on ordinary media, the other ex- 
treme was taken of attributing too much to it. 

The bacilU previously present in the intestines can, either by an 
increase in virulence in them or by a lowered resistance in the person, 
invade the tissues in which their toxins act, causing injury to the in- 
testinal tract. Thus in the case of ulceration in typhoid fever the 
colon bacilli enter the blood, or in perforation produce peritonitis. 
In dying conditions they at times pass through the intact mucous 
lining. In the gall-bladder or urinary tract the spread of bacilli from 
the intestines may cause disease. The specific serum reaction in the 
body is a sign of infection, but great care has to be observed in deciding 
that it is present, as group agglutinins also occur. Up to the present 
time it is very difficult to state in any colon infection whether the 
bacilli were previously present in the intestines or were derived from 
outside sources through water, food, or direct contact with other cases. 

Intestinal Lesions. — ^The lesions present in intestinal infection are 
those of enteritis; the duodenum and jejunum are found to contain 
fluid, the spleen is somewhat enlarged, and there are marked hyperaemia 
and ecchymosis of the small intestines, together with swelling of 
Peyer's patches. 

Virulence of Oolon Bacilli from Normal and Diseased Intestines. — 
The virulence varies with the culture and the time since its recovery 
from the intestines. Other things being equal, it is more virulent 
from an intestinal inflammation. From severe diarrhoea the colon 
bacilli in 0.25 c.c. bouillon culture may kill guinea-pigs if given 
intraperitoneally, while from the healthy bowel 2.5 c.c. are usually 
required. 

Increase of Virulence Outside the Body. — It has been found by 
several observers that in fermenting faecal matter a marked increased 
virulence takes place, so that infection is produced when received by 
man. 

Colon Bacillus in Sepsis. — In lesions of the intestinal mucous 
membranes or in colon cystitis, pyelitis, or cholecystitis, there is fre- 
quently just before death a terminal dissemination of the bacilli and 
consequent septicaemia. Here special symptoms of intoxication may 
occur, such as diarrhoea, changes in temperature, heart weakness, and 
hemorrhages. In most of these cases infection proceeds from the 
intestines, but in not a few from the wounded urethra or bladder. 
The colon septicaemia is detected by blood cultures. At times very 
few bacilli are found, and then the blood infection may be less im- 
portant than the local one. Cases occurring in typhoid and cholera 
are often observed, especially in relapses in typhoid. In very young 
infants a malignant septicaemia with tendency to hemorrhages is due 
to colon septicaemia. In a few cases in which colon but no typhoid 



264 PATHOGENIC MICRO-ORGANISMS. 

bacilli were present the course of the disease has been similar to 
typhoid fever. An epidemic due to colon infection of water has been 
noted. Infection through food and water are usually brought about 
by other closely allied bacilU not belonginj^to the colon group. 

Colon Bacillus in DiarrhoBa. — In diarrhoea we find increased peri- 
stalsis, less absorption of foodstuff, increased and changed intestinal 
secretions. Tissier observed that under treatment with cathartics 
the colon varieties increased, while the anaerobic forms are inhibited. 
In diarrhoea exciting conditions are active, inhibiting causes are 
lessened, and increased mucus and serum are poured out into the 
canal. This is notably seen in typhoid fever. In diarrhoea, although 
the common colon varieties are met with, there is usually seen a 
difference in that uncommon varieties and more typhoid-like bacteria 
are also found. Much more investigation is needed on this complex 
subject of variation in types between health and disease. 

Lesage, in 1898, stated that 25 per cent, of 770 cases reported by 
him of breast-fed children were due to pure colon infection, while 
the others were from mixed infection in which the significance of the 
colon bacilli present was more doubtful, as there are other, slightly 
different, microorganisms, colon-like in their characteristics, which 
produce infection. The reasons for this opinion are given by Escherich 
and Pfaundler as follows: 

1. Animals are certainly affected by epidemic infections of bacteria 
closely allied to the colon group — e. g., diarrhoea of calves and cows, 
hog-cholera, enteritis with ulceration in horses, etc. 

2. The histories of attacks of acute diarrhoea in men after eating 
food of such infected animals, and the presence of the serum reaction 
afterward. These bacteria are colon-like, though classed with the 
enteritidis group. 

3. The diseases of typhoidal nature are due to the closely allied 
paracolon or paratyphoid bacilli, and others are due to the dysentery 
group, in which the inflammatory and necrotic process localizes itself 
mostly in the lower colon and rectum. 

Numerous epidemics of acute diarrhoea in children from one to 
five years of age have been noted in which almost pure cultures of 
colon bacilli have been found. The symptoms begin with high fever 
which often rapidly falls, and frequent stools only watery or con- 
taining mucus and streaks of blood. These symptoms may quickly 
abate or go on to a toxic state characterized by heart weakness and 
drowsiness. This may lead to lung complications or death. In 
many such cases in America when blood has been present we have 
found one of the mannite fermenting types of the dysentery bacillus. 

B. Coli in Peritonitis. — Here the lesions must be considered as being 
due to mixed infection. 

Experimental evidence goes to show that the injection of virulent 
cultures of any of the varieties of colon bacilli into the peritoneal cavity 
produces intense and fatal peritonitis. Not only perforation of the 
intestines in man, but injury to the intestinal walls, allows colon infec- 



THE COLON BACILLUS GROUP, 265 

tion of the peritoneum to take place, and if foreign bodies are present 
in the peritoneum, or the epithelium injured, or absorption interfered 
with, such acute general peritonitis is very probable. At first most 
of these cases were believed to be a pure colon infection, but now it is 
known that this idea came largely from the overgrowth of colon bacilli 
in the cultures. More careful investigations, through cultures and 
smears, have demonstrated the fact that streptococci, and less fre- 
quently staphylococci and pneumococci, are also usually present in 
peritonitis arising from intestinal sources. The colon bacilli found 
even in the same case commonly comprise many varieties. 

The Ck>lon Group in Inflammation of the Bile Tract. — ^The normal 

healthy gall-bladder is usually sterile. This is true in spite of the 
fact that bile is apparently a good culture medium for the colon group. 
Simple tying of the neck of the gall-bladder usually causes a colon 
infection to take place within twenty-four hours. Obstruction of the 
bile-duct through various causes is fairly common in man. The gall- 
bladder then becomes infected, and following the inflammation of 
the mucous membranes there is often the formation of gall-stones. 
Some cases of jaundice are believed to be due to colon inflammation 
of the gall-ducts. Atypical varieties of B, Coli are frequently isolated 
from gall-bladder infections. 

Inflammation of the Pancreas. — Welch was the first to record a 
case of pancreatitis with multiple fat necroses due to colon infec- 
tion. A few more cases have since been reported due to members 
of the colon group, either alone or in conjunction with the pyogenic 
cocci. 

Inflammation of the Urinary Tract. — As far back as 1879 Bouchard 
noted cystitis due to bacilli of the colon group. After injury of the 
bladder mucous membrane, or by ligature of the urethra, it is possible 
to excite cystitis in animals by injection of colon bacilli. When 
cystitis is established the bacterial infection frequently spreads to 
the pelvis of the kidneys, causing a pyelitis or suppurative nephritis. 
The same process often occurs in man. In most cases of chronic 
cystitis the ureters and pelves of the kidneys become involved; any 
malformation of the ureters aids the process. From the pelvis the 
bacteria push up into the urinary tubules and excite inflammation 
and multiple abscesses. Colon infection of the different parts of the 
urinary tract may occur at any age, from infancy upward. Instead 
of starting in the bladder it may begin in the kidney itself, the colon 
bacilli coming from the blood or peritoneum. In many of these 
cases the bacilli isolated from the urine are clumped in high dilutions 
of the blood from the patient. 

Although other bacteria — the pyogenic cocci, the proteus, the 
typhoid bacillus, etc. — may excite cystitis, still in 90 per cent, of all 
cases some of the colon group are found, and this percentage is even 
higher in young children. The clinical picture of colon infection is 
very variable. The lightest cases progress under the guise of a bac- 
teriuria. The urine is passed a little more frequently and shows a 



266 PATHOGENIC MICRO-ORGANISMS. 

fine granular cloudiness. The reaction is acid. The cell elements 
are but little increased. There is an excess of mucus. Albumin 
is absent or present in only a trace. The condition may last for 
weeks or months and then spontaneously disappear or grow worse. 
With a somewhat more severe infection there is painful urination, 
perhaps tenesmus, increase of pus cells and sUght fever. In a conical 
glass a sediment of pus cells forms at the bottom, and clear urine 
remains above. If the infection passes to the kidney colicky pain and 
tenderness over the region of the kidneys is usually present. The 
most important symptom of pyelitis is an irregular intermittent fever 
resembling malaria. The albumin is increased in the urine and red 
blood cells may be seen. If a general nephritis arises the symptoms 
are all intensified and an ansemic condition may develop. Septicaemia 
may finally result. 

In most of these cases the microscopic examination is suflScient 
to make a probable diagnosis, since the bacteria are so abundant. 
The variety of colon bacillus present can, of course, only be told by 
cultures and other means. In the urine they appear as diplobacilli, 
or partly in short, almost coccus, forms, partly in long threads. As 
a rule, motility is absent. Not infrequently the cultures appear to 
be identical with those of the BaciUiis aerogenes. 

The characteristics of the urine itself have much to do with the 
probability of infection; the more acid urines being less likely to 
afford a propef soil for growth. Some urines are bactericidal even 
when they are neutral. The substances producing this condition are 
not known. The colon bacilli in the urine produce no appreciable 
effect on the reaction, but give up some of their toxins, which upon 
absorption cause the deleterious local and general effects. The 
serum of the patient usually agglutinates the cultures from the urine 
in 1 : 20 or 1 : 50 dilutions, but this property is sometimes absent, 
especially in light cases. 

In all cases in addition to the introduction of the colon bacillus a 
predisposing condition must be present, such as more or less marked 
retention of urine by an enlarged prostate or stricture, any unhealthy 
state of the mucous membrane or general depression of vitality. 

The B. Coli as Pus Formers. — Members of this group are frequently 
the cause of abscesses in the region of the rectum, urethra, and kidney. 
They rarely produce pus in other locations. 

The Colon Oroup in Inflammation Not Previously Mentioned. — 

Broncho-pneumonia, lobar pneumonia, and pleurisy have occasionally 
been caused by colon bacilli, probably from blood sources. Not 
a few cases of meningitis and spinal meningitis in infants, following 
localized colon infections, are due to colon bacilli. The symptoms 
are not well developed, as a rule. Some cases of endocarditis have 
also been noted. 

Treatment. — Prophylactic. — Immunization against colon bacillus 
infection can be produced, as in typhoid bacillus infection, by giving 
one injection of 300 millions followed in ten days by 500 millions. 



THE COLON BACILLUS GROUP. 267 

The serum can be prepared but is not at present employed therapeu- 
tically. It would have to be made by [injecting horses with many 
different strains. 

Curative Vaccine Treatment. — Localized inflammations due to the 
B, coli have been treated quite successfully by injections of dead or- 
ganisms. An injection of 25 millions can be made daily or 150 to 
300 millions every three to seven days. Autogenous vaccines should 
always be prepared if possible. 

Methods of Isolation. — While the isolation of typhoid bacilli from 
faeces, water, dust, etc., is attended, as a rule, with difficulty, pure 
cultures of colon bacilli can usually be obtained from such sub- 
stances by the simplest procedures. The following methods may be 
recommended : 

1. Inoculate 10 c.c. of fluid 2 per cent, lactose neutral litmus 
agar with diluted faces or suspected material. The melted agar 
should be at a temperature of about 41° C. After shaking pour in 
Petri dish. Several dilutions should be made. After eighteen hours 
at 37° C. examine the plates and inoculate the contents of a number 
of tubes containing 2 per cent, lactose agar with any colonies showing a 
red color. The colon bacilli will produce gas and acid (see page 256). 

2. Inoculation of increasing quantities of the material (water) in 
2 per cent, dextrose nutrient bouillon and 2 per cent, lactose peptone, 
solution or lactose peptone bile contained in fermentation tubes. The 
presence of colon bacilli in the inoculated portion produces after twelve 
to twenty-four hours active fermentation. 

Bacillus [Lactis] Aerogenes. — This organism resembles very closely 
the colon bacillus, and in ordinary tests is not differentiated from it. 
Furthermore, the two organisms are often found together in the intes- 
tines and in infections elsewhere. B. aerogenese is found frequently 
in milk (especially in sour milk) where it usually develops a capsule. 

Another capsule -forming bacterium which may be placed in this 
group is 

THE PNEUMOBAOILLUS OF FRIEDLANDER.— B. PNEUMONIS, 

B. [MU008U8] 0AP8ULATU8. 

This bacillus discovered by Friedlander (1883) is now known to 
occur frequently as a mixed infection in cases of phthisis, fibrinous 
pneumonia, and in rare instances as the only exciting factor in pneu- 
monia. It is also not infrequently found in the mucous membranes of 
the mouth and air passages of healthy individuals. 

Morphology. — Short bacilli with rounded ends, often resembling 
micrococci, especially in recent cultures; commonly united in pairs 
or in chains of four, and, under certain circumstances, surrounded 
by a transparent capsule. This capsule is not seen in preparations 
made from artificial culture media, but is visible in well-stained 
preparations from the blood of an inoculated animal. 

Friedlander's bacillus stains readily with the aniline colors, but is 
not stained by Gram's method. 



268 PATHOGENIC MICRO-ORGANISMS, 

Biology. — An aerobic, non-motile, non-liquefying bacillus; also faculta- 
tive anaerobic; does not form spores. In gelatin stick cuUwrea it presents 
the *' nail-shaped" growth first described by Friedlander, which is not, how- 
ever, peculiar to this bacillus, and in old cultures the gelatin acquires a dis- 
tinct brownish coloration. This latter characteristic distinguishes the 
growth of this bacillus from that of the Bacillus aerogenes^ which is otherwise 
very similar to it morphologically. On gelatin plates colonies appear at the 
end of twenty-four hours as small white spheres, which rapidly increase in 
size. These colonies, when examined by a low-power lens, present a some- 
what irregular outline and a slightly granular appearance. The growth on 
agar is in quite large and moist grayish colonies. The growth on potato is 
luxuriant — a thick, yellowish-white, glistening layer rapidly covering the 
entire surface. Milk is not coagulated. Indol is produced in bouillon or 
peptone solutions. Milk-sugar and glucose are fermented. Growth occurs 
at 16"* to 20° C, but is more rapid at 37° C. 

Pathogenesis. — ^Friedlander's bacillus is pathogenic for mice and 
guinea-pigs, less so for dogs, and rabbits are apparently immune. On 
autopsy after death due to inoculation into the lungs, the pleural 
cavities are found to contain a seropurulent fluid, the lungs are in- 
tensely congested, and in places show limited areas of red hepatiza- 
tion; the spleen is considerably enlarged, and bacilli are present in 
the lungs, the pleuritic fluid, and the blood. 

Friedlander's bacillus has been found in man, not only in patients 
suffering from croupous pneumonia and other respiratory diseases, 
but in healthy individuals, and also in the outside world. Netter 
observed it in 4 . 5 per cent, of the cases examined by him in the saliva 
of healthy individuals, and Pansini in cases of pulmonary tuberculosis 
in the sputum. In 129 cases of pneumonia examined by Weichselbaum 
this bacillus was found in only 9. The cases which are due primarily 
to the pneumo-bacillus are distinguished, according to Weichselbaum 
and Netter, by their peculiarly malignant type and by the viscidity of 
the exudate produced. This bacillus is also probably concerned, 
primarily or secondarily, under certain circumstances, in the produc- 
tion of pleurisy, abscess of the lungs, pericarditis, endocarditis, otitis 
media, and meningitis, in all of which it has at times been found to be 
present. Vaccines have been used successfully in treatment. 

The ''bacillus of rhinoscleroma" (see Fig. 18), found frequently in 
the lesions of an infectious-granuloma type of disease in the nose, is 
very similar to the B, capsulatus. Some investigators do not consider 
it possible to differentiate the two organisms. Some believe that the 
bacilli found in rhinoscleroma are secondary invaders. The rhino- 
scleroma bacilli do not always produce gas in sugar media and they 
are only slightly pathogenic for experimental animals. 

INTERMEDIATE MEMBERS OF THE TYPHOID -COLON GROUP OF 

BACILLI. 

Gartner's discovery in 1888 of the Bacillus enteritidis, in association 
with epidemics of meat poisoning, first gave impetus to the study of a 
number of parasitic bacteria resembling in many characteristics the 
colon or typhoid bacilli. These bacilli are fequently termed inter- 



THE COLON BACILLUS GROUP. 269 

mediates. Nocard's work showing that Bacillus psittacosis caused 
infection in parrots followed in 1892. In 1893 Gilbert introduced the 
terms "paracolon" and ** paratyphoid" to designate bacilli of this 
group resembling more nearly in biological characters the colon 
bacillus on the one hand and the typhoid bacillus on the other. 

The intermediates include Bacillus enteritidis and similar organ- 
isms recovered from cases of epidemic meat poisoning, the gas-pro- 
ducing typhoid-like bacilli of various observers obtained from cases 
suffering from typhoidal symptoms, Bacillus psittacosis, Bacillus 
cholerce suis (hog-cholera), bacillus of swine plaguCy Bacillus icteroides, 
Bacillus alcaligenes (Tables, pp. 256, 257). 

The paracolon and paratyphoid can be distinguished without diffi- 
culty from the typhoid bacilli. They produce gas in glucose media, 
and in this respect they differ from typhoid, but, unlike Bacillus coli, 
they do not produce gas from lactose, coagulate milk, or, as a rule, form 
indol. 

The main points of difference between the two varieties are that the 
paracolons turn milk and whey alkaline after a short initial acidity 
and form gas freely in glucose media, while with the paratyphoids there 
is in milk and whey an initial acidity, but no or very slight subsequent 
alkalinity; the gas production in glucose media is much less pronounced. 
Neutral red agar also differentiates between the two groups. Like 
Bacillus coli, all the intermediated reduce the color to yellow in twenty- 
four to seventy-two hours, but with the paratyphoids after four or 
five days the red color begins to return from above downward until 
in two or three weeks the medium is again red throughout. With the 
paracolons the yellow color is permanent. (Refer to table pp. 255, 
256, for chief differential points of whole group.) 

Agglutination tests applied to the intermediates show that the mem- 
bers of the paracolon group do not all show mutual reactions, and 
the group must, therefore, be composed of a number of distinct races, 
as in the case with Bacillus coli. The paratyphoids, on the other hand, 
most of which have been isolated from cases simulating typhoid fever 
belong chiefly to two strains; that is to say, an active serum prepared 
from either strain of the bacilli will agglutinate all the others of that 
strain. These are designated as type A and type B. 

RelatiTe Frequency of Paratyphoid Infections. — Gwyn's case was 
the only one of 265 cases which failed to give Widal reaction. Schott- 
miiller and Kurth from a total of 180 cases which had been looked 
upon as typhoid, were able in 12 cases to isolate a paratyphoid bacillus. 
Johnston's 4 cases were found among 194, and Hewlett's 1 in a series 
of 26 cases of typhoid fever. Hiinermann has reported an epidemic 
of 38 cases of paratyphoid infection occurring in the garrison at 
Saarbnick. Falcioni reports 5 cases out of 100 cases of supposed 
typhoid fever. The proportion of negative Widal reactions is low in 
the statistics, but there is a source of error here in that until very 
recently the tests have not been made in high enough dilutions — that 
is, at least as high as 1 : 40. 



270 PATHOGENIC MICRO-ORGANISMS. 

Post-mortem Findings. — Autopsies were performed on 3 fatal cases 
(Strong, Longcope, Tuttle). The interest in these autopsies naturally 
centres on the condition of the intestines. Strong states that both 
the large and the small intestines were normal throughout except for 
moderate catarrh and a few superficial hemorrhages. The solitary 
and agminated follicles showed no lesions. The mesenteric lymphatics, 
however, and some along the small intestines, were hemorrhagic. 
In Ivongcope's case the intestines showed no changes either on gross 
or microscopic examination. The spleen in both cases was enlarged. 
The other pathological changes were those common to febrile con- 
ditions. In Tuttle's case a few erosions just above the ileocaecal valve 
were present. 

Source of Infecting Bacilli. — ^Tuttle's case happened to be a labora- 
tory employ^ in the service of the Department of Health and was 
carefully investigated by us. We found that two families consisting 
of eleven members drank water from an open uncovered tank. During 
the summer the tank was not cleaned and was only occasionally filled 
by pumping in water from the city supply. Sometimes the water 
was the color of tea. During a single week four members of one 
family and three of the other were stricken with a typhoid-like fever. 
The two families had no social intercourse with each other. 

Symptomatology. — It is a significant fact that many of the reported 
cases of paratyphoid infection were considered to be genuine typhoid 
fever without the Gruber-Widal reaction until a bacteriological study 
revealed their character. Tuttle's case had severe hemorrhages and 
was considered in the hospital as true typhoid infection until the 
cultures proved it to be paratyphoid. The average course, lasting 
frequently only 12 to 18 days, is milder. The cases due to the paracolon 
bacilli are apt to run a course more like those due to the Bacillus 
enteriiidis in meat poisoning. 

The Serum Reaction in Cases of Paratyphoid Infection. — Since the 
introduction of serum reactions as a means of diagnosis, it has been 
a well-recognized fact that a small proportion of cases which are 
clinically typhoid fever fail to give the reaction. Brill, adding to 
Cabot's statistics, finds that of 4879 cases 4781, or 97.9 per cent., 
gave the reaction. Gwyn gives 99.6 per cent, as the percentage of 
positive reactions in the Johns Hopkins Hospital. On the other 
hand, in most of the reported cases of paratyphoid infection a reaction, 
except with low dilutions, against the Bacillus typhosus has been 
absent. It is probable, then, that some at least of the typhoid cases 
with negative reactions were really paratyphoid infection. 

Still it cannot be assumed that all cases clinically typhoid fever, 
which have been reported as giving the Gruber-Widal reaction, were 
cases of genuine typhoid infection. The brilliant work of Durham on 
the typhoid-colon group of bacilli and its serum reactions has brought 
out the fact that certain members of this group may be mutually 
interacted upon by sera of infected patients and of immunized animals. 
This is especially true of sera in low dilution. Since in the earlier 



k 



THE COLON BACILLUS GROUP, 271 

years of the Gruber-Widal reaction the technique had not been worked 
out, and dilutions were more frequently low than not, some of the 
cases reported as typhoid fever may have been infections with para- 
typhoid bacilli. 

Diagnosis. — The only reliable criteria for diagnosis are absence of 
the Gruber-Widal reaction in proper dilution (not less than 1:40) 
with a positive reaction against a known paratyphoid bacillus or the 
recovery of a paratyphoid bacillus from the blood, urine, or compli- 
cating inflammatory process. 

The clinical type of the disease is of little value in a single case. 
It has already been stated that the reported cases of paratyphoid in- 
fection have been both mild and severe. 

The cases of paratyphoid infection are too few to state what the 
prognosis should be. It can only be said that the majority of the 
cases have been mild, though there have been about 9 per cent, of 
deaths among the cases reported. The differential diagnosis between 
infections due to the typhoid bacillus and to those due to the para- 
typhoids and more rarely the paracolons is of importance mainly from 
the etiological side. If a specific serum therapy is ever successfully 
instituted the differentiation would be of more importance. 

Epidemic Meat-poisoning Tsrpe. — Gartner announced his discovery 
of Bacillus enieritidis as the cause of epidemic meat poisoning in 1888. 
A cow sick for two days with profuse diarrhoea had been slaughtered 
in Saxony and the meat sold for food. Of the persons who ate of the 
meat 57 became ill, and 1 died. Gartner recovered the bacillus from 
the meat and from the organs in the fatal case. 

Previous to Gartner's discovery the cause of meat poisoning had 
been held to be bacterial products, and while this is true of certain 
instances it is the exception. All cases in which a thorough bacteri- 
ological examination has not been made must be excluded. 

Two kinds of bacilli are concerned in the production of meat poi- 
soning: 1. Bacillus enieritidis of Gartner, including the different 
strains of this organism. 2. Anaerobic Badllvs hotidinus of Van Erm- 
inghem, a saprophyte (see later under anaerobic bacilli). 

True Meat Poisoning. — ^This form of meat poisoning is due to Bacil- 
lus enieritidis f and in every instance the animal is diseased at the time 
of the slaughter. It may be contracted by eating sausage, since the 
meat of diseased animals is sometimes surreptitiously put on the market 
in the form of sausage. 

Durham makes BaciUus enieritidis the chief type of the intermediates 
and proposes the name "the enteritidis group.'* Buxton classes 
the bacillus with the paracolons. It does not ferment lactose; milk 
becomes more alkaline; it ferments dextrose with a production of gas 
containing about one-third COj, two-thirds H, and it also ferments 
mannite, maltose, and dextrin. 

Bacillus enieritidis is pathogenic for cows, horses, pigs, goats, 
mice, and guinea-pigs, but not for dogs and cats. 

The Infected Meat. — In many epidemics Bacillus enieritidis has 



272 PATHOGENIC MICRO-ORGANISMS. 

been isolated not only from the organs of fatal eases, but from the 
suspected meat. The meat does not differ in appearance or taste 
from that of healthy animals. It has already b.een stated that it may 
be made into sausages, and one epidemic at least has been caused by 
eating ** dried meat" consisting of large pieces of the flesh of sheep 
and goats nearly dried in the sun and eaten cooked or merely softened 
by soaking. Cooking does not always destroy the bacilli, as the 
thermal death point may not be reached in the interior of the meat. 
Infected meat which is not eaten immediately after it has been cooked 
is especially dangerous. 

The meat has always come from animals sick at the time of slaughter. 
The meat of cows and calves have most often caused the epidemics, 
though that of horses, pigs, and goats have also been responsible. 
Durham says that no known case has come from mutton, and that 
the pig has been implicated in only one outbreak which has been 
studied bacteriologically. In this connection it is interesting to recall 
that Theobald Smith has insisted on the similarity between the hog- 
cholera bacillus and Bacillus enieritidis. 

The animals from which the infected meat has come have suffered 
during life from puerperal fever and uterine inflammations, navel 
infection in calves, septicaemia, septicopysemia, diarrhoea, and local 
suppurations, and have not infrequently been killed because of their 
unsound condition. How animals become infected is not known. 

Durham thinks milk may be a source of infection in man, but 
states that bacteriological evidence of it is incomplete. Bacillus en- 
teritidis has been found, however, in the milk of markedly infected 
guinea-pigs. 

Transmission to Man. — ^The disease may be transmitted to man in 
two ways: (1) by eating the infected meat, and this is by far the most 
common means, and (2) from man to man according to Gartner, Van 
Erminghem, and Fischer. Fischer thinks transmission may take place 
through the excreta. B, psittacosis has also been transmitted to man. 

Epidemics of meat poisoning may occur in any season, but are more 
frequent during the warm months. 

Symptomatology. — While the characteristic symptoms of sausage 
poisoning relate to the nervous system, in true meat poisoning they 
are gastrointestinal. Fischer divides meat poisoning into three clinical 
forms: (1) typhoidal; (2) choleraic; (3) gastroenteric. 

Prevention. — Since neither appearance nor taste affords any clue to 
the noxious quality of the infected meat, its unfitness for food can 
only be told through bacteriological examination or a knowledge of 
its source. Thorough cooking will kill the bacilli, but it must be re- 
membered that in this process the thermal death point of the bacilli 
may not be reached in the innermost portions of the meat. 

BACILLUS ALCAUGENES. 

This bacillus resembles somewhat a colon bacillus which has lost 
its power to ferment sugars. Morphologically and culturally it is 



THE COLON BACILLUS GROUP, 273 

more like the typhoid bacillus. It ferments no sugars. It is fre- 
quently present in the intestines and may have pathogenic properties, 
which facts have already been mentioned in speaking of the interme- 
diate group of bacilli (see table p. 257). 

BACILLUS OF HOG CHOLERA (B. CH0LERAE-8UIS). 

This is an actively motile bacillus. Grows vigorously in bouillon. 
Renders milk at first slightly acid then strongly alkaline, and dissolves 
casein. Ferments dextrose with gas production (see table p. 256). 

This bacillus is found almost regularly present in hogs sick with 
cholera, but is known now not to be the essential exciting factor, since 
this is a virus which passes through a fine filter. Even though now 
considered not to be the essential factor in exciting hog cholera it is 
believed to be of importance as an added infection. It is pathogenic 
for hogs causing, when fed, fatal enteritis. 

BACILLUS OF SWINE PLAGUE. 

This, a non-motile bacillus which grows feebly in bouillon, does not 
coagulate milk, and ferments glucose without production of gas. 
When fed to pigs it does not usually cause illness (see table p. 256). 
This bacillus is closely related to the hemorrhagic septicemia group. 



i8 



CHAPTER XXI. 

THE DYSENTERY BACILLUS— THE PARADYSENTERY BACILLI 

(MANNITE FERMENTING TYPES). 

Dysentery may be divided into acute and chronic. Amoebee ap- 
pear to be the chief exciting factor in most cases of chronic dysentery, 
though bacilli of the colon group also play a part. 

In temperate climates acute dysentery is but very rarely due to 
amoebae, but usually to the bacilli identified by Shiga or to allied 
strains identified by Kruse, Flexner, and Park. The usual summer 
diarrhoeas are not excited by the dysentery bacilli. 

Historical Note. — In 1897 Shiga found in the stools of cases of 
dysentery a bacillus which had not been before identified. This ba- 
cillus had many of the characteristics of the colon bacillus, but dif- 
fered from it, lacking motility and failing to produce gas from the 
fermentation of sugar. It also was entirely distinct in its aggluti- 
nation characteristics and in its pathogenic properties. Shiga found 
this bacillus present in all the cases of epidemic dysentery that he 
examined. It was most abundant during the height of the disease 
and disappeared with the return of faecal stools. It was not found in 
the stools of healthy persons. He found that the blood of dysenteric 
patients contained substances which agglutinated the bacilli that 
he had isolated. The serum from healthy individuals did not aggluti- 
nate the bacilli to any such degree as the serum from those sick with 
dysentery. When the mucous membrane of the colon was examined 
in fatal cases dying in the height of the disease, the bacilli were found 
in the superficial layers in almost pure cultures. A criminal fed 
with a culture of the bacillus developed typical dysentery. Certain 
animals, such as dogs, when subjected to treatment which made them 
more susceptible, were attacked with dysentery after feeding on cultures. 
This was fairly similar to that in man. 

Morphological and Oultnral Characteristics of Dysentery Bacilli. — 

Microscopic. — Similar to bacilli of the colon group. 

Staining. — Similar to bacilli of the colon group. 

Motility. — No definite motility has been observed. The molecular 
movement is very active. 

Flagella. — True flagella have not been observed by most examiners. 
On a very few bacilli in suitable smears Goodwin demonstrated what 
appeared to be terminal flagella. Spores are not formed. 

Appearance of Cultures. — On gelatin the colonies appear more like 
the typhoid than the colon bacilli. Gelatin is not liquefied. On 
agar, growth is somewhat more delicate than that of the average colon 
cultures. 

274 



THE DYSENTERY BACILLUS GROUP. 275 

Oft Potato.^-X delicate growth just visible or distinctly brownish. 

In Bouillon. — Diffuse cloudiness with slight deposit and sometimes 
a pellicle. Indol not produced ot in a trace only. 

In glucose bouillon no production of acid or gas. 

Neutral red agar is not blanched. 

In Litmus Milk. ^After twenty-four to forty-eight hours this be- 
comes a pale lilac.' Later, three to eight days, there is a return to 
the original pale blue color. The milk is not otherwise altered in 
appearance. 

PathogenesiB. — Animal Teats.— No characteristic lesions have fol- 
lowed swallowing large quantities of bacilli. Dogs at times have 
had diarrhoea with slimy stools, but section showed merely a hypersemia 
of the small intestine. 

Many animals are very sensitive to bacilli injected into vein or 
peritoneum; 0.1 mg, of agar culture injected intravenously produced 




Colony of dyecnteiy bacilli in lelalia. 
X *0 dism. 

diarrhcea, paralysis, and death; 0.2 mg. under the skin have killed, 
and the same amount in (he peritoneum has caused bloody peritonitis, 
with lowered temperature and diarrhcea. Both small and large animals 
are very sensitive to killed cultures. 

The autopsy of animals dying quickly from Injection into the per- 
itoneum of living or dead bacilli shows the peritoneum to be hyper- 
femic, the cavity more or less filled with serous or bloody serous exu- 
date. The liver is frequently covered with fibrinous masses. The 
spleen is moderately or not at all swollen. The small intestine is filled 
with fluid, the large intestine is usually empty. The mucous mem- 
brane of both is hypereemic and sometimes contains hemorrhages. 
Conradi found ulcer formation in one case. 

Subcutaneous injections of dead or living cultures are followed by 
infiltration of tissues and frequently by abscess formation. The 
dysentery bacilli produce toth extracellular and cellular toxins, the 



276 PATHOGENIC MICRO-ORGANISMS. 

latter being the most abundant. The elimination of these toxins 
from the body is supposed to take place through the intestines, and 
this gives rise to the intestinal lesions in animals injected intravenously 
or intraperi tone ally. The dysentery bacilli are not found in the 
blood or organs of animals. 

In Man. — In the onset acute dysenteiy is sudden and ushered in 
by cramps, diarrhcea, and tenesmus. The stools, at first feculent, then 
seromucous, become bloody or composed of coffee-ground sediment. 
At the height of the disease there are ten to fifty stools in the twenty- 
four hours. After two to seven days the blood usually disappears. 
In temperate climates the mortality varies from 5 to 20 per cent. 
Bacillary dysentery is a disease especially of the mucous membrane 
of the large intestines. The epithelium is chiefly involved. In the 
lightest cases a catarrhal inflammation is alone present, in the more 
severe the lymph follicles are swollen and some necrosis of epithelium 
takes place. 

In severe cases in adults the lesions are of a diphtheritic character 
and may be very marked. The entire lumen of the intestines may 
be filled with a fibrinous mass of pseudomembrane. In young chil- 
dren, even in fatal cases, the lesions may be more superficial. The 
following macroscopic and microscopic report of the intestinal find- 
ings on a fatal case of bacillary dysentery in an infant is a typical 
picture: 

SmaU InUslines. — Slightly distended. Mesenteric glands large and red. 
Peyer's patches are swollen slightly without ulceration. 

Large InUstines. — Outer surface vessels congested and prominent, on 
section, covered with a yellowish mucus. Mucous membrane seems to be 
absent in places. Solitary follicles are elevated and enlarged, especially in 
the region of sigmoid flexure. In some instances the centres of the follicles 
are depressed and appear to be necrotic. 

Large Intestine. — Mucous glands arc for the most part normal, but over the 
solitary follicles they have broken down somewhat and contain polynuclear 
leukocytes. The ioterglandular stroma in these places has undergone 
necrosis. The necrotic area extends down into the submucosa in the region 
of the solitary follicles. The capillaries of the solitary follicles are much 
dilated and congested. The submucosa is thickened and slightly (sdematous. 
The connective-tissue cells appear to have undergone a slight hyfdine degenera- 
tion. The musculature is not affected, neither is the peritoneal coat. 

Small Intestines. — Normal. , 

Paradysentery Bacilli as Exciters of Dysentery.— In 1900 Flexner 
and Strong, when in the Philippine Islands, isolated bacilli from dysen- 
ich were identical with the Shiga cultures. At first 
re supposed to be of the Shiga type, but later, among 
bacilli were found, which differed from Shiga's in 
ristics. In the same year Kruse, in Germany, ob- 
'senteric cases in an asylum bacilli which appeared to 
irally like those isolated by Shiga, but to differ in their 
haraeteristics. These, like those isolated by Flexner, 
i to differ in many characteristics. In 1902 Duval and 



THE DYSENTERY BACILLUS GROUP. 277 

Bassett, in Baltimore, thought they had found the Shiga bacilli in the 
stools of a number of cases of summer diarrhoea. These later proved to 
be identical with some of the bacilli isolated by Flexner in Manila. Dur- 
ing the same summer Park and Dunham isolated a bacillus from a 
severe case of dysentery occurring during an epidemic at Seal Harbor, 
Mt. Desert, Maine, which they showed to differ from the Shiga bacillus in 
that it produced indol in peptone solution and differed in agglutinating 
characteristics.^ They at first considered it identical with the Philip- 
pine culture given them by Flexner, but in January, 1903, it was shown 
by Park to be a distinct variety, and later found by him to be the exciting 
factor in a large number of cases in several widely separated epidemics. 

Martini and Lentz^ published the results of their work in December, 
1902. They showed that the Shiga type of bacilli obtained from several 
separate epidemics in Europe agreed with the original Shiga culture 
in that it did not ferment mannite. The cultures of this type agreed 
with each other in agglutinating characteristics. When the bacilli 
from Flexner, Strong, Kruse, Park, Duval, and others, which differed 
from the Shiga culture in their agglutinins, were tested they were all 
found to ferment mannite. Martini and Lentz considered that the 
Shiga bacillus was the true dysentery type and that the mannite-fer- 
menting variety or varieties might be mere saprophytes, or perhaps 
be a factor in the less characteristic cases. 

In January, 1903, Hiss^ and Russell, independently of others, 
showed that a bacillus isolated by them from a characteristic stool 
differed from Shiga's bacillus in the same characteristics as mentioned 
by Martini and Lentz. 

The German observers at first considered the Shiga type as the only 
one which had established its causal relation to dysentery, while 
the American observers generally considered both types to have equal 
standing and some* of them considered these differences as snot im- 
portant and perhaps not permanent. This latter opinion seems to 
have been held by Shiga.* 

We took up the investigation at this point with the object of care- 
fully studying the bacilli isolated by us from acute dysentery, which 
occurred in a number of widely separated epidemics. We hoped 
thus to determine whether the bacilli exciting acute dysentery in the 
Eastern States belonged to a few distinct types or were divided into 
a large number of varieties. 

In the most extensive epidemic that has recently occurred in the 
region of New York City there were in all some 500 cases of acute 
typical dysentery. Whole families were attacked with the disease. 

The majority of the cases were of moderate severity, the dysenteric 
discharges lasting from one to two weeks. There were a number of 
light cases, but all had dysenteric stools containing mucus and blood. 

* New York University Bulletin of the Medical Sciences, October, 1902, p. 187. 

* Zeitschrift f. Hygiene u. Infectionskrank., 1902, xli., 540 and 559. 

* Medical News, 1903, Ixxxii., 289. 

* University of Pennsylvania Medical Bulletin, July and August, 1903. 

* Zeit^schrift f . Hygiene u. Infectionskrank., 1902, xli., 356. 



278 PATHOGENIC MICRO-ORGANISMS, 

The mortality was about 6 per cent. Judging from the cases investi- 
gated by us, over one-half of those attacked seem to have been infected 
by the Shiga type, and these were, as a rule, the most severe cases. 
Most of the cases in two severe, though localized, epidemics in a 
Pennsylvania town and at Sheepshead Bay were also due to this 
type. The mortality was higher in these epidemics. The facts pub- 
lished abroad indicate that this variety has been found in the chief 
epidemics in Europe and Asia. We have never yet succeeded in 
isolating bacilli which had all the characteristics of the Shiga variety 
from any diarrhoea cases in which no dysenteric symptoms appeared. 

We turn now to the mannite-fermenting varieties, whose relation- 
ship to dysentery is still doubted by some. 

The cultures isolated by us from over forty cases were found to fall 
largely into two distinct types, one of which differs from the Shiga 
bacillus more radically than the other. 

The variety nearer to the Shiga bacillus has the characteristics of 
the culture, which was isolated by us at Seal Harbor, Maine, in August, 
1902. The other variety is represented by the Flexner Philippine 
type. 

The first type differs from the Shiga bacillus in its agglutinating 
characteristics and in that it produces considerable indol in peptone 
solution and ferments mannite with the production of acids. The 
second type differs in these points and in addition in its agglutinating 
characteristics and in fermenting chemically pure maltose in peptone 
solution. 

Besides the epidemic at Seal Harbor, numerous cases of moderately 
severe or slight dysentery due to the first type were met with in the 
extensive epidemic which has been already alluded to in the towns 
north of New York City. A few characteristic and many slightly 
developed cases of dysentery in New York City during the past two 
summers were caused by this type of bacillus. A great many cases 
are also due to the Philippine type. A number of rather severe cases 
of dysentery developed in Orange, N. J. Cultures from two cases 
were made, and this latter type alone obtained. 

At Riker's Island dysentery broke out in the penitentiary. A con- 
siderable number of the inmates including the attendants and doctor 
in charge came down with the disease. They usually had a short, 
sharp attack with a quick recovery. Large amounts of blood were 
passed by some. Those of the infected who were able to work were 
sent to the kitchen. This fact and the facts that open closets were near 
and that there were immense numbers of flies about were probably 
responsible for the spread. At the time of the epidemic, a contractor 
and some workmen were filling in the lower part of the island, about 
half a mile from the penitentiary. They were not allowed within 
the penitentiary inclosure. Not one of them contracted the disease. 

A large proportion of the bacteria isolated from these dysentery cases 
were bacilli of the Philippine type. No other type of dysentery bacilli 
was found in any of the cases in this epidemic. 



THE DYSENTERY BACILLUS GROUP. 279 

Charlton and Jehle report a series of cases occurring in Vienna, in 
which mannite-fermenting types were alone present. 

Summer DiarrhoBa. — Cases of ordinary enteritis without the symp- 
toms of dysentery are not excited by any of the the types of dysen- 
tery bacilli. 

Specific or Group Agglutinins Produced by the Three Types.— 

These are interesting as showing that cultures of each type selected 
from widely separated sources were identical with each other. 

Table I. — Agglutination of biicilli belonging to the three types in the serum of a 
young goat injected with the bacillus isolated by Shiga, in Japan. 

Source. 
Type I. Shiga. 

1. Original, Japan — Shiga, 

2. New Haven — Duval, 

3. Tuckahoe — Carey, 

4. Conev Island — Collins, 

5. Mt. Vernon. Case 1. — Collins, 
Type II. 

o. Original. Mt. Desert — Park, 

7. New York City — Goodwin, 

8. Hospital. New York— Collins, 

9. Foundling Hospital — Hiss, 

10. Mt. Vernon. Case I. — Collins, 
Type III. 

11. Origmal. Manila — Flexner, 

12. Baltimore — Duvid, 

13. New York City— WoUstein, 

14. Orange — Collins, 
16. Bikers Island — Ooodwin, 
The serum of this goat before injection did not agglutinate any of the above bacilli in 1 : 10 dilution. 

+ + —complete reaction. + —good reaction. I —slight reaction. 

+ I —very good reaction. ± —fair reaction. — —no reaction. 

When the agglutinating characteristics of these bacilli, and their 
susceptibility to immune sera are studied carefully, we find that each 
of the three types differs from the others. The mannite and the 
maltose types, since in animals they stimulate abundant common 
agglutinins and immune bodies, seem more closely allied to each other 
than to the Shiga type. 

This is seen in the accompanying tables, in which bacilli from a 
number of cases obtained from different sources are tested in sera from 
animals which have each received a single type of dysentery bacillus: 

Table II. — Showing agglutination of members of three types in the serum of animals 

injected with bacilli of Type if. 

Goat injected with No. 4. Rabbit injected with No. 6. 







Dilutions of Serum. 






1:20 


1:50 


1:100 


1:200 


1:500 


1:2000 


1:5000 


+ + 


+ + 


+ -H 


+ + 


+ + 


+ 4- 


+ 


+ + 


+ + 


+ + 


+ + 


+ + 


+ + 


+ 


+ 1 


+ + 


+ + 


+ + 


+ + 


+ 1 


± 


+ + 


+.+ 


+ + 


+ + 


+ + 


+ + 


+ 


+ + 


+ + 


+ -I- 


+ + 


+ + 


+ 1 


+ 


+ 1 


+ 1 


1 


_ 


,— 






+ 1 


+ 1 


1 


— 


— 






+ 1 


+ 1 


1 


— 


— 






4- I 


+ 1 


1 


— 


— 






+ 1 


1 


— 


— 


— 






+ 


+ 


+ 


± 


_— 






+ 1 


+ 1 


+ 


± 


— 






+ + 


+ 


± 


1 


— 






+ 


+ 


± 


— 


— 






+ + 


+ + 


+ 


1 


— 







Source. 1:20 1:50 1:100 1:500 1:1000 1.-20 1:50 1:100 1:500 1:800 
Type I. Shiga. 

1. Japan — Shiga, 4. — _ — _ |___>_ 

2. New Haven — Duval, + — — — — |___>_ 

3. Tuckahoe— Carey. -|.__-_. |____ 

Type II. 

4. Mt. boertr— Park. ++++++ ++ ++ ++++++ + I 

5. Mt. Vernon— ++++++ ++ ++ ++++++ + + 
Collins. 

6. New York— Rise, ++++++ ++ ++ ++++++ + -* 

Type III. 

7. Manila— Flexner. ++++ + - - ++++++ - - 

8. Baltimore— Duval ++++ + - - ++++++ - - 

9. Riker'»— Goodwin, ++++ + - - ++++++ - - 

The serum of the above animals previous to immunisation did not agglutinate any of the above 
bacilli in a 1 :20 dilution. 



280 PATHOGENIC MICRO-ORGANISMS. 

Table III. — Showing iigglutinationa of members of three types in the serum of 

animals injected with bacilli of Type III. 

Rabbit injected with Baltimore, DuvaL 



10 50 100 500 1.000 5.000 10.000 

Type I. 

1. Japan — Shiga, and 5 other cultures, +++ 4. — — — — 

TVpe II. 

6. Mt. Desert — Paric, and 5 other cultures, ++++ 4- — — — — 

TVpe III. 

Manila — Flexner. and 5 other cultures, +4- ++ ++ -I-+ ++ +-H + 

Previous to immunisation the serum agglutinated the bacilli of Type III. in 1 : 20 dilution but 
none of the others even in 1 : 10. This is one of the few animals in which agglutinins for Type I. 
developed through the injections of bacilli of the other types. 

Table IV. — Showing how Type III. is unable to absorb the agglutinins produced 
through injections of Type II. Serum from rabbit inoculated with Mt. Vernon 
culture^ Type II. 

Agglutinins exhausted with 



Serum 
t>efor( 

bbson 
tion. 



j[{JJ2I?_ Baltimore, Duval. Mt. Vernon, cc.s 



1:20 1:50 1:100 1:200 1:400 1:20 1:50 1:100 



Shiga, 5 other cultures, 1:10 — '— — — — — — — 

Type II. 

MtTDesert, 5 other cultures, 1 :600 ++++++ +1 I - - — 

Type III. 

Manila, 5 other cultures, 1:100 — — — — _^.+ | _ 

Before injections this rabbit's serum agglutinated Types II. and 
III. in 1:20 dilutions. 

The considerable amount of common agglutinins affecting Type 
II. and Type III. is seen to be absorbed by the bacilli of either type. 
The larger amount of specific agglutinin is left in the serum when 
any bacillus other than one of identical characteristics with the bacillus 
used in the immunization is employed. 

Table V. — Showing that horses injected with Shiga and Philippine types develop 
specific agglutinins for the bacilli belonging to these tu)o types and common 
agglutinins for the varieties included under Type II. 

Serum 
after Same serum after saturation with cultures of 

injec- • » 

Cultures. tionsfor Shiga Type III. Type II. Pyocy- Typhoid. Colon. 

several type. aneus. 

months. 
Type I. 

Shiga, original, and 4 others. +1500 - 10 +400 +700 +1000 +300 +300 

Type II. 
Park, original, and 4 others. +600-10 - 10 - 10 +600+30 +50 



TVpe h. (B.) 
Brooklyn 



Brooklyn +600+20+10+60+300+100+50 

Type II. (C.) +300-10 - 10 +50 +50+10 +20 

Type II. (D.) +600-20 - 10 +50 +100+30 +60 

Type III 

Flexner, original, and 4 othere. -1200 +400 -10 +600 +800+300 +600 



The manipulation necessary in making dilutions and filtering, as 
well as the effect of standing, cause a certain amount of destruction of 
agglutinins. 

Summary. — The great majority of the bacilli which have been 
isolated from cases of dysentery not due to amoebae, and which must 
be considered as being exciting factors in that disease, are included 
in three distinct varieties of types. 

The type most frequently found in severe epidemics is that of the 
first culture isolated by Shiga. Bacilli identical in biochemical and 
agglutinating characteristics with this bacillus have been isolated 



THE DYSENTERY BACILLUS GROUP. 281 

from cases of dysentery in many parts of the world. None of the 
bacilli belonging to this type produce indol, except, perhaps, in a 
trace, or ferment mannite, maltose, or saccharose. Animals injected 
with this type produce specific agglutinins for this type in abundance 
and only very little that combines with the others (table I, page 279). 

The second type ferments mannite with the production of acid, 
but does not split maltose or saccharose in peptone solution or agar. 

It produces indol. Animals, after inoculations with it, develop im- 
mune bodies and agglutinins specific for the type (table II). 

The third type is nearest to the colon group, since it not only pro- 
duces indol and actively ferments mannite, but also acts energetically 
upon pure maltose and feebly upon saccharose. 

These two mannite-fermenting types are widely scattered over the 
world, and certainly cause characteristic cases and epidemics of dysen- 
tery, although on the average the disease caused by them is milder 
than when due to the Shiga bacillus. One or the other of these two 
types also appears at times in small numbers in mixed infections where 
dysenteric symptoms are almost or entirely absent. 

Although the majority of bacilli obtained have had the characteristics 
of one of the above types, a moderate number of bacilli have also been 
met with which differ slightly in biochemical as well as agglutinating 
characteristics. Some of these approach very closely the colon bacilli. 

It seems, therefore, that these three types should be considered as 
the characteristic representatives of three groups. 

In consideration of all the above facts, it seems to us incorrect to 
name the mannite-fermenting groups as pseudodysentery bacilli. If 
we call them all dysentery bacilli, we must classify them as dysentery 
bacilli of the Shiga group, of the group fermenting mannite, but not 
maltose, and of the one fermenting both mannite and maltose. 

This manner of differentiating the groups would be very confusing, 
and it seems to us more convenient, and better, to restrict the name 
dysentery to bacilli having the characteristics of the bacillus isolated 
by Shiga, and give the name paradysentery to the other two groups 
which approach more closely to the colon group in that they produce 
indol and have a greater range of activity in fermenting carbohydrates. 

An additional reason for the use of the prefix para, beyond that of 
convenience, is the less average severity of the disease due to these 
types, and the probability that there will be found, in occasional 
sporadic cases and epidemics of dysentery, bacilli which have a 
causal relation to dysentery and exhibit more pronounced character- 
istics of the colon group than any of the varieties so far isolated. 

Serum Treatment. — In characteristic cases the polyvalent serum 
is of considerable value. The serum is given subcutaneously in 
20 c.c. doses once or twice a day for several days, or until convales- 
cence is established. In cases of the usual summer diarrhoea the 
serum is not indicated. 



CHAPTER XXII. 
THE TYPHOID BACILLUS. 

This organism was first observed by Eberth, and independently by 
Koch, in 1880, in the spleen and diseased areas of the intestine in 
typhoid cadavers, but was not obtained in pure culture or its princi- 
pal biological features described until the researches of GaflFky 
in 1884. The methods of identification employed by Gaffky were 
found insuflBcient to separate the typhoid bacillus from other bacilli 
of the colon-typhoid group. The absolute identification of the 
bacillus only became possible with the increase of our knowledge 
concerning the specific immune substances developed in the bodies of 
immunized' animals. Its etiological relationship to typhoid fever has 
been particularly difficult of demonstration, for, although pathogenic 
for many animals when subcutaneously or intravenously inoculated, 
it has been impossible to produce infection in the natural way or pro- 
duce gross lesions corresponding closely to those occurring generally in 
man. Nevertheless the specific reactions of the blood serum of typhoid 
patients, the constant presence of the Bacillus typhosus in the intestines 
and some of the organs of the typhoid cadavers, the very frequent 
isolation of this bacillus from the roseola, spleen, blood, and excretions 
of the sick during life, the absence of the bacilli in healthy persons, 
unless they have at some time been directly exposed to, or are convales- 
cent from, typhoid infection, all these have demonstrated scientifically 
that this bacillus is the chief etiological factor in the production of the 
great majority of cases designated as typhoid fever. 

Morphology and Staidng. — Typhoid bacilli are short, rather 
plump rods of about l/£ to 3fi in length by 0.5/£ to 0.8/£ in diameter, 
having rounded ends, and often growing into long threads. They 
are longer and somewhat more slender in form than most of the 
members of the colon group of bacilli (Figs. 99 and 100). 

The typhoid bacilli stain with the ordinary aniline colors, but a 
little less intensely than do most other bacteria. Like the bacilli of 
the colon and paratyphoid groups, they are decolorized by Gram's 
method. Bi-polar staining is sometimes marked. 

Biology. — ^The typhoid bacillus is a motile, aerobic, facultative, 
anaerobic, non-liquefying bacillus. It develops best at 37° C; 
above 40° and below 30° growth is retarded; at 20° it is still moderate; 
below 10° it almost ceases. It grows slightly more abundantly in 
the presence of oxygen. It does not form spores. 

Resistance. — When a number of typhoid bacilli are dried most of 
them die within a few hours and a few frequently remain alive for 
months, but sometimes all the bacilli die very quickly. In their 

282 



THE TYPHOID BACILLUS. 283 

resistance to heat and cold they behave like the average non-spore- 
bearing bacilli. With rare exceptions they are killed by heating to 
60° C. for one minute. 

Motility. — Typhoid bacilli, when living under favorable condi- 
tions, are very actively motile, the smaller ones having often an un- 
dulating motion, while the larger rods move about rapidly. In different 
cultures, however, the degree of motility varies. 



Typboid twciUi froi 



Flacells, hnvily Ksined, sttuhed to Typhoid bsdilug with (ainlly slained 

bmcilli, (Viu EnneDgen'i metbod.) BaaelU. (Lo«ffler'9 meihod.) 

VUgalk, — These are often numerous and spring from the sides as 
well as the ends of the bacilli, but many short rods have but a single 
terminal flagellum (Figs. 101 and 102). 

Cultivation. — Its growth on most sugar-free culture media is quite 
similar to that of the Bacillus coli, but it is somewhat slower and not 
quite so luxuriant. 

Growth on 0«Utlii Plates (Fig. 103). — The colonies growing deep 
down in this plate medium have nothing in their appearance to dis- 
tinguish them from submerged colonies of the colon group; they ap- 
pear as finely granular round points with a sharp margin an<] a yellow- 



284 PATHOGENIC MICRO-ORGANISMS. 

ish-brown color. The superficial colonies, however, particularly 
when young, are often quite characteristic; they are transparent, bluish- 
white in color, with an irregular outline, not unlike a grape-leaf in shape. 
Slightly magnified they appear homogeneous in structure, but marked 
by a delicate network of furrows. Surface colonies from some varieties 
of colon bacilli give a similar picture. 

In slick cultures in gelatin the growth is mostly on the surface, appear- 
ing as a thin, scalloped extension, which gradually reaches out to the 
Pig ,03 sides of the tube. In the track 

of the needle there is but a hmited 
growth, which may be granular 
or uniform in structure, and of a 
yellowish-brown color. There 
is no liquefaction. 

Qrowtji in Bouillon.— This me- 
dium is uniformly cloudeil by 
the typhoid bacillus, but the 
clouding is not so intense as bv 
the colon bacillus. When the 
lx)uillon is somewhat alkaline a 
delicate pellicle is sometimes 
formed on the surface after 
eighteen to twenty-four hours' 
growth. 

Growth on Agu. — The streak 
cultures on agar are not distinc- 

typhoid bacilli in gelatin. X 26diaineli!rB. 11^^, 8 iransparcni, UUIOrm, 

grayish streak is formed. 

Orowth on Potato. — The growth on this medium was formerly of 
great importance in i<lentification, but now other media, giving more 
specific charactersitics, have been discovered. When characteristic, 
the growth is almost invisible but luxuriant, usually covering the 
surface of the medium, and when scraped with the needle offers a 
certain resistance. In some cases, however, the growth is restricted 
to the immediate vicinity of the point of inoculation. Again, the 
growth may be quite heavy and colored yellowish-brown, and with 
a greenish halo, when it is very similar to the growth of the colon 
bacillus. These differences of growth on patoto appear to be chiefly 
due to variations in the substance of the potato, especially in its re- 
action. For the characteristic growth the potato should be slightly 
acid. A new lot of potato should always be tested with a typical 
typhoid bacillus as a control. 

Indol Roaction.^It does not, as a rule, produce even a trace indol 
in peptone-water solution. This test was proposed by Kitasato for 
differentiating the typhoid bacillus from other similar bacilli such as 
those of the colon group, which, as a rule, give the indol reaction. 

The typhoid bacillus, like the colon bacillus, produces alkaUne 
substances from peptone. 



THE TYPHOID BACILLUS, 285 

Neutral Red. — In stick cultures in glucose agar the typhoid bacillus 
produces no change, while the colon bacillus decolorizes the medium 
and produces gas. 

Effect of Inhibiting Substances in Culture Fluids. — The typhoid bacil- 
lus is inhibited by weaker solutions of formaldehyde, carbolic acid, 
and other disinfectants than is the colon bacillus. Most typhoid-like 
bacilli resemble the typhoid bacillus in this respect. Some sub- 
stances, such as malachite green, inhibit the colon bacillus more. 

Action on Different Sugars. — The determination of the action upon 
sugars of any bacillus belonging to the typhoid or colon group is one 
of the most important of all the cultural differential tests. It has 
been considered in detail in connection with the colon group. 

While the typhoid bacillus does not produce gas from dextrose 
galactose, and levulose, it does produce acid from these substances. 

Milk. — The typhoid bacillus does not cause coagulation when grown 
in milk. In litmus whey the neutral violet color becomes more red 
during the first forty-eight hours; the fluid, however, remains clear. 

Production of Disease in Animals. — It is impossible experimentally 
to produce the characteristic lesions usually met with in human 
typhoid fever in animals. Sickness or fatal results without the appear- 
ance of the typical pathological changes have regularly followed 
animal inoculations, but in most cases they could easily be traced 
to the toxaemia produced by the substances in the bodies of the bacilli 
injected. Typhoid bacilli, freshly obtained from typhoid cases and 
introduced subcutaneously in animals, rapidly die. In the peritoneal 
cavity they may increase, causing a fatal peritonitis with toxic poison- 
ing. By accustoming bacilli to the animal body a certain degree of 
increased virulence for the animal can be obtained, so that smaller 
amounts of culture may prove fatal. Among the most successful 
efforts in this direction are the experiments of Cygnaeus and Seitz, 
who, by the inoculation of typhoid bacilli into dogs, rabbits, and mice, 
produced in the small intestines conditions that were histologically, 
and to the naked eye, analogous to those found in the human subject. 
Their results, however, were not constant. Very similar results 
followed inoculation of virulent strains of colon bacilli. 

Distribution of Bacilli in the Human Subject. Toxic Effects.— 

Typhoid fever belongs to that class of infectious diseases in which the 
specific bacilli are constantly passing into the blood. They thus 
pass to all parts of the body and become localized in certain tissues, 
such as the bone marrow, lymphatic tissues and spleen, liver and 
kidneys. Wherever found in the tissues the typhoid bacilli are usually 
observed to be arranged in groups or foci; only occasionally are they 
found singly. These foci are formed, during life, as is proved by the 
degenerative changes often seen about them; but it is possible that 
the bacilli may also multiply somewhat after death. 

Important Primary Gharacteristic Lesions in Man.— The lesions of the 
intestines which are most pronounced in the lower part of the ileum consist 
of an inflammatory enlargement of the solitary and agminated lymph nodules. 



286 PATHOGENIC MICRO-ORGANISMS. 

Necrosis with ulceration frequently follows the hyperplasia in the more severe 
cases. In the severest cases the ulceration and sloughing may involve the 
muscular and peritoneal coats and perforation may occur. Peritonitis and 
death usually follow. In rare cases the perforation is closed by adhesions. 

The minute changes are an hyperplasia of normal elements of the lym- 
phatic tissue, namely, the lymph cells and the endothelium of the trabecule and 
sinuses. In severer forms necrotic changes are apt to intervene. These 
changes are attributed to the toxic substances formed by the typhoid bacilli, 
but may be directly brought about by the occlusion of the nutritive blood 
vessels, as pointed out by Mallory (Jour. Experimental Medicine, Vol. iii, 
p. 611). 

The mesenteric lymph nodes undergo changes similar to those in the ileum. 
The spleen is enlarged because of congestion and hyperplasia. The liver and, 
to a less extent, the kidneys are apt to show foci of cell proliferation. 

In typhoid fever, as in other infectious diseases, toxic poisoning may be 
manifested by disturbances in the circulatory, respiratory, and heat-regtilating 
mechanism as well as by manifest lesions. In a few cases the intestinal 
lesions are absent. Some of the inflammatory complications which occur in 
typhoid fever are due to the growth of the bacillus in excessive numbers in 
unusual places in the body; but many of them are due to a secondary infec- 
tion with other bacteria, especially the pyogenic cocci and bacilli of the colon 
group. 

Unusual Location of Typhoid Lesions Occurring as Complications 

of T]^hoid Pever. — Cases of sacculated and general peritonitis, ab- 
scess of the liver and spleen, subphrenic abscess, osteomyelitis, peri- 
ostitis, and inflammatory processes of other kinds have been reported 
as being due to the typhoid bacillus. In certain cases of typhoid 
pneumonia, serous pleurisy, empyema, and inflammations of the 
brain and spinal cord or their membranes, typhoid bacilli exclusively 
have occurred. The inflammation produced may or may not be 
accompanied by the formation of pus. There are indeed a number 
of cases now on record in which the typhoid bacillus has played the 
part of pus producer. 

Such cases, however, are of comparatively rare occurrence, because 
only exceptionally do the bacilli mass together in such numbers as to 
become pus producers. 

The Importance of Mixed Infection.— Frequently when* complica- 
tions occur in typhoid fever they are due to secondary or mixed injection 
with the staphylococccus, pneumococcus, streptococcus, pyocyaneus, 
and colon bacillus. Frequently these bacteria are found side by side 
with typhoid bacilli; in such cases it is difficult to say which was the 
primary and which was the secondary infection. 

Elinunation of T]rphoid Bacilli from Body. — Not infrequently 
typhoid bacilli are found in the secretions. They are present in the 
urine in about 20 per cent, of the cases in the third and fourth weeks 
of typhoid fever. Slight pathological lesions in the kidneys almost 
always occur in typhoid fever, but severe lesions also sometimes occur. 
In some cases the urine is crowded with typhoid bacilli. 

In cases of pneumonia due to the typhoid bacillus it is abundantly 
present in the sputa, and care should be taken to disinfect the expec- 
toration of typhoid patients. In typhoid fever the bacilli are almost 



THE TYPHOID BACILLUS, 287 

always present in the gall-bladder. The bacilli are usually eliminated 
by the faeces, being derived from the ulcerated portions of the intestines; 
their growth within the intestinal contents is, with few exceptions, 
not extensive. 

Not only do the very great majority of cases examined bacterio- 
logically and pathologically, but the epidemiological history of the 
disease, proves that the chief mode of invasion of the typhoid bacillus 
is by way of the mouth and stomach. The infective material is dis- 
charged principally by means of the excretions and secretions of the 
sick — namely, by the faeces, the urine, and occasionally by the sputum. 

Occurrence in Healthy Persons. — ^The bacilli usually disappear 
from the body in the fourth or fifth week, but may remain for months 
or exceptionally years in the urine and throughout life in the gall- 
bladder. They have been found in deep abscesses one year after 
recovery from typhoid fever. 

Typhoid Ganiers. — Examinations of convalescent typhoid cases 
show that about 1 to 5 per cent, continue to pass typhoid bacilli for 
years, perhaps for life. Petruschky in 1898 reported that typhoid 
bacilli sometimes remained in the urine of typhoid convalescents 
for months. Gushing soon after observed a case who had had ty- 
phoid fever five years before. In 1902 Frosch, and a little later 
Conradi and Drigalski, reported persons who passed typhoid-infected 
faeces months after recovery from typhoid fever. Some bacilli carriers 
did not know either that they had had typhoid fever or been in contact 
with it, and others knew only that they had been in contact with it. 
Lentz in 1905 found out of a large number of examinations that about 
4 per cent, of persons convalescent from typhoid fever were typhoid 
carriers. In our laboratory we have found six in one hundred and 
forty institution convalescents. The focus of infection is believed 
to be in either the gall-bladder, chronic ulcers of the intestines, or the 
normal intestinal tract. The majority are women. 

A remarkable case of a cook has been under our care for the past 
three years. A visitor of the family in which this woman was cook 
developed typhoid fever some ten days after entering the household. 
This was in 1901. The cook had been with the family 3 years and it is 
diflScult to judge which infected the other. The cook went to another 
family. One month later the laundress in this family was taken ill. 

In 1902 the cook obtained a new place. Two weeks after arrival 
the laundress was taken ill with typhoid fever; in a week a second case 
developed and soon seven members of the household were sick. 

In 1904 the cook went to a home in Long Island. There were 
4 in the family as well as 7 servants. Within 3 weeks after arrival, 
4 servants were attacked. 

In 1906 the cook went to another family. Between August 27th 
and September 3d, 6 out of its 11 inmates were attacked with typhoid. 
At this time the cook was first suspected. She entered another family 
on September 21st. On October 5th, the laundress developed typhoid 
fever. 



288 PATHOGENIC MICRO-OHGANISMS. 

In 1907 she entered a family in New York City, and two months 
after her arrival two cases developed, one of which proveil fatal. 
Altogether during five years this cook is known to have been the cause 
of 26 cases of typhoid fever. 

The cook was removed to the hospital March 19, 1907. Cultures 
taken every few days showed bacilli off and on for three years. 
Sometimes the stools contained enormous numbers of typhoid bacilli 
and again for days none would be found. We recently traced some 
hundreds of cases of typhoid fever to a milk supply produced at a 
farm, looked after by a typhoid carrier who had typhoid fever forty- 
seven years ago. 

TreatmAnt of Typhoid Oairiers. — Medicinal treatment or immuni- 
sation seems so far to have yielded only slight results. Urotropin 
in very large amounts is reported to have cured one case, in which 
operation alone had failed. 

Duration of Life Oatside of the Body. — It is of importance to 
know for what length of time the typhoid bacillus is capable of liv- 
ing outside of the body; but, unfortunately, owing to the great diffi- 
culties in proving the presence of this organism in natural conditions, 
our knowledge on this point is still incomplete. In fteces the length 
of life of the typhoid bacilli is very variable, depending on the composi- 
tion of the fieces and on the varieties of bacteria present; sometimes 
they live but a few hours, usually a day, exceptionally for very long 
periods. Thus, according to Levy and Kayser, in winter typhoid 
bacilli may remain alive in fieces for five months. Foote says that 
they can be found in living oysters for a month at a time, but in nu- 
merous experiments we have not been able to find them after five days. 
Their life in privies and in water, however, is usually very much 
shorter. As a rule, they can be detected in river water no longer than 
seven days after introduction, and often not after forty-eight hours. 
The less the general contamination of the water, the longer the bacilli 
are apt to live. The life of the typhoid bacillus varies according to the 
abundance and varieties of the bacteria associated with it, and accord- 
ing to the presence or absence of such injurious influences as deleterious 
chemicals, high temperature, light, desiccation, etc., to which it is 
known to be sensitive. Good observers claim to have found bacilli 
very similar to typhoid bacilli in the soil in a region where no typhoid 
fever was known to exist. The previously mentioned typhoid carriers 
could account for this. In ice typhoid bacilli rapidly die, none prob- 
ably ever live as long as six months (see pp. 305-307). 

dommnnicability. — The bacilli may reach the mouth by means of 
infected fingers or articles of various kinds, or by the ingestion of 
milk, water, etc., or by more obscure ways, such as the 
oysters and clams or the contamination of food by 
r insects, or by inhalation through the mouth. Of the 
■tance, however, is the production of infection by com- 
nking-water or milk. In a verj' large number of ca,ses 
of this mo<le of infection has been afforded by finding 



THE TYPHOID BACILLUS. 289 

that the water had been contaminated with urine or faeces from a 
case of typhoid. In a few instances the proof has been direct — 
namely, by finding typhoid baciUi in the water. Examples of in- 
fection from water and milk have frequently come under our direct 
observation. The following instances may be cited: A large force 
of workmen obtained their drinking-water from a well near where 
they were working. Typhoid fever broke out and continued to spread 
until the well was filled up. Investigation showed that some of the 
sick, in the early stages of their disease, repeatedly infected the soil 
surrounding the well with their urine and faeces. Another example 
occurred in which typhoid fever broke out along the course of a creek 
after a spring freshet. It was found that, far up near the source 
of the creek, typhoid faeces had been thrown on one of its banks and 
had then been washed into the stream. 

In the late epidemic at Ithaca some 1500 cases developed among 
those using the infected water supply of the town. The students 
and townspeople not drinking the infected supply escaped. The epi- 
demic at Scranton, Pa., during the winter of 1907 was most interesting. 
A little over 1 per cent, of the inhabitants were attacked. No pollu- 
tion of the water with typhoid infected faeces or urine could be discov- 
ered, although typhoid bacilli were isolated from the water of a small 
intercepting reservoir by Dr. Fox. This was only accomplished by 
using large quantities of water. The bacillus isolated was identical by 
all known tests with the typhoid cultures from cases of typhoid fever. 

An instance of milk infection secondary to water infection was in 
the case of a milk dealer whose son came home suffering from ty- 
phoid fever. The faeces were thrown into a small stream which ran 
into a pond in which the milk cans were washed. A very alarm- 
ing epidemic of typhoid developed, which was confined to the houses 
and asylums supplied with this milk. During the Spanish-Ameri- 
can war not only water infection, but food infection was noticed, as 
in the case of a regiment where certain companies were badly in- 
fected, while others nearly escaped. Each company had its sepa- 
rate kitchen and food supply, and much of the infection could be traced 
to the food, the contamination coming partly through the flies. Several 
epidemics have been traced to oysters. 

Individual Susceptibility. — In this, as in all infectious diseases, 
individual susceptibility plays an important r6le in the production 
of infection. Without a suitable soil upon which to grow, the seed 
cannot thrive. There must in manv be some disturbance of the di- 
gestion, excesses in drinking, etc., or a general weakening of the power 
of resistance of the individual, caused by bad food, exposure to heat, 
over-exertion, etc., as occurs with soldiers and prisoners, for example, to 
bring about the conditions suitable for the production of typhoid fever. 

The supposition that the breathing of noxious gases predisposes to 
the disease, though possibly true to a certain extent, as some animal 
ex(>eriments already referred to would seem to indicate, has not yet 
been conclusively proven; nor do Pettenkofer*s investigations into 

19 



290 PATHOGENIC MICRO-ORGANISMS. 

the relation of the frequency of typhoid fever to the ground-water 
level satisfactorily explain the occurrence of the disease in most cases, 
whether sporadically or in epidemics. 

Immunization. — After recovery from typhoid fever a considerable 
immunity is present which lasts for years. This is not absolute, as 
about 2 per cent, of those having typhoid fever have a second attack, 
which is usually a mild one. Specific immunization against experi- 
mental typhoid infection has been produced in animals by the usual 
method of injecting at first small quantities of the living or dead typhoid 
bacilli and gradually increasing the dose. The blood serum of animals 
thus immunized has been found to possess bactericidal and feeble 
antitoxic properties against the typhoid bacillus. These charpcteris- 
tics have also been observed in the blood serum of persons who are 
convalescent from typhoid fever. The attempt has been made to 
employ the typhoid serum for the cure of typhoid fever in man, but, 
although a number of individual observers have reported good results 
with one or another of the sera, most consider that little or no good is 
derived from serum. 

Vacdnation Against Typhoid.— The use of killed typhoid bacilli as 
vaccines has been advocated by Wright and tried upon some 8000 
persons who expected to be subjected to danger of infection. Two in- 
jections are usually given. The first of 500 millions and the second, 
ten days later, of 750 millions. If it is impossible to count the 
number 0.1 c.c. and 0.3 c.c. of a bouillon culture can be given. The 
bacilli are heated to 60° C. for thirty minutes or killed by \ per 
cent, lysol or carbolic acid. For a day or two the injection produces 
a slight fever and local pain, followed in a few days by the develop- 
ment of bactericidal substances in the blood, apparently suflScient in 
amount to give some immunity lasting for a year or more. A second 
injection adds to the degree of immunity. In 49,600 individuals 
under observation in India and Africa, 8600 were thus treated. The 
disease appeared in them to the extent of 2.25 per cent., with a case 
mortality of 12 per cent. In the 41,000 uninoculated there was a case 
percentage of 5.75 per cent., and a case mortality of 26 per cent. 
The use of protective vaccines in the shape of dead cultures, would, 
therefore, seem to be advisable where danger of typhoid infection 
exists. 

Vaccination During Typhoid Fever. — The results obtained by Rich- 
ardson and others do not show any definite effect except that relapses 
seem to be less. 

Diagnosis by Means of the Widal or Serum Reaction. — The chief 

practical application of our knowledge of the specific substances de- 
veloped in the blood of persons sick with typhoid fever has been as an 
'aid to diagnosis. 

In 1894-95 Pfeiffer showed that when cultures containing dead 
or living cholera spirilla or typhoid bacilli are injected subcutaneously 
into animals or man, specific protective substances are formed in 
the blood of the individuals thus treated. These substances confer 



• THE TYPHOID BACILLUS. 291 

a more or less complete immunity against the invasion of the living 
germs of the respective diseases. He also described the occurrence 
of a peculiar phenomenon when some fresh culture of the typhoid 
bacillus on agar is added to a small quantity of serum from an animal 
immunized against typhoid bacilli and the mixture injected into the 
peritoneal cavity of a non-immunized guinea-pig. After this procedure, 
if from time to time minute drops of the liquid be withdrawn in a capil- 
lary tube and examined microscopically, it is found that the bacteria 
previously motile and vigorous and which remain so in control animals 
inoculated without the specific serum, rapidly lose their motility and 
die. They are first immobilized, then they become somewhat swollen 
and agglomerated into balls or clumps, which gradually become paler 
and paler, until finally they are dissolved in the peritoneal fluid. This 
process usually takes place in about twenty minutes, provided a suf- 
ficient degree of immunity be present in the ^animals from which the 
serum was obtained. The animals injected with the mixture of the 
serum of immunized animals and typhoid cultures remain unaffected, 
while control animals treated with a fluid containing only the serum of 
non-immunized animals mixed with typhoid cultures die. Pfeiffer 
claimed that the reaction of the serum thus employed is so distinctly 
specific that it could serve for the differential diagnosis of the cholera 
vibrion or typhoid bacillus from other vibrions or allied bacilli, such 
as Finkler's and Prior's or those of the colon group respectively. 

In March, 1896, Pfeiffer and KoUe published an article entitled 
''The Differential Diagnosis of Typhoid Fever by Means of the 
Serum of Animals Immunized against Typhoid Infection," in which 
they claimed that by the presence or absence of this reaction in the 
serum of convalescents from suspected typhoid fever the nature of 
the disease could be determined. It was further found, if the serum 
of an animal thoroughly immunized to the typhoid bacillus was 
diluted with 40 parts of bouillon, and a similar dilution made of the 
serum of non-immunized animals, and both solutions were then 
inoculated with a culture of the typhoid bacillus and placed in the 
incubator at 37° C, that after the expiration of one hour macroscopic 
differences in the culture could be observed, which increased in dis- 
tinctness for four hours and then gradually disappeared. The reaction 
occurring is described as follows: In the tubes in which the typhoid 
culture is mixed with typhoid serum the bacilli are agglomerated in 
fine, whitish flakes, which settle to the bottom of the tube, while the 
supernatant fluid is clear or only slightly cloudy. On the other hand, 
the tubes containing mixtures of bouillon with cholera or coli serum, 
or the serum of non-immunized animals, inoculated with the typhoid 
bacilli, become and remain uniformly and intensely cloudy. These 
serum mixtures, examined microscopically in a hanging drop, show 
distinct differences. The typhoid serum mixture inoculated with the 
typhoid bacilli exhibits the organisms entirely motionless, lying 
clumped together in heaps; in the other mixtures the bacilli are actively 
motile. 



292 PATHOGENIC MICRO-ORGAN I SMB. 

Similar obsen^ations were made independently by Gruber and 
Durham, who maintained, however, that the reaction described by 
Pfeiffer was by no means specific, and that when the reaction is positive 
the diagnosis still remains in doubt, for the reaction is qumdiicdive 
only, and not qualitative. They concluded, nevertheless, that these 
investigations would render valuable assistance in the clinical diagnosis 
of cholera and typhoid fever. 

Oruber-Widal Test. — The first application of the use of serum, 
however, for the early diagnosis of typhoid fever on a more extensive 
scale was made by Widal, and reported with great fullness and detail 
in a communication published in June, 1896. Widal confirmed the 
reaction as above described, proved that the agglutinative reaction 
usually occurred early, elaborated the test, and proposed a method by 
which it could be practically applied for diagnostic purposes. Since 
then the serum test for the diagnosis of typhoid fever has come into 
general use in bacteriological laboratories in all parts of the world, 
and though the extravagant expectations raised at the time when 
Widal first announced his method of applying this test have not 
been entirely fulfilled, it has, nevertheless, proved to be of great 
assistance in the diagnosis of obscure cases of the disease, and is 
now one of the recognized tests for the differentiation of the typhoid 
bacillus. 

It should also be mentioned that to Wvatt Johnson, of Montreal, 
belongs the credit of having brought this test more conspicuously 
before the public, by introducing its use into municipal laboratories, 
suggesting that dried blood should be employed in place of blood 
serum (Widal having previously noticed that drying did not destroy 
the agglutinating properties of typhoid blood); and that in October, 
1896, the serum test was regularly introduced in the New York Depart- 
ment of Health Laboratory for the routine examination of the blood 
serum of suspected cases of typhoid fever. Since then numerous 
health departments have followed the example set by those of Montreal 
and New York. 

Use of Dried Blood. — Directions for Preparing Specimens of Blood. 
— The skin covering the tip of the finger or the ear is thoroughly 
cleansed, and is then pricked with a needle deeply enough to cause 
several drops of blood to exude. Two fair-sized drops are then placed 
on a glass slide, one near either end, and allowed to dry. Glazed 
paper may also be employed, but it is not as good, for the blood soaks 
more or less into it, and later, when it is dissolved, some of the paper 
fibre is apt to be rubbed off with it. The slide is placed in a box for 
protection. 

Preparation of Specimen of Blood for Examination. — In preparing 
the specimens for examination the dried blood, if accuracy is desired, is 
first weighed and then brought into solution by adding to it the quantity 
of normal salt solution to make the desired dilution, remembering of 
course to allow for the loss in water through drying; then a minute 
drop of this decidedly reddish mixture is placed on a cover-glass, and 



THE TYPHOID BACILLUS. 293 

to it is added a similar drop of an eighteen to twenty-four-hour-old 
bouillon culture of the typhoid bacillus, which, if it has a slight pellicle, 
should be well shaken. The drops, after being mixed, have in a 1 : 10 
dilution a distinct reddish color and in 1:20 a faint pink tinge. The 
cover-glass with the mixture on the surface is inverted over a hollow 
slide (the edges about the concavity having been carefully smeared 
with vaselin, so as to make a closed chamber), and the hanging drop 
then examined under the microscope by either dayhght or artificial 
light, a high-power dry lens being used, or, somewhat less serviceably, 
a yj oil-immersion lens. Ordinarily the dried blood is not weighed, 
but the measure of dilution is estimated by the color of the drop. To 
judge this the beginner must carefully make dilutions of fluid blood 
and notice the depth of color in 1 :10 and 1 : 20 dilutions. Besides 
the faulty judgment of the dilution color by the examiner, the variation 
in depth of color of diflierent specimens of blood makes the estimation 
of dilutions more or less inaccurate, but fortunately this does not 
greatly interfere with the value of the test 

The Reaction.— If the reaction takes place rapidly the first glance 
through the microscope reveals the reaction almost completed, most 
of the bacilli being in loose 

clumps and nearly or altogether ^'°' '*** 

motionless (Fig. 104). Between 
the clumps are clear spaces con- 
taining few or no isolated bacilli. 
If the reaction is a little less 
complete a few bacilli may be 
found moving slowly between 
the clumps in an aimless way, 
while others attached to the 
clumps by one end are apparently 
trying to pull away, much as a 
fly caught on fly-paper struggles 
for freedom. If the agglutinat- 
ing substances are present, but 
still less abundant, the reaction 

may be watched through the timber-Wirtal reaclion. BuHIIi Kathcred inl« 

whole course of its development. bSct!™ wilig mot'iS?e» or"Cmi*it.™ "" 
Immediately after mixing the 

blood and the culture together it will be noticed that the bacilli move 
more slowly than before the addition of serum. Some of these soon 
cea.se all progressive movement, and it will be seen that they are 
gathering together in small groups of two or more, the individual 
bacilli being still somewhat separated from each other. Gradually 
they close up the spaces between them, and clumps are formed. 
According to the completeness of the reaction, either all of the bacilli 
may finally become clumpe<l and immobilized or only a small portion 
of them, the rest remaining freely motile, and those clumped may 
appear to be struggling for freedom. With blow! containing a large 



294 PATHOGENIC MICRO-ORGANISMS. 

amount of agglutinating substances all the gradations in the intensity 
of the reaction may be observed, from those shown in a marked and 
immediate reaction to those appearing in a late and indefinite one, by 
simply varying the proportion of blood added to the culture fluid. 

Psendoreactions. — If too concentrated a solution of dried blood from 
a healthy person is employed a picture is often obtained which may 
be mistaken for a reaction. Dissolved blood always shows a varj'- 
ing amount of detritus, partly in the form of fibrinous clumps, and 
prolonged microscopical examination of the mixture of dissolved 
blood with a culture fluid shows that the bacilli, inhibited by sub- 
stances in the blood, often become more or less entangled in these 
clumps, and in the course of one-half to one hour very few isolated 
motile bacteria are seen. The fibrinous clumps alone, especially if 
examined with a poor light by a beginner, may be easily mistaken 
for clumps of bacilli. Again, the bacilli may become fixed after 
remaining for one-half to two hours, by slight drying of the drop 
or the effect of substances on the cover-glass. The reaction in 
typhoid is chiefly due to specific substances, but clumping and inhi- 
bition of movement similar in character may be caused by other sub- 
stances such as exist in normal horse and other serums. This is 
a very important fact to keep in mind. (For details of technique 
see pages 42-46.) 

In psendoreactions Wilson has noticed that many free bacilli are 
apt to be gathered at the margin of the hanging drop. 

Mode of Obtaining Serum from Blood or Blisters for Examination. — 
Fluid blood serum can easily be obtained in two ways: First, the 
serum may be obtained directly from the blood, thus: Ihe tip of the 
finger or ear is pricked with a lancet-shaped needle, and the blood 
as it issues is allowed to fill by gravity a capillary tube having a cen- 
tral bulb. The ends of the tube are then sealed by heat or melted 
wax, or candle-grease, and as the blood clots a few drops of serum 
separate. To obtain larger amounts of serum for a microscopic 
examination the blood is milked out from the puncture into a small 
homoeopathic vial or test-tube. One cubic centimetre of blood can 
easily be collected in this way. The vial is then corked and placed 
on the ice to allow the serum to separate. As a rule, one or two drops 
of serum are obtainable at the end of three or four hours. Second, 
the serum may be obtained from blisters. This gives more serum, 
but causes more or less delay. The method is as follows: A section 
of cantharides plaster, the size of a 5-cent piece, is applied to the skin 
at some spot on the chest or abdomen. A blister forms in from six 
to eighteen hours. This should be protected from injury by a vaccine 
shield or bunion plaster. The serum from the blister is collected in 
a capillary tube, the ends of which are then sealed. Several drops of 
the serum can easily be obtained from a blister so small that it is prac- 
tically painless and harmless. The serum obtained is clear and 
admirably suited for the test. A piece of blotting-paper soaked in 
*rong ammonia when placed on the skin and covered by a watch- 



THE TYPHOID BACILLUS, - 295 

glass or strips of adhesive plaster will quickly raise a blister. A little 
vaselin should be smeared on the skin surrounding the blotting-paper. 

Advantages and Disadvantages of Serum, Dried Blood, and Fluid 
Blood for the Senun Test. — The dried blood is easily and quickly ob- 
tained, and does not deteriorate or become contaminated by bacterial 
growth. It is readily transported, and seems to be of nearly equal 
strength with the serum in its agglutinating properties. It must in use, 
however, be diluted with at least five times its bulk of water, other- 
wise it is too viscid to be properly employed. The amount of dilution 
can only be determined roughly by the color of the resulting mixture, 
for it is impossible to estimate accurately the amount of dried blood 
from the size of the drop, and it is generally considered too much 
trouble to weigh it accurately. Serum, on the other hand, can be 
used in any dilution desired, varying from a mixture which contains 
equal parts of serum and broth culture to that containing 1 part of 
serum to 100 parts of culture or more, and this can be exactly measured 
by a graduated pipette or, roughly, by a measured platinum loop. 
The disadvantages in the use of serum are entirely due to the slight 
difficulty in collecting and transporting it, and the delay in obtaining 
it when a blister is employed. If the serum is obtained from blood 
after clotting has occurred a greater quantity of blood must be drawn 
than is necessary when the dried-blood method is used; if it is obtained 
from a blister, a delay of one to eighteen hours is required. The trans- 
portation of the serum in capillary tubes presents no difficulties if tubes 
of sufficiently thick and tough glass are employed and placed in tiny 
wooden boxes. For scientific investigations and for accurate results, 
particularly in obscure cases, the use of fluid serum is to be preferred to 
dried blood. Practically, however, the results are nearly as good for 
diagnostic purposes from the dried blood as from the serum. 

Fluid Blood. — When properly obtained this gives good results. The 
Thoma-Zeiss blood pipette is very useful. Lance finger-tip or ear 
and draw the blood into the pipette to the mark 0.5. Then distilled 
water is sucked up in sufficient amount to make the desired solution. 
One loop of this is added to one loop of bouillon culture. 

The Culture to be Employed. — It is important that the culture em- 
ployed for serum tests should be a suitable one, for although all cul- 
tures show the reaction, yet some respond much better and in higher 
dilutions than others. Cultures freshly obtained from typhoid cases 
are not as sensitive as those grown for some time on nutrient media 
Those kept for a long time on artificial media sometimes show a decided 
tendency to spontaneous agglutination. Decrease in virulence is apt 
to be accompanied by increase of capacity for agglutination. For the 
past fifteen years we have used a culture obtained from Pfeiffer. A 
broth culture of the typhoid bacillus developed at 25° to 35°' C, not 
over twenty-four hours old, in which the bacilli are isolated and actively 
motile, has been found to give us the most satisfactory results. Cul- 
tures grown at temperatures over 38° C. are not apt to agglutinate 
so well as those grown at lower temperatures. Stock cultures of typ- 



296 PATHOGENIC MICRO-ORGANISMS. 

phoid bacilli can be preserved on nutrient agar in sealed tubes and kept 
in the ice-box. These remain alive for months or even years. From 
time to time one of these is taken out and used to start a fresh series of 
bouillon cultures. 

Dilution of the Blood Serum to be Employed and Time Required for 
the Development of Reaction. — The serum test, as has been pointed out, 
is quantitative and not qualitative. By this it is not meant to assert 
that all the agglutinating substances produced in the blood of a pa- 
tient suffering from typhoid infection are the same as those present 
in small amount in normal blood, or those produced in the blood of 
persons sick from other infections. It is true, however, that the ap- 
parent effect upon the bacilli of specific and group agglutinins is iden- 
tical, the difference being that in typhoid fever, as a rule, the specific 
substances which cause this reaction are usually far in excess of the 
amount of the non-specific which ever appears in non-typhoid blood, 
so that the reaction occurs after the addition to the culture of far smaller 
quantities of serum than in other diseases, or when the same dilution is 
used it occurs far more quickly and completely with the typhoid serum. 
(See chapter on agglutinins.) It is most important to remember that 
it is purely a matter of experience to determine in any type of infection 
what agglutinating strength of a serum is of diagnostic value. 

The results obtained in the Health Department laboratories, as 
well as elsewhere, have shown that in a certain proportion of cases 
not typhoid fever a slow reaction occurs in a 1:10 dilution of serum 
or blood; but very rarely does a complete reaction occur in this dilution 
within fifteen mmnies. When dried blood is used the slight tendency 
of non-typhoid blood in 1: 10 dilution to produce agglutination is in- 
creased by the presence of the fibrinous clumps, and perhaps by other 
substances derived from the disintegrated blood cells. 

From many cases examined it has been found that in dilutions of 
1 : 20 a quick reaction is almost never produceil in any febrile dis- 
ease other than due to typhoid or paratyphoid bacillus infection, while 
in typhoid fever such a distinct reaction often occurs with dilutions of 
1 : 100 or more. It is possible that some cases of paratyphoid infection 
give a prompt reaction in 1 : 20 dilutions, but if this is so, it is not a serious 
drawback. The very rare cases of persons who though never having 
had typhoid fever yet are typhoid bacillus carriers usually have specific 
agglutinins in their blood. 

The mode of procedure, therefore, as now employed is as follows: 
The test is first made with the typhoid bacillus in a 5 per cent, solu- 
tion of serum or blood. In the case of serum, one part of a 1 : 10 dilution 
is added to one of the bouillon culture. With dried blood, a solution of 
the blood is first made, and the dilution guessed from the color. To 
obtain an idea of the dilution bv the color, known amounts of blood are 
dried and then mixed with definite amounts of water; the colors result- 
ing are fixed in the memory as guides for future tests. If there is no 
reaction — that is to say, if within five minutes no marked change is 
noted in the motility of the bacilli, and no clumping occurs — nothing 



THE TYPHOID BACILLUS. 297 

more is needed; the result is negative. If marked clumping and 
immobilization of the bacilli immediately begin and become complete 
within five minutes, this is termed a marked immediate typhoid reaction, 
and no further test is considered necessary, though it is always advisable 
to confirm the reaction with higher dilutions up to 1 : 50 or more, so as 
to measure the exact strength of the reaction. If in the 1 :20 dilution a 
complete reaction takes place within thirty minutes, the blood is consid- 
ered to have come from a case of typhoid infection, while if a less com- 
plete reaction occurs it is considered that only a probability of typhoid 
infection has been established. By many the time allowed for the 
development of the reaction with the high dilutions is from one to 
two hours, but to us twenty minutes with the comparatively low dilution 
of 1 : 20 seems safer and more convenient. Positive results obtained 
in this way may be considered conclusive, unless there be grounds for 
suspecting that the reaction may be due to a previous fairly recent at- 
tack. The failure of the reaction in one examination by no means 
excludes the presence of typhoid infection. If the case clinically re- 
mains doubtful, the examination should be repeated every few days. 

Use of Dead Gultores. — ^Properly killed typhoid bacilli respond well 
to the agglutination test. For the physician at his office the dead 
bacilli offer many advantages. The reaction is slower than with 
the living cultures and is observed either macroscopically or micro- 
scopically. A number of firms now supply outfits for the serum 
test. These outfits consist of a number of small tubes containing 
an emulsion of dead typhoid bacilli. Directions accompany the outfit. 

Proportion of Gases of Typhoid Fever in which a Definite Reaction 
Occurs, and the Time of its Appearance. — As the result of a large num- 
ber of cases examined in the Health Department Laboratories, it 
was found that about 20 per cent, give positive results in the first 
week, about 60 per cent, in the second week, about 80 per cent, in the 
third week, about 90 per cent, in the fourth week, and about 75 per 
cent, in the second month of the disease. In 88 per cent, of the cases 
in which repeated examinations were made (hospital cases) a definite 
typhoid reaction was present at some time during the illness. 

Persistence of the Reaction. — In persons who have recovered from 
typhoid fever this peculiar property of the blood serum may persist 
for a number of months. Thus a definite typhoid reaction has been 
observ^ed from three months to a year after convalescence, and a 
slight reaction, though much less than sufficient to establish a diagnosis 
of typhoid infection, from one to fifteen years after the disease. In 
persons in whom the typhoid bacilli persist the serum reaction may 
last as long as the bacilli remain in the body. 

Reaction with the Blood Serum of Healthy Persons and of Those HI 
with Diseases other than Typhoid Fever. — In the blood serum of over 
one hundred healthy persons examined in the Health Department 
lal)oratories an immediate marked reaction has not been observed in 
a 1:10 dilution. In several hundred cases of diseases, eventuallv 
not believed by the physicians in charge to be typhoid fever, only 



298 PATHOGENIC MICRO-ORGANISMS. 

very rarely did the serum give a marked immediate reaction in a 1: 10 
dilution. In the light of past experience, I believe a typhoid or 
paratyphoid infection, though not a typical typhoid fever, to have 
existed in these cases. These results have been confirmed by others, 
the question of dilution having recently been made the subject of 
elaborate investigations, with the view of determining, if possible, at 
what dilution the typhoid serum would react while others would not. 
Thus, Schultz reports that among 100 cases of non-typhoid febrile 
diseases apparently positive results were obtained in 19 with dilutions 
of 1:5, in 11 of these with 1:10, in 7 with 1:15, in 3 with 1:20, and 
in 1 a very faint reaction with 1 :25; whereas, in as many cases of true 
typhoid he never failed with dilutions of 1:50. In these experiments 
it must be noted, however, that the time limit was from one to two 
hours. A faint reaction with a 1:25 dilution with a time limit of two 
hours indicates less agglutinating substance than an immediate 
complete reaction with a 1 : 10 dilution. 

From an experience with the practical application of the serum 
test for the diagnosis of typhoid fever extending over many years, it 
may be said that this method of diagnosis is simple and easy of per- 

• formance in the laboratory by an expert bacteriologist, but it is not 
to be recommended for routine employment by practising physicians 
as a clinical test unless they have had experience; that with the modi- 
fications as now employed, and due regard to the avoidance of all 
possible sources of error, it is as reliable a method as any other bac- 
teriological test at present in use; and that as such the Gruber-Widal 
test is an indispensable, though not absolutely infallible, aid to the 
clinical diagnosis of irregular or slightly marked typhoid fever. 

Isolation of Typhoid Bacilli from Suspected Feces, Urine, Blood, 
Water, etc. — In the bacteriological study of typhoid infection for 
diagnostic and other purposes, attempts have been made to isolate the 
specific bacilli from the blood, rose spots, sweat, urine, fseces, and by 

• spleen puncture. Although the results obtained by puncture of the 
spleen have b^en encouraging and have thrown light upon the dis- 
tribution of the organism in the body during life, yet as a regular 
means of diagnosis it is to be discouraged, on account of the possible 
danger to the patient. The results of the examination of the blood 
and rose spots of typhoid patients have until recently proved un- 
satisfactory, investigations of some of the later observers have given 
a large percentage of positive results from the blood. The examination 
of the urine and faeces of typhoid patients has often given positive 
results, and these positive results have become more frequent and 
satisfactory as the methods for differentiating the Bacillus typhosus 
have grown more exact and refined. 

Several media recently devised for the isolation and identification 
of the typhoid bacillus are much better than any of those formerly 
used. These are the Hiss, Capaldi, Endo and the Drigalski-Conradi 
media. In the hands of trained bacteriologists they give satisfactory 
results. 



THE TYPHOID BACILLUS. 299 

The ffisB Madia: Thair OompoBitioii and Pr«p»ration.' — Two media are 
used: one for the isolation oC the typhoid bacillus by plate culture, and one 
for the different iation of the typhoid bacillus from all other forms in pure 
culture in tubes. 

The plating medium is composed of 10 grams of agar, 25 grams of gelatin, 
5 grams of sodium chloride, 5 grams of Liebig's beef extract, 10 grams of 
glucose, and 1000 c.c. of water. When the agar is thoroughly melted the 
gelatin is added and completely dissolved by a few minutes' boiling. The 
medium is then titrated, to determine its reaction, phenolphthalein being 
used aa the indicator. The reijuisite amount of normal hydrochloric acid or 
sodium hydrate solution is added to ■ 
bring it to the desired reaction^i. «., Fiq. ids 

a reaction indicating 2 per cent, of 
normal acid. To clear the medium add 
one or two eggs, well beaten in 25 c.c. of 
water, boil for forty-five minutes, and 
filter through a thin filter of absorbent 
cotton. Add the glucose, after clearing. 
The reaction of the medium is most 
important; it should never contain less 
than 2 per cent, of normal acid. 

The lube medium contains agar, 5 
grams; gelatin, 80 grams; sodium chlo- 
ride, 5 grams; meat extract, 5 grams, 
and glucose, 10 grams to the litre of 
water, and reacts 15 per cent, acid by 
the indicator. The mode of prepara- 
tion is the same as for the plate 
medium, care being taken always to 

add the gelatin after the agar is 

thoroughly melted, so as not to alter 

this ingredient by prolonged exposure to high temperature. The glucose is 

added after clearing. The medium must contain 1.5 per cent, of normal acid. 

Growth of the Colonies. — The growth of the typhoid bacilli in plales 
made from the medium as above described gives rise to small colonies with 
irregular outgrowth and fringing threads (Fig, 105). The colon colonies, 
on the other hand, are much larger, and, as a rule, are darker in color and do 
not form threads. The growth of the typhoid bacilli in lubes produces uni- 
form clouding at 37° C. within eighteen hours. The colon cultures do not 
give the uniform clouding, and present several appearances, probably depend- 
ent upon differences in the degree of their motility and gas-producing proper- 
ties in media. Some of the varieties of the colon bacillus grow only locally 
where they were inoculated by the platinum needle. Others grow diffusely 
through the medium, but owing to the production of gas and the passage of 
(jas-bubbles through the medium, clear streaks ramify through the otherwise 
diffusely cloudy tube contents. This characteristic appearance is not pro- 
duced when the medium is incorrect in reaction or in consistency. With un- 
tried media it is always well to insert a platinum wire into the tube contents 
and stir them about : if any gas is liberated the culture is not one of the 
typhoid bacillus and the medium is not correct. 

Method or Making the TEST.^The usual method of making the test is 
to take enough of the specimen of fteces or urine—/, e., from one to several 
loops— and transfer it to a tube containing broth. From this emulsion in 
broth five or six plates are generally niade by transferring one to five loops 

' This (icBcription is taken from an article by Dr. Philip Hanson Hiss, Jr., " On 



300 PATHOGENIC MICRO-ORGAKISMS. 

of the emulsioQ to tubes containing the melted plate medium, and then pour- 
ing the contentx of these tubes into Petri dishes. These dishes are placed 
in the incubator at 37° C. and allowed to remain for eighteen to twenty-four 
hours, when they may be examined. If typical thread-forming colonies are 
found the tube medium is inoculated from them, and the growth in the tubes 
allowed to develop for about eighteen hours at 37° C. If these tubes then 
present the characteristic clouding, experience indicates that the diagnosis of 
typhoid may be safely made, for the typhoid bacillus alone, of all the organ- 
isms investigated, has displayed the power of giving rise both to the thread- 
forming colonies in the plating medium and the uniform clouding in the tube 
medium when exposed to a temperature of 37° C. The oiganisms isolated 
in this manner have been subject«d to the usual test^ for the recognition of 
the Bacillus typhosus, and have always corresponded in all their reactions to 
those given by the typical typhoid bacillus. 

The Oipaldi Plate Hadium.— In his original paper, Capaldi gives the 
following recipe: 

Aqua dest 1000 

Witte's peptone 21) 

Gelatin 10 

Mannite or grape-sugar , 10 

Soilium chloride and potassium chloride each 5 

Boil, filter, add 2 per cent, agar and 10 e.c. of normal sodic hydrate solution; 
boil, fjlter, and sterilize. 

In making up the medium for work the only variation was that in the origi- 
nal recipe the agar was added when the gelatin was put in, now the gela- 
tin is added after the first filtration. 

The Capaldi medium is usually employed for surface cultures, but can be 
inoculated while melted in the 
"*' tubes. Plates may be made be- 

forehand, so that they are ready 
for use when the specimen comes 
in. As these plates are to be kept 
at 37° C, the difficulties in regard 
to temperature are avoided; but, 
unlike the Eisner plates, other 
organisms besides the colon and 
typhoid develop and may cause 
.'wme confusion. In making the 
plates one or two are inoculated 
by gently carrying across their 
surface a platinum loop of fteces 
or urine. Others are then inocu- 
lated with a loop of urine or much 
diluted fajccs. In this way we are 
apt to have some plates with just 
the right amount of colonies. 
Appearance of the Colonies. 
foioniri of colon hnciiii on {'npaiili mrriiiim — Capaldi thus describes the differ- 
-i..h.iv ra^ifi^j. Tvu^j,w«ion,» of some s,« entiation: Typhoid— Small, glis- 
tening, transparent, almost color- 
reflected light, blue). Colon— Large, milky colonies 

ium it was found that even in a pure plate of typhoid the 
li in sixe and appearance, while different typhoids show 
[■es in growth. In general, a medium-sized, gray-vhite 
refractive granulen, is the tvphoid. However, it is often 
it the refra<'tive granules; sometimes with a nuclear cen- 



THE TYPHOID BACILLUS. 301 

tre, and sometimes of equal consistency throughout. Streptococci simulate 
typhoid, but a high-power lens will show the coccus. 

Colon colonies are usually much larger than the typhoid — a decided brown 
color, very large, refractive granules, and in general quite different in ap- 
pearance (Fig. 106). 

The best way to work with the Capaldi medium is to make several plates 
with different typhoid cultures, observe carefully all the variations in the 
colonies, and bear these in mind when working with the mixed plates. After 
these precautions have been taken the medium will be found very satisfactory. 
The colonies, as a rule, appear characteristically in twelve to eighteen hours, 
and thus give a quick method of diagnosis. 

We found that the two media (Capaldi and Hiss) work excellently together, 
as one is an aid to the other. When many colonies of the typhoid bacilli were 
present the points of differentiation were usually easily seen upon both media, 
and the two together made diagnosis almost certain. The bacilli from the 
suspected typhoid colonies can be quickly tested, sufficiently for practical 
purposes, on the Hiss tube medium, and by the reaction between the bacilli and 
the serum from an immunized horse. 

Typhoid Medium of von Drigalski and Oonradi. — These authors 
modified lactose litmus agar by adding to it nutrose and crystal violet 
and by using 3 per cent, of agar instead of 2 per cent. The crystal 
violet strongly inhibits the growth of many other bacteria, especially 
cocci, which would also color the medium red; the 3 per cent, agar 
makes the diffusion of the acid which is formed more difficult. 

Three pounds of chopped beef are allowed to stand twenty-four 
hours with 2 litres of water. The meat infusion is boiled one hour and 
filtered. Twenty grams Witte's peptone, 20 grams nutrose, and 10 
grams of salt are then added, and the mixture boiled another hour. 
After filtration and the addition of 60 grams agar the mixture is 
boiled for three hours, alkalized and filtered. In the meantime 300 
c.c. litmus solution (Kahlbaum) are boiled for fifteen minutes with 
30 grams lactose. Both solutions are then mixed and the mixture, 
which is now red, faintly alkalized with 10 per cent, soda solution. 
To this feebly alkaline mixture 4 c.c. hot sterile 10 per cent, soda 
solution are added and 20 c.c. of a sterile solution (0.1: 100) of crystal 
violet Hochst B. A substitute for Kahlbaum's litmus solution can be 
made as follows: 

One pound of litmus cubes are ground in mortar to a fine powder and 
extracted three times with boiling alcohol — 500 c.c. each time. This is twice 
extracted with boiling water — 1000 c.c. each time. 

The extract is evaporated down to a saturated solution and made acid with 
hydrochloric acid. It is then placed in a dialyzing bag and dialyzcd for six 
days in running water. It is again evaporated down to a saturated solution 
and 10 per cent, absolute alcohol added when it is cool. 

Enough one one-hundredth normal HCl is added so that one drop more 
brings about a distinct red color. 

Plates are inoculated on the surface only. The material to be 
examined (stools first diluted with ten volumes of 0.8 per cent, salt 
solution) is spread directly on the surface of the plates, and these in- 
verted are allowed to stand slightly open for about half an hour in 
order that they may dry somewhat. They are then placed inverted 



302 PATHOGENIC MICRO-ORGANISMS. 

into the incubator for from sixteen to twenty-four hours. Typhoid 
colonies are small (1 to 3 mm.), transparent, and blue; colon colonies 
are red, coarser, less transparent, and larger. The colonies of fresh 
alkaligenes are blue and usually larger. The suspected colonies can 
at once be tested for agglutination with a high-grade typhoid serum. 

In general this method has withstood critical tests and it is nowa- 
days regarded as perhaps the very best. 

As to the comparative merits of the three media, it is probably safe 
to say that any one of them will, in the hands of one accustomed to 
them, reveal the typhoid bacilli, except perhaps when they exist in 
only the most minute numbers. The Hiss plate medium has the ob- 
jection that it is a diflScult medium to prepare. If the acidity is not 
just right the thread outgrowths do not appear. Indeed, the only 
sure way is to test a new batch of medium with a pure culture and 
alter the reaction until the culture grows correctly. A very few 
strains of the typhoid bacillus do not produce typical thread out- 
growths from the colonies. In the Drigalski medium the typhoid 
colonies are easily separated from those of the colon bacilli, but there 
are other intestinal bacteria which grow fairly like them. 

The Capaldi medium has the objection that some of the typhoid 
and some of the colon colonies frequently look much alike. If one, 
however, will always pick out the colonies which look most like the 
typhoid, it will usually turn out that typhoid bacilli have been ob- 
tained if any were present. Personally, for general use, I prefer 
the Drigalski medium for the plate cultures and the Hiss tube me- 
dium for the first step in identifying the bacilli obtained. Through 
these media and specific agglutinating serum we are now in a posi- 
tion to obtain and make a fairly accurate identification of typhoid 
bacilli from faeces, urine, etc., within forty-eight hours. 

Endo Medium for Tjrphoid Differentiation.^ — Fuchsin solution prepared 
by adding 10 grams fuchsin (not acid) to 100 c.c. 96 per cent, alco- 
hol. Shake and allow to stand for twenty hours, decant and filter 
supernatant fluid. Always filter before using. 

Make 4 per cent, nutrient agar as follows: 1 liter water, 5 grams 
sodium chloride, 10 grams Liebig's meat extract, 10 grams peptone; 
dissolve by heating, cool and add 40 grams agar; cook in Arnold 
three hours and then filter through cotton or perforated funnel 
(Buchner) by aid of vacuum, neutralize to litmus-paper with NajCO, 
solution and add 10 c.c. sterilized 10 per cent. NajC03 solution. to 
alkalinity; add 10 grams C. P. lactose (important to have C. P.); 
add 5 c.c. of above alcoholic fuchsin solution; add 50 c.c. freshly 
made and sterilized 10 per cent, sodium sulphite solution; tube and 
sterilize for a short time in Arnold. 

The medium after cooling should be nearly colorless to transmitted 
light and rose- or flesh-colored to reflected light. The lactose^ 

»Endo, Centblt.f. Bakt., 35, 1904, p. 109. Klinger, Arb. a. d. Kais. Ges., 1906, 
p. 52. Willson, J. of Hyg., 1905, p. 429. Kayser, Munch, med. Woch., 1906, 
pp. 17-18. 



THE TYPHOID BACILLUS. 303 

fuchsin, and sodium sulphite solutions must be added to the melted 
agar just before it will be used. The plates are poured and allowed 
to stand twenty minutes uncovered in the incubator in order to do 
away with water of condensation and to obtain a good surface. The 
plates should be neither too moist nor too dry. "v^rZ^i? ^^^fi hry 

Organisms which split lactose restore the red fuchsin and appear 
as deep red sharply limited opaque colonies with a greenish surface 
shown. 

The typhoid organism produces smaller transparent colonies re- 
sembling a small drop of water. 

Typhoid Bacilli in Faeces. — Recently numerous investigations have 
been carried out to discover how frequently and at what period in 
typhoid fever bacilli are present in the faeces and urine. Hiss some 
time ago examined the faeces of 43 consecutive cases, 37 of which were 
in the febrile stage and 6 convalescent. In a number of instances 
only one stool was examined, but even under these adverse conditions 
the average of positive results in the febrile stage was 66.6 per cent. 
Out of 26 cases of typhoid fever examined in hospitals, 21 were in 
the febrile stage and 5 convalescent. In the febrile cases in 17 the 
presence of typhoid bacilli, often in great numbers, was demonstrated. 
Thus in these carefully followed cases the statistics show over 80 per 
cent, of the febrile cases positive. The bacilli were isolated from 
these cases as early as the sixth day, and as late as the thirtieth day, 
and in a case of relapse on the forty-seventh day of the disease. The 
convalescent cases examined gave uniformly negative results, the 
earliest examination having been made on the third day after the 
disappearance of the fever. The bacilli seemed to be more numerous 
in the stools from about the tenth or twelfth day on. These ob- 
servations, with regard to the appearance of the baciUi in the stools 
during the febrile stage and their usually quick disappearance, except 
in the permanent typhoid carriers, after the defervescence, have 
been confirmed bv others. The bacilli were isolated in several cases 
in which no Widal reaction was demonstrated. Between the seventh 
and twenty-first days of the disease, experience seems to indicate that 
the bacilli may be obtained from about 25 per cent, of all cases on 
the first examination and from about 75 per cent, after repeated exami- 
nations. In some samples of faeces typhoid bacilH die out within 
twenty-four hours; in others they remain alive for days or even weeks. 
This seems to depend on the bacteria present in the faeces and upon 
its chemical character. Probably the presence of typhoid bacilli in 
some stools and their absence in others must be explained largely 
by the characteristics of the intestinal contents. The short life of 
the typhoid bacillus in many specimens of faeces suggests that stools 
be examined as quickly as possible. In fact, unless the physician 
wishes to take the trouble to have the sample of faeces sent immediately 
to the laboratory, it is hardly worth while for the bacteriologist to take 
the trouble to make the test. 

Typhoid Bacilli in the Urine. — Of great interest is the frequent 



304 PATHOGENIC MICRO-ORGANISMS. 

occurrence of typhoid bacilli in large numbers in the urine. The 
results of the examinations of others as well as our own indicate that 
the typhoid bacilli are not apt to be found in the urine until the be- 
ginning of the third week of the fever, and may not appear until much 
later. From this on to convalescence they appear in about 25 per cent, 
of the cases, usually in pure culture and in enormous numbers. Of 
9 positive cases examined by Richardson* 2 died and 7 were discharged. 
At the time of their discharge their urine was loaded with typhoid 
bacilli. We have observed similar cases. In one the bacilli persisted 
for five weeks. Undoubtedly in exceptional cases they persist for 
years. When we think of the chances such cases have to spread 
infection as they pass from place to place, we begin to realize how 
epidemics can start without apparent cause. The more we investigate 
the persistence of bacteria in convalescent cases of disease, the more 
difficult the prevention of their dissemination is seen to be. The 
disinfection of the urine should always be looked after in ti-phoid 
fever, and convalescents should not be allowed to go to places where 
contamination of the water supply is possible, without at least warning 
them of the necessity of great care in disinfecting their urine and fseces 
for some weeks. Richardson made the interesting discovery that 
after washing out the bladder with a very weak solution of bichloride 
of mercury the typhoid bacilli no longer appeared in the urine. 

Typhoid BacUU in Blood. — In many cases typhoid bacilli are found 
in small numbers in the blood early in the course of the disease. They 
continue to be present until the height of the fever, when they de- 
crease, owing to the increase of bactericidal substances. Thus the 
early bacteriological examination of the blood may be an important 
aid in early diagnosis. 

The following methods are recommended for this blood examin- 
ation: (1) Schottmuller's method: 1.5-2 c.c. of blood grown in 
100 c.c. of nutrient broth. (2) CcnradVs bile-enriching method: 
2-5 c.c. of blood grown in 10 c.c. bile mixture (beef bile H- 10 per 
cent, peptone + 10 per cent, glycerin). (3) Meyer stein's^ enriching 
method with concmirated bile salts: 2-3 c.c. of blood are well shaken 
with 2-3 drops of bile salts solution (20-40 pef cent, of pulverizeil 
bile-acid salts in equal parts of glycerin and distilled water). (4) 
Rosen-Rujige's method: 1 per cent, sodium glycocholate added to nu- 
trient agar. In each tube containing 10-15 c.c. of this medium 
melted, 2 c.c. of blood is added and plates are poured. 

Of these four methods, Meverstein\s was found by Bohne' to be the 
most satisfactory. 

Bile media are supposed to allow a good growth of t\^hoid bacilli 
and at the same time to inhibit the growth of possible contaminations. 

Detection of Typhoid Bacilli in Water.— There is absolutely no 

* Journal of Experimental Medicine, May, 1898. 

=^Meyerstein, W. Ueber Typhusanreicherung. Munch, med. Woch., 1006, 
liii. pp. 1864 and 2148. 

' Bohne, A. Vergleichende bakteriologische Blut-, Stuhl- und Urinuntersuch- 
ungen bei Typhus abdominalis. Zeitschr. f. Hyg., etc., 1908, Ixi, 213. 



THE TYPHOID BACILLUS. 305 

doubt that the contamination of streams and reservoirs is a frequent 
cause of the outbreak of epidemics of typhoid fever, but the actual 
finding and isolation of the bacilli is a very rare occurrence. This 
is often due to the fact that the contamination has passed away before 
the bacteriological examination is undertaken, and also to the great 
difficulties met with in detecting a few typhoid bacilli when they are 
associated with large numbers of other bacteria. The greater the 
amount of contamination entering the water, and the shorter the 
time which elapses between this and the drinking of the water, the 
greater is the danger. A recent isolation of the typhoid bacillus is 
that from the small storage reservoir supplying Scranton. In this 
city of over 100,000 inhabitants more than 1 per cent, were infected 
during the epidemic of the winter of 1907. The bacillus was isolated 
by Fox from about 1 liter of water. Tested alongside of a culture 
from one of the Scranton cases it seemed identical. 

The Importance of Ice in the Production of Typhoid Fever. — 

We may endeavor to settle this question directly by determining 
whether epidemics or scattered cases of typhoid fever have been 
traced to ice, or, failing in this, we may try to estimate the probability 
of such infection by learning the duration of life of the typhoid bacillus 
after freezing. 

The total number of instances of typhoid fever which have been 
directly traced to ice infection are remarkably few. One was in 
France, where a group of officers placed ice made from water polluted 
by a sewer in -their wine and afterward a large percentage developed 
typhoid fever, while those of the same company not using ice escaped. 
A second case was a small epidemic which occurred in those who used 
ice from a pond. It was found that water directly infected with typhoid 
ffleces had flowed over its frozen surface and been congealed there. If 
typhoid fever is communicated through ice, except under exceptional 
conditions, it is remarkable that so few cases are traced to it. 

The fact that freezing kills a large percentage of typhoid bacilli 
makes it indeed possible to conceive that ice from moderately infected 
water contains so few living typhoid bacilli that only the exceptional 
person here and there becomes infected, and so the source of the 
infection remains undetected. 

If this be true and scattered cases occur, there should be at least 
some increase on some or every year in March, April, and May in 
such a citv as New York, where four-fifths of all the ice consumed is 
from the Hudson River, which is known to be contaminated with 
typhoid bacilli. The people of New York use ice very freely and 
most of them put it directly in their water or place their vegetables 
on it. The new ice from the Hudson River is gathered in January 
or February and stored on top of the left-over ice, and thus shipments 
to the city are immediately begun. It is an established fact that 
typhoid bacilli in ice are most abundant during the days immediately 
after freezing. At the end of two months less than 0.1 per cent, of 
the original number survive. 

20 



306 PATHOGENIC MICRO-ORGANISMS. 

If Hudson River ice produced an appreciable amount of typhoid 
fever, this would then be noticeable in March and in April and per- 
haps in May. 

When we examine the records for the past ten years we find no in- 
crease of typhoid fever in Greater New York during those months, 
with the one exception of 1907, when we had in the borough of ^lan- 
hattan a sharp outbreak lasting four weeks. This outbreak did not 
occur at all in Brooklyn. As the people of Brooklyn drank different 
water, but received ice from the same places of the Hudson River as 
those of Manhattan, this directed attention to the water or milk rather 
than the ice. Examination of the Croton watershed at the time showed 
that a small epidemic of typhoid existed there and that pollution of 
the water was probable. This suggested still more strongly that the 
water and not the ice was the cause of the typhoid infection. 

It happened that most of the cases occurred in those living in the 
section of the upper West Side, where only well-to-do people live. An 
investigation showed that the majority of the infected had used only 
artificial ice and several had used no ice in their water at all. 

Let us now turn our attention to the life of the typhoid bacillus in 
ice in laboratory experiments. The first important investigation 
was that of Prudden, who showed that typhoid bacilli might live for 
three months or longer in ice. This experiment is frequently wrongly 
interpreted, as when a recent writer states: **It has been amply demon- 
strated that the germs of typhoid fever are not killed by freezing and 
that they have been known to live in ice for long periods of time." 

It is true that in Prudden's experiment a few typhoid bacilli re- 
mained alive for three months, when the experiment was terminated, 
but those were but a small fraction of 1 per cent, of the original num- 
ber. Following Prudden's experiment Sedgwick and Winslow in 
Boston and Park in New York City carried on independently a series 
of experiments. These led to the same conclusions. A table sum- 
marizing a final experiment of ours in which twenty-one different 
strains, mostly of recent isolation, were subjected to the test is given 
below: 

Life of Twenty-one Strains of Typhoid Bacilli in Ice. 

Average number of Percentage typhoid 
bacilli in ice. • bacilli living. 

Before freezing 2,560,410 100 

Frozen three days 1,089,470 42 

Frozen seven days 361,136 14 

Frozen fourteen days 203,300 8 

Frozen twenty-one days 10,280 0.4 

Frozen twenty-eight days 4,540 0.17 

Frozen five weeks 2,950 0.1 

Frozen seven weeks 2,302 0.09 

Frozen nine weeks 127 0.005 

Frozen sixteen weeks 107 0.004 

P>ozen twenty-two weeks 

In these experiments twenty-one different flasks of Croton water 
were inoculated each with a different strain of typhoid bacilli. In 



THE TYPHOID BACILLUS. 307 

one a little of the faeces rich in typhoid was directly added. The in- 
fected water in each flask was then pipetted into thirty tubes. These 
tubes were placed in a cold-storage room in which the temperature 
varied from 20 to 28° F. At first tubes were removed and tested twice 
a week, later once a week. The object of using so many different 
strains was because it has become evident that some cultures live 
longer than others. 

At the end of five weeks the water infected with six cultures was 
sterile, at the end of sixteen weeks only four strains remained alive. 

Interesting investigations of Hudson River ice were carried out in 
1907 by North. 

There was noticed a considerable difference between the number of 
bacteria in the top, middle, and bottom layers of ice. This is natural, 
since while water in freezing from above downward markedly purifies 
itself, 75 per cent, of the solids and a fair proportion of bacteria being 
eliminated, yet this cannot happen in the case of the snow blanket 
which becomes flooded by rain or by cutting holes through the ice. 
Here all impurities, such as dust and leaves which have fallen on the 
surface and dirt which may come from the water, remain with the 
bacteria which they carry, since all are retained in the porous snow. 
The bacteria in freshly cut bottom ice generally show the least destruc- 
tion by freezing. 

Dr. North, in his investigation, examined the ice from forty spots 
between Hudson and Albany. He took samples from the top, middle 
and bottom of each cake and the water of the river. 

The river water in the forty specimens averaged 1,800 bacteria 
per c.c, the top ice 306, the bottom ice 36, and the middle ice 14. 
Only four specimens of top ice had over 500 bacteria per c.c, none of 
the specimens of middle or bottom ice. 

Thirty-three of the specimens of water had over 500 and 23 over 
1000. Colon bacilli were obtained from but one specimen of the 
middle ice, two from the bottom ice, and twelve from the top 
ice. 

The great destruction by freezing is noticeable in these figures. 
Even the top ice soiled by the horses and men gathering it contained 
but 16 per cent, as many bacteria as the water from which it was 
obtained. The bottom ice, the last to be frozen, had but 2 per cent, 
of those in the water. 

Conclusions in Regard to Ice Pollution. — ^The danger from the use 
of ice produced from polluted water is always much less than the use 
of the water itself. Every week that the ice is stored the danger be- 
comes less, so that at four weeks it has become as much purified from 
typhoid bacilli as if subjected to sand filtration. At the end of four 
months the danger becomes almost negligible, and at the end of six 
months quite so. The slight danger from freshly cut ice, as well as 
the natural desire not to put even sterilized and diluted frozen sewage 
in our water, suggests that portions of rivers greatly contaminated, 
such as the Hudson within three miles of Albany, should be con 



308 PATHOGENIC MICRO-ORGANISMS. 

demned for harvesting ice for domestic purposes — such ice alone to 
be used where there is absolutely no contact with food. 

Differential Diagnosis. — The typhoid bacillus and the bacilli of 
the colon group resemble each other in many respects. It is neces- 
sary to remember that there are many varieties of bacilli differing in 
both cultural and agglutinating reactions which are grouped under 
the general name of the colon bacillus. By comparing what has been 
said of the BadUiLS coli and the Bacillus typhosus it will be seen that 
while certain varieties of each simulate each other in many respects, 
the characteristic varieties of each still possess individual character- 
istics by which they may be readily differentiated : 

1. The motiHty of the 5. coli is, as a rule, much less, marked than 
that of the B. typhosus. The colon bacillus is also shorter, thicker, 
and has fewer flagella. 

2. In gelatin the colonies of the colon bacillus develop more rap- 
idly and luxuriantly than those of the typhoid bacillus. 

3. On potato the growth of the colon bacillus is usually rapi d, 
luxuriant, am^vjgjjjle, though not invariably so; while that of the 
tvphoid harilliLs is ordinarjly jnvisilple. 

47The chafactenstic colon bacillus coap^ulate*^ milk in from thirty- 
six to forty-eight hours in ^the incubator, with acid reaction. The 
t yphoid_ ba cilhis does not cau se coagulatio n . 

o. lEecoloii' bacillus is conspicuous for its power of causing fer- 
mentation, with the production of gas in media containi ng gl u cos e. 
The typhoid bacillus never does this. 

6. In nutrient agar or gelatin containing lactose and litmus tinc- 
ture, and of a slightly alkaline reaction, the color of the colonies of 
the colon bacillus is pi.ukj and the surrounding medium becomes red; 
while the colonies of the typhoid bacillus are blqe, and there is little 
or no reddening of the surrounding medium. The same points hold 
true on the Drigalski-Conradi medium. 

7. The colon bacillus possesses the_property of producing indol in 
culturesTn bqiiIlloj[i or peptone; the characteristic typhoid bacillus 
does not produce indol in these soluti oiis . 

8. The colon bacillus rarely produces thread outgrowths in properly 
prepared Hiss plate medium. The typhoid bacillus produces thread 
outgrowths and smaller colonies in this medium. In the Hiss tube 
medium the colon bacillus produces either a growth limited to the 
area inoculated or a diffuse growth streaked with clear lines and spaces. 
The typhoid bacillus produces a diffuse growth, evenly clouding the 
entire medium. 

9. On the Capaldi medium the colon colonies are more granular 
and darker than those of the typhoid bacilli. 

10. Finally, on adding the typhoid bacilli to the serum of animals 
immunized to the typhoid bacillus, the typhoid bacilli are found to 
absorb all the agglutinin acting on the typhoid bacilli, while the colon 
bacilli absorb little or none of it. 

None of these tests alone except perhaps the absorption test can 



THE TYPHOID BACILLUS. 309 

be depended upon for making a diflFerential diagnosis of the atypical 
colon bacillus which does not ferment sugars with the formation of 
gas from the typhoid bacillus or other similar bacilli. 

Unfortunately, also, in most of these characteristics certain de- 
grees of variation may often be observed and these may lead to con- 
fusion. For instance, the morphology may vary considerably, at 
times even when growth on the same culture media, and the motility 
is not always equally pronounced; the flagella may vary; the rapid- 
ity of growth may differ, especially between freshly made and old 
cultures; the grape-leaf appearance of the surface colonies on gelatin, 
which is usually characteristic, may vary with the composition of the 
gelatin, at times no typical colonies at all being presented; the threads 
in the Hiss media may be lacking; in rare instances the typhoid bacillus 
produces indol; the growth on potato is not to be depended on, often 
being visible and not characteristic; the virulence of both the bacilli 
is so little characteristic that it can hardly be used for diagnostic pur- 
poses; and finally, the serum test is not to be depended on unless the 
agglutinins in the serum have been properly tested, for there is abun- 
dant agglutinin for some of the colon bacilli in the serum of many 
untreated animals. This is less true of rabbits than of horses and of 
young than older animals. 

In spite, however, of these difficulties it is very easy suflBciently to 
identify the typhoid bacillus for all practical purposes. A bacillus 
which grows typically in the Hiss tube media, and shows agglutina- 
tion with a high dilution of the serum of an animal immunized to the 
typhoid bacillus, is in all probability the typhoid bacillus. If this 
bacillus absorbs the specific typhoid agglutinins it is undoubtedly 
the typhoid bacillus. The same could probably be said of a bacillus 
which grew characteristically in glucose bouillon and nutrient gela- 
tin, besides showing the specific serum reaction. A still further test 
is to inoculate animals with several doses of the dead bacilli whose 
identification is sought, and note whether there is produced a serum 
which specifically agglutinates undoubted typhoid bacilli. 



CHAPTER XXIII. 
THE BACILLUS AND THE BACTERIOLOGY OF TUBERCULOSIS. 

A KNOWLEDGE of phthisis was certainly present among men at the 
time from which our earliest medical descriptions come. For over 
two thousand years many of the clearest-thinking physicians have 
considered it a communicable disease; but it is only within compara- 
tively recent times that the infectiousness of tuberculosis has become 
an established fact in scientific medicine. Villemin, in 1865, by 
infecting a series of animals through inoculations with tuberculous 
tissue, showed that tuberculosis might be induced, and that such 
tissue carried the exciting agent of the disease. He also noticed the 
difference in virulence between tuberculous material of human and 
bovine sources, and says that not one of the rabbits inoculated ^ith 
human material showed such a rapidly progressive and widespread 
generalization as those receiving material from the cow. Baumgarten 
demonstrated early in 1882, bacilli in tissue sections which are now 
known to have been tubercle bacilli. But these investigations and 
those of others at the same time, though paving the way to a better 
knowledge of the disease, proved to be unsatisfactory and incom- 
plete. The announcement of the discovery of the tubercle bacillus 
was made by Koch in March, 1882. Along with the 'announcement 
satisfactory experimental evidence was presented as to its etiological 
relation to tuberculosis in man and in susceptible animals, and its 
principal biological characters were given. He submitted his full re- 
port in 1884. Innumerable investigators now followed Koch into this 
field, but their observations served only to confirm his discovery. 

Distribution of Bacilli.— They are found in the sputum* of per- 
sons and animals suffering from pulmonary or larjmgeal tubercu- 
losis, either free or in the interior of pus cells; in miliary tubercles 
and fresh caseous masses in the lungs and elsewhere; in recent tuber- 
culous cavities in the lungs; in tuberculous glands, joints, bones, 
serous effusions, mucous membranes, and skin affections. They are 
also found in the faeces of those suffering from tuberculous disease of 
the intestines or of those swallowing tuberculous sputum. 

Morphology. — The tubercle bacilli are slender, non-motile rods of 
about . 3// in diameter by 1 . 5 to 4// in length. (Plate I., Figs. 1 and 2.) 
The morphology is extremely variable, especially on culture media, and 
varies with the type of medium used. Commonly they occur singly or 
in pairs, and are then usually slightly curved; frequently they are ob- 
served in smaller or larger bunches. Under exceptional conditions 
branching and club-shaped forms are observed. The tubercle bacillus 
clearly belongs among the higher forms of bacteria and is closely allied 

310 



PLATE I 



FIG. 1 



FIG. 2 



/ 



» 

r 



\''\- 



f -r 



/ 



Tuberculosis bacilli 
Human. 



Tubercle bacilli in red. 
Tissue in blue. 



X lOOO diameters. 



X llOO diameters. 



FIG. 8 



FIG. A 



/ 



/ 



/ 






\ 



/ 



/ 



-1 \ 



Leprosy bacilli in nasal secre- 
tion of person suffering from 
nasal lesions. (Hansen.) 



Short smegma bacilli in red, 
rest of material in blue. 



X BOO diameters. 



X 1100 diameters. 



THE BACILLUS OF TUBERCULOSIS. 311 

to nocardia. In stained preparations there are often seen un- 
stained portions, From two to six of these vacuoles may sometimes 
be noticed in a single rod. In old cultures irregular forms may de- 
velop, the rods being occasionally swollen at one end or presenting 
lateral projections. Here also spherical granules appear which stain 
vnth more difficulty than the rest of the bacillus and also retain the 
stain with greater tenacity. The bacilli, however, containing these 
bodies are not appreciably more resistant than those not having them; 
therefore they cannot be considered true spores. (See, however, 
nocardia.) 

The bacilli have a thin capsule, shown in one way by the fact that 
they appear thicker when stained with fuchsin than with methylene 
blue. The capsule is believed to contain the greater portion of the 
wax-like substance peculiar to the bacillus. The characteristics of 
different stains are given below. 

Staining Peculiarities. — These are very important, for by them its 
recognition in microscopic preparations of sputum, etc., is rendered 
possible. Owing to the waxy substance in its envelope it does not 
readily take up the ordinary aniline colors, but when once stained it 
is very difficult to decolorize, even by the use of strong acids. The 
more recently formed bacilli are much more easily stained and decol- 
orized than the older forms. The details of methods of staining are 
given on pages 342, 343. 

Biology. — The bacillus of tuberculosis is a parasiticy aerobic, non- 
motile bacillus, and grows only at a temperature of about 37° C, 
limits 30° to 42° C. It does not form true spores. 

ResiBtance. — The bacilli, on account of the nature of their capsule, it 
has been assumed, have a somewhat greater resisting power than 
most other pathogenic bacteria, since frequently a few out of a great 
number of bacilli resist desiccation at ordinary temperatures for 
months; most bacilli die, however, soon after drying. This, however, 
may be the case with any pathogenic organism and it is doubtful 
if there is a greater resistance shown by the tubercle bacillus than by a 
considerable number of other non-sporebearing bacilli. Upon cul- 
tures the bacilli do not live longer than three months, unless the media 
be favorable, such as egg or serum; transplants after this time may fail 
to grow, showing that at least the majority of the bacilli are dead. A 
few bacilli, sufficient to infect guinea-pigs, may persist much longer. 
They frequently retain their vitality for several weeks in putrefying 
material, such as sputum. Cold has little effect upon them. When 
dry, some of the organisms stand dry heat at 100° C. for twenty min- 
utes but are dead in forty-five minutes; but when in fluids and 
separated, as in milk, they are quickly killed — viz., at 60° C. in twenty 
minutes, at 65° C. in fifteen minutes, at 70° C. the great majority in 
one minute, all in five minutes, at 80° C. the great majority in one- 
half minute, all in one minute, and at 95° C. in one-half minute. There 
are reports of experiments which indicate that tubercle bacilli may 
withstand heat to a greater extent than the above figures indicate. It 



i 



312 PATHOGENIC MICBO-ORGANISMS. 

is possible when masses of enormous numbers, especially in coagulated 
clumps, are tested one or two bacilli may resist the exposures noted. 
One reason why in some experiments they appear to withstand high 
temperature is, as pointed out by Theobald Smith, that when heate<l 
in a test-tube in the usual way the cream which rises on heating is 
exposed on its surface to a lower temperature than the rest of the milk, 
and as this contains the greatest percentage of the bacteria some of 
them are exposed to less heat than those in the rest of the fluid 
receive. Rosenau points out, however, that where reports seem to 
indicate that the tubercle bacillus is more resistant than the average 
pathogenic organism the foUow- 
"■ ' ' ing is the cause: If a moderate 

number of killed bacilli are injected, 
limited lesions will arise and case- 
ation may follow. On killing and 
autopsying the animals, tubercle 
bacilli can then be demonstrated in 
smears from the lesions, and the 
Inoculation is considered positive. 
If, however, this material is rein- 
jected into a second pig, the latter 
wilt show nothing on autopsy. 
This capacity of dead bacilli to 
cause macroscopic lesions has long 

Tubercle bBcilli. Imprewion prptunition been kuOWU, having been shoWU 

'n.SS.'"'"""'"*'"'™*"'"**''^""'" by Prudden and Hodenpyl. Its 
importance, however, is not suffi- 
ciently consideretl. Cultures are not suitable to test the viability of 
the bacillus, inoculations into guinea-pigs are resorted to and another 
animal should be inoculated from the first one. 

The resisting power of this bacillus to chemical disinfectants, dry- 
ing, and light is considerable, but not as great as it is apt to appear, 
for, as in sputum, the bacillus is usually protected by mucus or cell 
protoplasm from penetration by the germicidal agent. It is not al- 
ways destroyed by the gastric juice in the stomach, as is shown by 
successful infection experiments in susceptible animals by fee<ling 
them with tubercle bacilli. They are destroyed in sputum in sii- 
hours or less by the addition of an equal quantity of a 5 per cent, so- 
lution of carliolic acid. Bichloride of mercury is less suitable for 
the disinfection of sputum as it combines with the mucus and forms 
a more or less protecting envelope. Iodoform has no effect upon cul- 
tures until 5 per cent, is added. The fumes from four pounds of 
burning sulphur to each HHK) cubic feet of air space will kill tubercle 
bacilli in eight hours when fully exposed to the action of the gas, 
providing they are moist, or abundant moisture is present in the air. 
Formaldehy<le gas is quicker in its action, but not much more effi- 
cient. Ten ounces of formalin should lie employed for each 1000 cubic 
feet of air space. 



THE BACILLUS OF TUBERCULOSIS. 313 

The tubercle bacillus in sputum when exposed to direct sunlight is 
killed in from a few minutes to several hours, according to the thick- 
ness of the layer and the season of the year; it is also usually destroyed 
by diffuse dayhght in from five to ten days when placed near a window 
in fine powder. Protected in cloth the bacilli survive exposure to 
light for longer periods. Tuberculous sputum expectorated upon 
sidewalks, etc., when left undisturbed in the shade may be infectious 
for weeks, but when exposed to the action of direct sunlight, will in 
many cases, especially in summer, be disinfected by the time it is in 
condition (o be carried into the air as dust, but not before children 




and flies get into it. This action of sunlight and other more impor- 
tant hygienic reasons, suggest that the consumptive patients should 
occupy light, sunny rooms. 

Dried sputum in rooms protected from abundant light has occa- 
sionally been found to contain virulent tubercle bacilli for as long as 
ten months. For a year at least it should be considered dangerous. 
The Roentgen rays have a deleterious effect on tubercle bacilli in cul- 
tures, but practically none upon those in tissues. 

Multiplication of' Tubercle Bacilli in Mature Takes Place Only in 
the Living Animal. — The tubercle bacillus is a .strict parasite — that is 
to say, its biological characters are such that it could scarcely find 
natural conditions outside of the bodies of living animals favorable for 
its multiplication. Under exceptional conditions, such as in freshly 
expectorated sputum, tubercle bacilli may increase for a limited time, 

Ooltiration of the Tubercle Bacillus'— On account of their slow 
growth and the special conditions which they require, tubercle ba- 
cilli cannot be grown in pure culture by the usual plate method on 
ordinary culture media. Koch first succeeded in cultivating and 
isolating this bacillus on coagulated beef serum, which he inoculated 
by carefully rubbing the surface with sections of tuberculous tissue and 
then leaving the culture, protected from evaporation, for several weeks 
in the incubator. Cultures are more readily obtained of human 
than of bovine bacilli. 



314 PATHOGENIC ."^ICRO-ORGAXISMS. 

Orowth on Ooagulated Dog or BoTins Seram or on Egg. — Un these, one 
of which is generally used to obtain the first culture, the growth is usually 
visible at the end of ten days at 37° C, and at (he end of three or tour weelcs 
a distinct and characteristic development has occurred. On serum small, 
grayish-white points and scales first appear on the surface of the medium. 
As development progresses there is formed an irregular, membranous-looking 
layer. On egg the growth is in the form of more or less elevated colonies 
which may become confluent. 

OroTlli on Kuttisnt 3-6 p«r cent. Olycsiin Agar.— Owing to the greater 
facility of preparing and sterilising glycerin agar, it is now usually employed 
in preference to blood serum for continuing to produce later cultures. Wnen 
numerous bacilli have been distributed over the surface of the culture medium, 
a rather uniform, thick, white layer, which subsequently acquires a slight 
yellowish tint, is developed; when the bacilli sown are few in number, or are 
associated in scattered groups, separate colonies are developed, which acquire 
considerable thickness and have more or less irregular outlines. The growth 
appears similar to that shown upon bouillon as seen in Fig, 110. 

Growth on Kntilsnt Veal or Beef Broth Oontaisiug 6 per cent, of 
Glycerin. — This is of importance, because in this way tuberculin is 
pnaduced. On these media (he tubercle bacillus abo grows readily 
if a very fresh thin film of growth from the glycerin agar is floated 
on the surface. Glycerin broth is used for the development of tu- 



l>erculin and must be neutral to litmus, viz., between 1.5 per cent 
to 2 per cent, acid to phenolphthalein. The small piece of pellicle 
removed from the previous culture continues to enlarge while it floats 
of the liquid, and in the course of three to six weeks 
■ as a single film, which on agitation is easily broken 
ttles to the bottom of the flask, where it ceases to de- 
The liquid remains clear. A practical point of im- 
quick growth is desired, is to remove for the new 
on of the pellicle of a growing bouillon culture, which 
I actively increasing. 

ato,— A good growth from cultures and sometimes even from 
e on potato, and this forms the niowt uniform medium for 
,\ftcr the potato is cut, soak in 1-1000 sodium carbonate so- 



THE BACILLUS OF TUBERCULOSIS, 315 

lution for twenty-four hours, drain, and then soak in 5 per cent, glycerin 
solution in distilled water for twenty-four hours. Tube and add the glycerin 
solution for moisture. The potato tubes are paraffined to lessen evaporation 
and may have at their lower end a bulb to hold sufficient fluid to prevent the 
potato from drying, though special tubes are not necessary. 

Obtaining of Pure Cultures of the Tubercle Bacillus from Sputum, 
Infected Tissue, and other Materials. — On account of the time re- 
quired and the difficulties to be overcome, this is never desirable ex- 
cept when careful investigations of importance are to be undertaken. 
The chief point of present interest is to determine the type of bacillus 
present in as many cases as possible to learn the frequency of 
bovine bacilli in man. Pure cultnres can be obtained directly from 
tuberculous material when mixed infection is not present, and a proper 
blood serum or egg culture medium is at hand; but it is difficult to get 
material free from other bacteria which grow ihuch more rapidly and 
take possession of the medium before the tubercle bacillus has had time 
to form visible colonies. Therefore, it is usually necessary first to 
inoculate guinea-pigs, subcutaneously or intramuscularly, preferably in 
the thigh, and then obtain cultures from the animals as soon as the tu- 
berculous infection has fully developed. From acute tuberculosis in 
man in other regions than the lungs, direct cultures on blood serum 
or egg may be made with some hope of success. Under the best con- 
ditions great care and patience are necessary if successful results are 
to be obtained. 

Animals inoculated usually die at the end of three weeks to four 
months. It is better, however, not to wait until the death of the ani- 
mals, but at the end of four to six weeks to kill a guinea-pig without vio- 
lence, using illuminating gas, chloroform, or ether in a closed tin or jar. 
(Animals which develop tuberculosis acutely are apt to have abundant 
tubercle bacilli and give successful cultures, while the chronic cases 
usually have few bacilli and frequently give unsuccessful cultures.) 
The animal after being killed is tied out in trays, and after washing 
with a 5 per cent, solution of carbolic acid, immediately autopsied. 
The skin over the anterior portion of the body having been carefully 
turned back, the inguinal nodes are removed with fresh instruments. 
The nodes on the side of injection are especially favorable for cultures. 
The abdomen is then opened and the spleen and retroperitoneal nodes 
removed. As the organs are removed they should be placed in petri 
dishes and thoroughly minced with knife and forceps. Fresh instru- 
ments should be used for each operation. The sternal nodes may be 
used for cultures, but the lungs are almost useless as the majority of 
cultures will be contaminated. The minced tissue is then placed on 
the surface of the culture media and evenly and thoroughly smeared 
over its surface, then the cotton plug is dipped in hot paraffin and the 
tube corked with a tightly fitting charred cork, to keep the media from 
drying. The tubes are incubated in the inclined position. On egg,* 
growth IS visible in from seven to ten days, and well marked at the end 
of three weeks. Many tubes should be inoculated as it is only with 



i 



316 PATHOGENIC MICRO-ORGANISMS. 

the dexterity acquired by practice that contaminations are avoided. As 
will be noted further on, the growth of the bovine type will be very 
sparse and on glycerin egg probably negative. 

Media for Isolation. — Egg media are the best, failure of growth even 
when the bacilli are few practically never occurs. This medium was 
first advised by Dorset, and has been variously modified. Absolutely 
fresh eggs should be secured. Wash clean of any adherept dirt, then 
wash with 5 per cent, carbolic, allow to partly dry; then with flamed 
forceps punch holes in both ends, rupturing the membrane at one end, 
this end is held over a sterile flask and the contents carefuUv blown 
out. If the blowing is done from the cheeks rather than from the 
lungs, spattering of saliva and blowing into the flask is avoided. To 
the eggs is then added 10 per cent, of water by volume of the weight 
of the eggs. The flask is carefully shaken and the contents mixed 
with a sterile rod. The mixture is then strained through cheese-cloth 
into a funnel, and tubed. This is done by tying cheese-cloth over a 
funnel just so it sags about two inches below the rim when pressetl. 
Over this is placed a common pie plate to protect from dust. To the 
end of the funnel is attached a rubber tube with drawn out piece of 
glass tubing for a tip and pinchcock put in place. To protect the tip 
from dust a piece of tubing is used about 3 inches long and wide enough 
to allow the test-tubes used to slip through. Into one end a perfo- 
rated rubber cork is inserted and the glass tip pushed through the cork 
half-way down the larger glass tube. The pinchcock having been 
loosened the whole is carefully wrapped and sterilized in the auto- 
clave. When ready for use it is carefully unwrapped, set in a ring 
stand, and the pinchcock tightened. The tin is tilted up and the mixed 
egg poured upon the cheese-cloth. It filters through by gravity. The 
tubes to be filled are then flamed and pushed up into the larger tube, 
and thus filled from the protected tip. 

Another modification of the egg medium is that of Lubenau. This 
consists of 10 eggs plus 200 c.c. of 5 per cent, glycerin bouillon neutral 
to litmus, treated as above. 

After the tubes are filled they are then inspissated at 70° C. for t^'o 
hours, and incubated for one week for sterility. In coagulating, the 
air should be thoroughly saturated with moisture, and if the Koch in- 
spissator is used preferably only one layer should be coagulated at a 
time. After the tubes are inspissated a few drops of sterile water 
should be added. To prevent evaporation push down the stopper, 
burn, and plug with a charred cork. 

Pathogenesis. — The tubercle bacillus is pathogenic not only for man, 
but for a large number of animals, such as the cow, monkey, pig, 
cat, etc. Young guinea-pigs are very susceptible, and are used 
for the detection of tubercle bacilli in suspected material. When 
inoculated with the minutest dose of the living bacilli they usually 
succumb to the disease. Infection is most rapidly produced by intra- 
peritoneal injection. If a large dose is given, death follows in from ten 
to twenty days. The omentum is found to be clumped together in 



THE BACILLUS OF TUBERCULOSIS. 317 

sausage-like masses and converted into hard knots, which contain many 
bacilli. There is no serous fluid in the peritoneal cavity, but generally 
in both pleural sacs. The spleen is enlarged, and it, as well as the liver 
and peritoneum, contains large numbers of tubercle bacilli. If smaller 
doses are given the disease is prolonged. The peritoneum and inte- 
rior organs — spleen, liver, etc., and often the lungs — are then filled 
with tubercles. On subcutaneous injection, for instance, into the 
thigh, there is a thickening of the tissues about the point of inocu- 
lation, which may break down in one to three weeks and leave a 
sluggish ulcer covered with cheesy material. The neighboring lymph 
glands are swollen, and at the end of two or three weeks may at- 
tain the size of hazel-nuts. Soon an irregular fever is set up, and 
the animal becomes emaciated, usually dying within four to eight 
weeks. If the injected material contained only a small number of 
bacilli the wound at the point of inoculation may heal up and death 
be postponed for a long time. On autopsy the lymphatic glands are 
found to have undergone cheesy degeneration; the spleen is very 
much enlarged, and throughout its substance, which is colored dark 
red, are distributed masses of nodules. The liver is also enormously 
increased in size, streaked brown and yellow, and the lungs are filled 
with grayish-white tubercles; but, as a rule, the kidneys contain 
no nodules. Tubercle bacilli are found in the affected tissues, but the 
more chronic the process the fewer the bacilli present. 

Injection into the thigh is to be preferred for diagnostic purposes, 
the swelling of the local lymph nodes being then palpable. As soon 
as this is appreciable the node may be removed with or without killing 
the pig, the presence or absence of tuberculous lesions noted, and smears 
made for the detection of tubercle bacilli, thus saving time. It must 
be remembered that the pig may not show the usual picture of gener- 
alized tuberculosis, but only a swelling of the local lymph nodes. 
Fortunately tubercle bacilli are usually easily demonstrable in smears 
made from the crushed nodes. If there is any doubt the remaining 
tissue should be emulsified and reinjected into a second set of pigs. 
Another point to be considered is that other organisms may, rarely, give 
a picture impossible to distinguish macroscopically from tuberculosis, 
as, for instance, streptothrix. To safeguard against error smears 
should be stained and tubercle bacilli demonstrated. 

Rabbits are very susceptible to tuberculosis of the bovine type, 
less so to that of the human type. This will be given more in detail 
under the differences between human and bovine tuberculosis. 

Monkeys are very susceptible to infection with both types of bacilli. 
Cats, dogs, rats, and mice are susceptible, the last two usually show 
no tuberculous lesions, but there is great multiplication of the bacilli 
in the tissues. 

Tubercle Toxins. — ^These comprise both endotoxins and extracellular 
poisons. Injections of endotoxins cause necrosis, abscess, and cheesy 
degeneration of tissues and general cachexia. The extracellular 
poisons produce fever and an acute inflammatory reaction of the 



318 PATHOGENIC MICRO-ORGANISMS. 

tissues. These poisons \\ill be considered in detail later in connec- 
tion with tuberculins. 

Action upon the Tissaes of the Poisons Produced by the Tubercle 
Bacillus. — Soon after the introduction into the tissues of tubercle bacilli, 
either Uving or dead, the cells surrounding them begin to show that 
some irritant is acting upon them. The connective-tissue cells 
become swollen and undergo mitotic division, the resultant cells 
being distinguished by their large size and pale nuclei. A small 
focus of proliferated epithelioid cells is thus formed about the bacilli, 
and according to the intensity of the inflammation these cells are sur- 
rounded by a larger or smaller number of the lymphoid cells. When 
living bacilli are present and multiplying the lesions progress, the 
central cells jlegenerate and die, and a cheesy mass results, which 
later may lead to the formation of cavities. Dead bacilli, on the other 
hand, unless bunched together, give off sufficient poison to cause the 
less marked changes only (Prudden and Hodenpyl). Of the gross 
pathological lesions produced in man by the tubercle bacilli the most 
characteristic are small nodules, called miliary tubercles. When 
young, and before they have undergone degeneration, these tubercles 
are gray and translucent in color, somewhat smaller than a millet 
seed in size, and hard in consistence. But miliary tubercles are not the 
sole tuberculous products. The tubercle bacilli may cause diffuse 
growth of tissue identical in structure with that of miliary tubercles, 
that is, composed of a basement substance, containing epithelioid, 
giant, and lymphoid cells. This diffuse tuberculous tissue also tends to 
undergo cheesy degeneration. 

Usual Point of Entrance of Infection. — Infection by the tubercle bacil- 
lus takes place usually through the respiratory tract or the digestive 
tract, including the pharynx and tonsils, more rarely through wounds 
of the skin. 

Tuberculosis may be considered to be caused chiefly by the direct 
transmission of tubercle bacilli to the mouth through soiled hands, 
lips, handkerchiefs, milk, etc., or by the inhalation of fine particles of 
mucous thrown off by coughing or loud speaking, or of tuberculous 
dust contaminated by sputum or faeces. 

Tuberculosis of Sldn and Mucous Membranes. — When the skin or 
mucous membranes are superficially infected through wounds there 
may develop lupus, ulceration, or a nodular growth. The latter two 
forms of infection are apt after an interval to cause the involve- 
ment of the nearest lymphatic glands, and then finally the deeper 
structures. 

Tuberculosis of Respiratory Tract. — The lungs are the most frequent 
location of tuberculous inflammation, in spite of the fact that on ac- 
count of their location they are greatly protected. Most of the bacilli 
are caught upon the nasal or pharyngeal mucous membranes. Only 
a small percentage can find their way to the larynx and trachea, and 
still less to the smaller bronchioles. From the examination of the 
lungs of miners as well as from experimental tests there is no doubt 



THE BACILLUS OF TUBERCULOSIS. 319 

but that some of the bacilli find their way into the deeper bronchi. 
The deeper the bacilli penetrate the more unlikely that they can be 
cast out. The lungs become frequently infected by indirect ways, for 
it is now well established that infection taking place through the intes- 
tine may find its way by the blood to the lungs and excite there the 
most extensive lesions with or without leaving any trace of its point 

of entrance. The nasal cavities are rarelv affected with tuberculosis, 

* 

but more often the retropharyngeal tissue. Tuberculosis of this tissue 
as well as that of the tonsils is apt to give rise to infection of the 
lymph nodes of the neck. It is believed that just as bacilli may pass 
through the intestinal walls to infect the mesenteric nodes, so bacilli 
may, without leaving any trace, pass through the tonsils to the nodes 
of the neck. 

Primary infection of the larynx is rare. Secondary infection is 
fairly common. The region of the vocal cords and the interarytenoid 
space are the special sites attacked. 

Infection by Inhalation of Dried and Moist Bacilli. — A common mode 
of infection is by means of tuberculous sputum, wj^ch, being coughed 
up by consumptives, is either disseminated as a fine spray and so 
inhaled, or, carelessly expectorated, dries and, broken up by tramping 
over it, sweeping, etc., distributes numerous virulent bacilli in the 
dust. As long as the sputum remains moist there is no danger of dust 
infection, but only of direct contact; it is when it becomes dry, as on 
handkerchiefs, bedclothes, and the floor, etc., that the dust is a source 
of danger. 

A great number of the expectorated and dried tubercle bacilli 
undoubtedly die, especially when exposed to the action of direct sun- 
light; but when it is considered that as many as five billion virulent 
tubercle bacilli may be expectorated by a single tuberculous individual 
in twenty-four hours, it is evident that even a much smaller pro- 
portion than are known to stay alive will suflSce in the immediate 
vicinity of consumptives to produce infection unless precautions are 
taken to prevent it. The danger of infection is greatest, of course, 
in the close neighborhood of tuberculous patients who expectorate 
profusely and indiscriminately, that is, without taking the necessary 
means for preventing infection. We found that of 100 tuberculous 
men admitted to one of the consumption hospitals, only 20 claimed 
to have taken any care to prevent the contamination of their surround- 
ings by their sputum. There is much less danger of infection at a 
distance, as in the streets for instance, where the tubercle bacilli have 
become so diluted that they are less to be feared. In rooms the sputum 
is not only protected from the direct sunlight, but it is constantly broken 
up and blown about by the walking, closing of doors, etc. In crowded 
streets on windy days infected dust must sometimes be in the air unless 
the expectoration of consumptives is controlled. 

Exhaustive experiments made by many observ^ers have shown that 
particles of dust collected from the immediate neighborhood of con- 
sumptives, when inoculated into guinea-pigs, produce tuberculosis 



320 PATHOGENIC MICRO-ORGANISMS. 

in a considerable percentage of them; whereas, the dust from rooms 
inhabited by healthy persons or dust of the streets does so only in 
an extremely small percentage. Flugge is probably right in think- 
ing that the dust which is fine enough to remain for a long time in 
suspension in the air is usually free from living bacilli. It is in the 
coarser though still minute particles, those in which the bacilli are 
protected by an envelope of mucus, that the germs resist drying 
for considerable periods. These are carried only short distances 
by air currents. Such results as those obtained by Straus, who, on 
examining the nasal secretions of twenty-nine healthy persons living 
in a hospital with consumptive patients, found tubercle bacilli in 
nine of them, must be accepted with some reserve, since we know that 
in the air there are bacilli which look and stain like tubercle bacilli 
and yet are totally different. It may be said that the danger of in- 
fection from slight contact with the tuberculous is not so great as it is 
considered by many, but that on this account it is all the more to be 
guarded against in the immediate neighborhood of consumptives. 
Those who are most liable to infection from this source are the families, 
especially young children, the nurses, the fellow- workmen, and 
fellow-prisoners of persons suffering from the disease. In this connec- 
tion, also, attention may be drawn to the fact that rooms which have 
been recently occupied by consumptives are not infrequently the means 
of producing infection (as has been clinically and experimentally 
demonstrated) from the deposition of tuberculous dust on furniture, 
walls, floors, etc. The danger is not apt to last beyond three months. 
Flugge has recently drawn attention to the fact that in coughing, 
sneezing, etc., very fine particles of throat secretion containing bacilli 
are thrown out and carried by air currents many feet from the pa- 
tient and remain suspended in the air for a considerable time. To 
encourage us we now have a mass of facts which go to show that when 
the sputum is carefully looked after there is very little danger of 
infecting others except by close personal contact. 

Tuberculosis of Digestive Tract. — Tuberculosis of the gums, cheeks, 
and tongue is rare. The tonsils and pharynx are somewhat more 
often involved. The stomach and oesophagus are almost never at- 
tacked. The small intestines are rather frequently the seat of infection 
from bacilli swallowed with the food or dust-infected mucus. In a 
striking case four previously healthy children died within a short period 
of one another. Their nurse was found to have tuberculosis of the 
antrum of Highmore, with a fistulous opening into the mouth. She 
had the habit of putting the spoon with which she fed the children 
into her mouth so as to taste the food before it was given to them. 
As already noted, the bacilli frequently pass through the mucous 
membrane to the lymph glands without leaving any lesions. 

Infection by Ingestion of Milk and Milk Products. — Milk serves 
as a conveyer of infection, whether it be the milk of nursing mothers or 
the milk of tuberculous cows. In this case evidence of infection is 
usually shown in the mesenteric and cervical lymph nodes or general- 



THE BACILLUS OF TUBERCULOSIS. 321 

ized tuberculosis may be caused, while the. intestinal walls are fre- 
quently not affected. Bacilli accompanied by fat much more readily 
pass through the intestinal mucous membrane or that of the tonsils and 
pharynx. The transmission of tubercle bacilli in the milk of tubercu- 
lous cows has been abundantly proved. 

Formerly it was thought that in order to produce infection by milk 
there must be a local tuberculous affection of the udder; but it is now 
known that tubercle bacilli may be found in the milk in small numbers, 
when adjacent tissue is infected and when careful search fails to detect 
any udder disease. Schroeder has shown that the faeces are a very 
dangerous factor in the dissemination of tubercle bacilli. He compares 
faeces in cattle to sputum in man, since the tubercle bacilli are swal- 
lowed by cattle and are to a great extent passed through the intestinal 
tract without destruction. He found that when milk from phthisical 
cows having healthy udders was obtained so as not to become infected 
by feces it was free from bacilli, but when obtained without special 
precautions it was frequently infected. The milk of every cow which 
has any well-developed internal tuberculous infection must there- 
fore be considered as possibly containing tubercle bacilli. Rabino- 
witsch, Kempner, and Mohler also proved beyond question that not 
only the milk of tuberculous cattle, which showed no appreciable udder 
disease, but also those in which tuberculosis was only detected through 
tuberculin, frequently contained tubercle bacilli. Different, observers 
have found tubercle bacilli in 10 to 30 per cent, of the samples of un- 
heated city milk. Butter may contain tubercle bacilli in higher per- 
centages of samples examined. When we consider the prevalence of 
tuberculosis among cattle we can readily realize that, even if the 
bovine bacillus infects human beings with diflBculty, there is danger to 
children when they are exposed to this source of infection. The 
milk from cattle suffering from udder tuberculosis usually contains a 
few hundred bacilli per c.c, but may contain many millions. It is 
also important to mention the fact that mixed milk from a herd, though 
tending to dilute the milk of cows excreting tubercle bacilli, may 
be badly infected from one cow, especially if this cow has udder 
disease. 

Taking the abattoir statistics of various countries, we find that 
about 10 per cent, of the cattle slaughtered were tuberculous. A less 
pcobable source of infection by way of the intestines is the flesh of 
tuberculous cattle. Here the danger is considerably less, from the 
fact that meat is usually cooked, and also because the muscular tissues 
are seldom attacked. In view of the finding of the bovine type of bacilli 
in a considerable percentage of the few cases of tuberculous children 
tested, the legislative control and inspection of cattle and milk would 
seem to be an absolute necessity. As a practical and simple method of 
preventing infection from suspected milk, suflBcient heating of the 
milk used as food must commend itself to all. Human tubercle bacilli 
may be found in milk as instanced by one sample of city milk ex- 
amined in the Research Laboratory by Hess. 

91 



322 PATHOGENIC MICRO-ORGANISMS. 

H«tbod of Ezamining Milk for Tuberds BadUl. — Thirtv c.c. of 
milk are centrifuged at high speed and 10 c.c. of the lower milk and 
sediment collected. Four cubic centimetres of the cream is thinned 
with a little sterile water and injected into two guinea-pigs. The sedi- 
ment is injected in amounts of 3 to 5 c.c. into other pigs. Larger 
amounts than this are apt to kill too many pigs from the associated 
bacteria. Subcutaneous injection is to be preferred. There are cer- 
tain precautions that must be taking in drawing conclusions as the dif- 
ferent types of acid-fast "butter bacilli" may cause lesions, and their 
presence will be noted in smears made from these lesions. To avoid 
this source of error, two methods are resorted to. If cultures are made 
from the suspected lesions on glycerin agar, these bacilli develop in a 
few days, whereas tubercle bacilli would not. When one is ready to 
kill the pigs, 2 c.c. of old tuberculin should be injected into each pig 
late in the day. The following morning the tuberculous pigs will be 
dead or dying. Autopsies should be done on all to confirm the test. 
The milk should be as fresh as possible to prevent the growth of 
bacteria. 

Bovine Xnfoction in Han. — Numerous investigations have been made 
on this point. To Ravenel probably belongs the credit of isolating the 
first bovine bacillus from a child. The following tables summa- 
rizing the results of three large series of cases give a fair idea of inci- 
dence of such infection. As will be seen, children are especially the 
ones infected, and usually the point of entry is clearly alimentary as 
shown by the lesions. Cervical adenitis and abdominal tuberculosis 
are the most frequent types of infection. Generalized tuberculosis 
due to bovine infection is less frequent. Bone and joint tuberculosis 
is almost exclusively of the human type. The meninges are less com- 
monly affected by the bovine type than by the human type. Infection 
of adults is very uncommon; and, though cases of pulmonary tubercu- 
losis due to the bovine type of bacillus have been reported, the evidence 
advanced is open to question. Such cases are certainly exceedingly 
rare. 

A careful study of all the factors leads us to estimate that about 
10 per cent, of all tuberculosis in children under five is due to 
bovine infection. 

The following tables give a summary of the results obtained in the 
larger investigations so far carried out: 



THE BACILLUS OF TUBERCULOSIS. 



323 



Table I. 

Tabulation of Cases* Reported by Kossel Weber, Heuss, and Taute, and 
Oehlecker (Kaiserliches Gesundheitsamt) and by the English 

Royal Commission.* 





1 
Adults C^ldren i 


1 
Children 


DiaguoBis of 
oases examined 


16 yrs. and over 


5 to 16 yrs. 


under 5 yrs. 

1 Not«« 


! 1 










Human 


Bovine 


Human BoArine 


Human 

1 


BoArine 




Pulmonary tuber- 


22 




1 
1 1 


5 




Cases diagnosed 


culosis. 








1 






clinically or by 
autopsy. Some 
showed abdomi- 






















1 






nal lesions (inges- 
















tion ?) but no true 














generalisation. 
















One case (age?)" 












1 human type. 


Tuberculous ad^ii- 









1 




tis (axillary). • 












t 


Tuberculous adeni- 


2 




8 5 


7 


4 One autf no age. * 


tis (cervical). 












human type. 


Abdominal tuber- 


6 




2 


. 4 


3 


7 


Lesions exclusively 


culosis. 














of abdominal or- 














sans as far as 
, known. 


Generalised tuber- 


3 


See notes 


2 


2 


6 


4 


Including cases 


culosis (alimen- 








See notes 






where generalisa- 


tary origin). 














tion has begun or 
is complete. 














One ease (30 yrs.) 














gave both types 
















1 n mesenteric 
















nodes, human 
















type in bronchial 














nodes. 
















One case, 5i yrs., 
















gave human tvpe 
from spleen, bo- 






























vine type from 
















mesenteric nodes. 


G^ieralised tuber- 




1 




1 


6 1 Two of the bovine 


culosis including 












See notes cases had cul- 


meninges ( a 1 i - 














turee from the 


mentary origin). 














menmges. 
One caaet 4 yrs., 
gave human type 
from menini^es 






























and bronchial 
















nodes, bovine 












^ 




from mesenteric 
nodes. 


Oeneraliaed tuber- 


15 







1 


3 




Pulmonary lesions 


culosis. 














predommant in 












most of cases. 


Generalised tuber- 






3 




10 




Eight cases had cul- 


culosis incl. men- 


i 












tures from the 


mges. 


1 1 






1 menmges. 


Tuberculosisof 


16 




15 


1 


1 14 




One case, age not 


bones and joints. 














stated, gave hu- 


m 


1 










man type. 


Genitourinary tu- 


4 














berculosis. 


, 


1 






1 

1 


Tuberculosis o f 


1 1 


1 








• 


skin. 
















Miscellaneous 


1 


1 
! 






Calcified mesenteric 
















and bronchial 






1 








nodes. C a r c i - 




1 1 


• 




j noma. 


Totals 


70 1 33 


13 


i 50 


21 1 fA nnn-tAhiilAt«d) 






See notes 

1 


1 


See notes 


See notes 


Total cases 193. 



* The cases were selected, that is, to include as many cases of alimentary origin as possible. 

* Exclusive of sputum feeding, two cases without culture and cases showing irregularity 
change in virulence, for which refer to original. 



or 



324 



PATHOGENIC MICRO-ORGANISMS. 



Table II. 

The Relative Proportion of Human and Bovine Tubercle Bacilli Infec- 
tions IN A Large Series of Unselected Cases' Examined at the 

Research Laboratory. 



AdultB Children 
Diagnoeis of j ^® y"' ^^ °^*^*" I ^ to 16 yra. 
cases examined i ' 



Children 
under 5 yrs. 



Notes 



Human Bovine Human Bovine i Human Bovine 



Pulmonary tuber- 
culosis. 



Tuberculous adeni- 
tis, inguinal and 
axillary. 

Tuberculous adeni- 
tis, cervical. 



Abdominal tuber- 
culosis. 

Generalised tuber- 
culosis, alimen- 
tary origin. 




Generalised tuber- 
culosis. I 



Generalised tuber- { 
culosis including 
meninges. 

I 

Tubercular menin-| 
gitis. I 



Tuberculosis o f 
bones and joints. | 

Genitourinary tu-' 
berculosis. 

Tuberculous a b -' 
scesses. 

Totals 



296 , 1 45 , 9 62 

* Unselected cases from the hospitals of New York City. 



Clinical diagnosis 
only known, and 
therefore no posi- 
tive detaib as 
to extent of tu- 
berculosis else- 
where than in 
lungs. 



(See next.) 



In two cases cultures 
were from axil- 
lary nodes, but 
primary focus 
was cervicaL 

One case died short- 
ly afterward 
with pulmonary 
tuberculosis. 



Only two cases are 

given under this 
eading. Many of 
of the cases in the 
following subdi- 
visions showed 
marked intestinal 
lesions and some 
possibly were of 
alimentary origin. 



2 1 1 I 1 12 4 

* 1 


1 1 18 1 

1 'See notes 

1 ' ' 


1 ' 1 1 14 ' 1 

1 1 1 ^ - 


1 1 10 ' 6 . » 

1 ' 1 


3 ' 1 1 1 1 

1 


1 1 , , [ 



One bovine case 
had tuberculous 
osteomyelitis of 
metatarsal bone. 

One cojte not includ- 
ed in table gave 
both t3rpes of 
bacillus. 

No autopsy. Ex- 
tent of lesions 
elsewhere. un- 
known. 



Possibly primary in 
bone. 



I 22 ' (1 non-tabulated) 
See notes' Total cases 436. 



THE BACILLUS OF TUBERCULOSIS. 325 

HypothedB of Transmissibility of Tubercle Bacilli to the FoBtas. — There 
is some evidence of the transmission of tubercle bacilli from the mother 
of the foetus in animals. With regard to tuberculosis in the human 
fcBtus the evidence is not so clear, though some twenty cases have been 
recorded of tuberculosis in newly bom infants, and about a dozen cases 
of placental tuberculosis. As to the infection of the foetus from the pa- 
ternal side, where the father has tuberculosis of the scrotum or seminal 
vessels, we have no reason to suppose that such can occur. There 
are, however, grounds for belief that infection in this way may take 
place from husband to wife. Thus, Gartner found, as a result of his 
experiments in animals, that a large majority of the guinea-pigs and 
rabbits which were brought together with males whose semen contained 
tubercle bacilli died of primary genital tuberculosis; but from the rarity 
of this affection in women and cows it may be assumed that tubercle 
bacilli occur very much less frequently in semen of men and cattle 
than in that of the smaller animals. 

Attenuation. — Tubercle bacilli when subjected to deleterious in- 
fluences or to growth on culture media very slowly decrease in viru- 
lence. Cultures grown at temperatures of 42^ C. become attenuated 
more quickly. 

Blized Infection. — In regions where tuberculous processes are on 
the surface, such as lung and skin infections, and also when the in- 
fection itself is multiple, as in disease of the glands of the neck from 
tonsillar absorption, the tubercle bacilli are frequently associated with 
one or more other varieties of organisms. Those of most importance 
are the streptococcus, pneumococcus, and influenza bacillus. Be- 
sides these many other varieties are met with occasionally in individ- 
ual cases. What the influence of this secondary or mixed infection 
is, under all circumstances, is not exactly known; but generally the 
effect is an unfavorable one. For the technique employed in examin- 
ing sputa for mixed infection see page 344. 

Individual Susceptibility. — It is believed by many that in demon- 
strating the possibility of infection in pulmonary tuberculosis its 
occurrence is sufficiently explained; but they leave out another and 
most important factor in the production of an infectious disease — 
individual susceptibility. That this susceptibility, or ** predisposi- 
tion," as it is improperly called, may be either inherited or acquired 
is now an accepted fact in medicine. It is even thought that the physi- 
cal signs and characters — the "phthisical habit — which indicate this 
susceptibility can be externally recognized. At first the inherited 
susceptibility was considered more important than the acquired, but 
now much that was attributed to the former is known to be explained 
by the fact of living in an infected area. The acquired susceptibility 
may arise from faulty physical development or from depression, sick- 
ness, overwork, excessive use of alcohol, etc. Uncjuestionably, vast 
differences exist in different individuals in the intensity of the tuber- 
culous process in the lung. That this does not depend chiefly upon 
a difference in virulence of the infection is evident from the fact 



326 PATHOGENIC MICRO-ORGANISMS. 

that individuals contracting tuberculosis from the same source are 
attacked with different severity, and that there is, as a rule, no great 
difference in degrees of virulence for animals in the tubercle bacilli 
obtained from different sources. As is seen from the results of post- 
mortem examinations in which, according to the completeness of the 
examinations, the remains of old tuberculous processes have been 
found in the lungs of about one-third to one-half of all the bodies ex- 
amined, many cases of pulmonary infection must occur without showing 
any visible evidence of disease, and heal of their own accord. The 
possibility of favorably influencing in many an existing tuberculosis 
by treatment also proves that, under natural conditions, there is a 
varying susceptibility to the disease. Clinical experience teaches, 
likewise, that good hygienic conditions, pure sir, good food, freedom 
from care, etc., increase immunity to phthisis. Animal experiments 
have shown that not only are there differences of susceptibility in vari- 
ous animal species, but also an individual susceptibility in the same 
species. The doctrine of individual susceptibility, therefore, is seen 
to be founded on fact, although the reasons for it are only partially 
understood. 

Various other tuberculous affections which are natural in man 
have been produced experimentally in animals, as, for instance, tuber- 
culosis of the joints, tuberculous abscess, etc. 

ImiQailiEation. — As in other infectious diseases, many attempts 
have been made to produce an artificial immunity against tuberculosis, 
but so far the results have been only fairly satisfactory. The great 
majority of mankind has in a varying degree some natural immunity 
against tuberculosis. Even the infant receives through the placental 
membranes a considerable amount of any immune substances present 
in its mother. In many individuals this immunity is only relative, 
and is maintained only as long as the health is kept at a high standard 
or the exposure to infection not too intense or prolonged. An un- 
favorable environment, the occurrence of some other infectious disease, 
overwork, dissipation, or, in fact, anything which tends to depreciate 
the nutrition of the body, is apt to render the individual, previously 
immune, susceptible to the tubercle bacillus. 

Acquired immunity against many bacterial diseases occurs within 

a very few days or weeks after the development of infection. This 

immunity may be complete or slight and vary greatly in its duration. 

There is little at first glance in the clinical history of tuberculosis 

which shows that acquired immunity occurs in this disease, for relapse 

•= *'•'■ '"i", and one attack does not seem to afford any protection against 

le. For this reason the production of an artificial immunity 

iberculosis has always been looked upon by many as a result 

lever to be achieved. The careful study of tuberculosis 

iwever, to indicate an attempt on the part of nature at 

iction of acquired immunity in this disease. It is known 

30 to 60 per cent, of cadavers show the healed lesions of 

ns. The small proportion of those which progres.sed to 



THE BACILLUS OF TUBERCULOSIS. 327 

serious lesions or became reinfected indicates a degree of acquired 
immunity. 

Artificial immunity is an attempt to imitate nature's methods, and 
is obtained by the inoculation of a modified living culture or of toxins 
and dead bacilli. The injection by Koch of the heat-resistant-toxins, 
as in his original tuberculin treatment, produced in animals a certain 
degree of acquired resistance to larger doses of toxins, but did not 
protect to any appreciable degree from subsequent living tubercle 
bacilli, or produce in animals an antitoxic serum. In 1892 Trudeau 
succeeded in producing in rabbits an appreciable immunity by inocu- 
lations of living avian cultures. The rabbits so treated supported, as 
a rule, inoculation of virulent tubercle bacilli in the anterior chamber 
of the eye, while in controls the eyes were invariably lost. Later, 
attenuated human cultures were used with the same results. De 
Schweinitz, McFadyan, Behring, Calmette and Pearson and Gillilaud 
have since reported successful results. The latter two treated a num- 
ber of cows by giving each of them intravenous injections of 1 to 6 c.c. 
of an emulsion of human tubercle bacilli. This was of an opacity 
equal to a twenty-four-hour broth culture of typhoid bacilli. They 
report from their investigations^ that the treatment had the effect not 
only of keeping in check the progress of the tuberculous process, but 
of causing in some cases a distinct retrogression. The bacilli remained 
alive in the encapsulated lesions. Calmette fed calves with tubercle 
bacilli and found that a very small amount of infectious material became 
arrested in the intestinal and mesenteric glands and resulted in im- 
munity, while a larger amount, though arrested for a time, later 
passed through and caused a general and fatal infection. Behring 
has had prepared an emulsion of attenuated human bacilli to use in 
cattle. This should be used in cattle within a month of its preparation 
since the bacilli gradually die and lessen the effect of the vaccination. 

The work already done is believed by Trudeau to establish the 
principle that the most successful protective inoculation is the living 
germ of such diminished virulence for the animal experimented upon 
as to produce a reaction ending in healing of the process at first set 
up by it. This is termed by Behring isopathic immunity. After 
the living culture the best results have been obtained with the unheated 
filtrate of bouillon cutlures or the ground-up protoplasm of the chem- 
ically unaltered bacilli. 

The avian and bovine bacilli immunize against infection from hu- 
man bacilli probably nearly as well as the attenuated human variety. 
This is strong evidence in favor of the genetic unity of all tubercle 
bacilli. 

The importance of time in the production of artificial immuniza- 
tion has also been thoroughly demonstrated. It seems that whatever 
degree of immunity it is possible to produce is produced only very 
slowly. Von Behring found that his vaccinated cows which received 
the virulent inoculation before three months had passed showed little 

• University of Pennsylvania Medical Bulletin, April, 1905. 



328 PATHOGENIC MICRO-OHGASISMS. 

immunity and generally died of the infection, while after three months 
they resisted a fatal dose of the virus. While the principle of artificial 
immunity seems to be fairly well established by animal experimenta- 
tion, it must be admitted that the laboratory evidence which bears 
on the production of immunity in animals, or the cure of experimental 
tuberculosis by tuberculin, is far from satisfactory. 

Trudeau obtained the best results in treating animals with tul>er- 
culin in the eye tuberculosis of the rabbit, which is naturally a chronic 
and almost always a purely localized process. Tuberculosis in the 
guinea-pig, on the other hand, is an acute progressive Infection, and 
experimental and clinical evidence are in perfect accord in demonstrat- 
ing that against the acute types of tuberculous infection tuberculin 
is powerless, whether it be employed in man or animals. 

The duration of immunity, such as has been successfully produced so 
far in animals, has not yet been definitely ascertained, but the evi- 
dence so far at hand points to the fact that as the most solid immunity 
is produced by hving though attenuated cultures, the immunity which 
lasts the longest is also brought about in this way, the an titonc immunity 
produced by bacterial products being of shorter duration. The period 
of immunity after inoculation probably lasts more than one year, but 
usually less than two years. 

Ohemical OonBtitnents of Taberde Bacilli. — The bacilli contain 
on an average 86 per cent, water. The dry substance consists of 
material soluble in alcohol and ether, of proteid substance extracted 
by warm alkaline solutions, and of carboydrates and ash. The al- 
cohol-ether extract equals about one-quarter of the dry substance and 
consists of 15 per cent, of a fatty acid, which is mostly combined 
with an alcohol to make a wax. No glycerin is present and, there- 
fore, no true fat. It is on the presence of this wax that the staining 
characteristics depend. Other substances produce abscess, necrosis, 
and cheesy degeneration. Lecithin and a convulsive poison are also 
present in the extract. 

;s left after the ether-alcohol extraction are mostly pro- 

A nucleic acid which contains phosphorus is present. 

red by many to be the specific endotoxin of the tu- 

r TniTn^iiiiMiig Snbstances Prepared from Taberde 

r Ooltures. — Tub«rctilin Original "T. 0." (Koch's). — 
contains not only the products of the growth of the 
in the nutrient bouillon which withstand heat as 

ces extracted from the bodies of the bacilli them- 
the materials contained in the l>ouillon, which have 

cted by the activities of the bacilli. 

In is prepared as follows: The tubercle bacillus is 
infusion of calf's flesh, or of beef flesh, or extract to 

it. of peptone and 3 to 5 per cent, of glycerin have 

• culture liquid being slightly alkaline. The inocula- 

on the surface from a piece of very thin pellicle from 



THE BACILLUS OF TUBERCULOSIS. 329 

a young bouillon culture, or, if the bouillon culture is unobtainable, 
with small masses from a culture on glycerin agar. These masses, 
floating on the surface, give rise in from three to six weeks, according 
to the rapidity with which the culture grows, to an abundant develop- 
ment and to the formation of a tolerably thick and dry, white crumpled 
layer, which finally covers the entire surface. At the end of four 
to eight weeks development ceases, and the layer after a time sinks 
to the bottom. Fully developed cultures, after having been tested 
for purity by a microscopic examination, are poured into a suitable 
vessel and steamed in an Arnold sterilizer for three hours. The 
bacilli are then filtered off and the liquid evaporated to one-tenth of 
its original bulk over a water-bath at a temperature of 70° to 100° C. 
The liquid is then filtered through chemically pure filter-paper and 
finally through a stone filter. The crude tuberculin thus obtained 
contains 30 to 50 per cent, of glycerin, albumoses, traces of peptone, 
extractives, and inorganic salts. The true nature of the toxic sub- 
stances is not known. It keeps well, retaining its activity indefinitely. 
\Mien used it is diluted with one-fourth per cent, carbolic acid solu- 
tion. This diluted tuberculin is not quite stable and should be used 
within a week's time. It is considered that 1 mg. of tuberculin equals 
1 c.c. of a 1 : 1000 dilution. 

Tuberculin Precipitation "T. P." — A quantity of old (concentrated) 
Koch's tuberculin is poured into two volumes of 95 per cent, alcohol, 
allowed to settle, and filtered per paper. The sediment is washed 
with 70 per cent, alcohol until the filtrate runs clear, then pressed 
between layers of filter-paper to remove excess of moisture, scraped 
into a dish, dried in vacuo over HjSO^, and broken up in a mortar. 
For the Calmette eye test solutions of the powder are made in sterile 
normal salt solution of \ and 1 per cent, by weight, boiled in a 
water-bath, filtered, diluted as required, distributed into small tubes 
containing about two drops, which are then sealed and boiled for ten 
minutes. 

Bacillus Emulsion "B. E." — This is Koch's latest product. It is an 
emulsion of the entire substance of the unaltered tubercle bacilli in 
20 per cent, of glycerin. The broth culture is poured into a filter 
and the broth filtered off. The bacilli are washed, pressed between 
absorbent paper and dried in exsiccator. They are then ground in a 
mortar until no formed bacilli are found on staining. The powder is 
taken up in 0.8 per cent, salt solution and added to 20 per cent, 
glycerin water so that 1 mg. of powder is contained in . 2 c.c. of the 
final preparation. Dilutions are made in 0.5 per cent, carbolic acid 
in 0.8 salt solution. As can be readily seen, in a preparation thus 
made, contamination is difficult to avoid, freedom from intact bacilli 
is uncertain. This preparation is, therefore, before marketing, usually 
subjected to heating at 60° C. 

Bouillon Filtrate Tuberculin. — This is the unheated filtrate from 
bouillon cultures of human tubercle bacilli. Its use was suggested 
by Denys. The last two preparations are for treatment only. After 



330 PATHOGENIC MICRO-ORGANISMS. 

six years of trial in the treatment of cases the results obtained from the 
use of the new tuberculin preparations, which are unheated or heated 
not over 60*^ C, are considered superior to those obtained from the 
older product. 

Many other tuberculins have been proposed during the past fifteen 
years, among which are Hunter's Modification B., von Ruck's Watery 
Extract, Landemann's Tuberculol, Denys' Bouillon Filtrate, Baraneck's 
Tuberculins, Spengler's Bovine B. F., and Behring's T. C. and Tulasa, 
which he claims immunizes cows as well as the living bacilli, but the 
value of which has not yet been put to a practical test in the treatment 
of human tuberculosis. These tuberculins are all vaccines, they are all 
made from either the body substance of the germ or the liquid medium 
in which it has grown, or both, and their aim is to stimulate the defensive 
resources of the system, and to induce antitoxic and antibacterial im- 
munity. They all produce, when given in suflScient doses, local 
reactions in tuberculous foci, and the well-known but little understood 
phenomena of general tuberculin reaction. These preparations are 
described in detail by Baldwin in Osier's "Practice of Medicine" 
(Vol. iii, page 160). 

The Use of Tuberculins in Treatment and Immunization.— Koch's 

old tuberculin, which was at first principally used, has of late been 
generally discarded for preparations which have not been subjected to 
heat at least not above 60°. The two most used are B. F., a filtrate of 
human cultures of recorded virulence to which a quarter of 1 per cent, 
carbolic acid has been added, or B. E., which is an emulsion in glycerin 
and water of the pulverized bodies of the virulent tubercle bacilli. 
With the B. E. habituation takes place with much more diflSculty 
than with B. F., and occasionally unexpected and sometimes violent 
reactions occur, even if the utmost caution in increasing the dose is 
exercised. It is possible that having obtained a certain degree of 
antitoxic immunity with a course of B. F., a secondary course in 
which B. E. is employed might prove more efficacious, and it is evi- 
dent we have much yet to learn about the production of the tuber- 
culous vaccines and their application in the treatment of disease. 
Time and experience alone can show us which tuberculin produces 
the best results. 

According to Koch, the substances produced in the body by the old 
tuberculin neutralize the tuberculous toxins, but are not bacteri- 
cidal. After a series of experiments he considered the difficulty to 
be due to the nature of the envelope of the tubercle bacillus, which 
made it difficult to obtain the substance of the bacilli in soluble form 
without so altering it by heat or chemicals that it was useless to pro- 
duce immunizing substances. He conceived that immunity was not 
produced in man for somewhat similar reasons — possibly the bacilli 
never giving out sufficient toxin to cause curative substances to be 
produced. He therefore decided to grind up the washed and dried 
bacilli and soak them in water, and thus obtain, if possible, without 
the addition of heat, a soluble extract of the body substance of the 



THE BACILLUS OF TUBERCULOSIS. 331 

bacilliy which he hoped would be immunizing. He also tried to 
eliminate as much as possible of the toxic products which produce 
fever. Buchner by a different method, through crushing under a 
great pressure tubercle bacilli mixed with sand, and thus squeezing 
out their protoplasm, obtained a very similar substance called plas- 
mine. Vaughan has tried the immunizing effect of the non-poisonous 
split products obtained from treated tubercle bacilli. 

Trudeau and others have formulated a schedule by which the 
initial dose, the intervals between injections, the rate of progression, 
and the ultimate dose to be attained are distinctly laid down, this 
schedule to be literally followed so long as the patient shows no evi- 
dence of intolerance, but modified at once, as soon as he does, to suit 
the requirements of each case. Many patients can be carried from 
beginning to end of the treatment — a period which, when no reac- 
tions occur, usually takes about eight months — without any symp- 
toms which call for any departure from the schedule itself, laid down. 
If this were always so the treatment would be simplicity itself, but un- 
fortunately in the majority of cases, at some period in the treatment, 
sometimes at the very be^nning, sometimes at the middle, and some- 
times even at the very last dose, symptoms of intolerance appear, and 
it is then that the physician requires certain definite rules to guide 
him in his conduct of the case. 

Experience has shown that it is essential to begin treatment with 
very small doses; that is, for afebrile cases, ^ ^ ^ ^ ^ milligram of fil- 
trate B. F., or Koch's B. E. (liquid measure, not solid substance), or 
ttjVtt milligram of old tuberculin. Denys makes use of eight solu- 
tions in giving B. F. No. 1 contains ^ ^^ ^ ^ milligram to each cubic 
centimeter. This is for febrile cases only. No. 2 contains x^Vir 
milligram to each cubic centimeter; No. 3, j-J^^; No. 4, ^^5 N^- 5, 
1 milligram; No. 6, 10 milligrams; No. 7, 100 to each c.c, and No. 8 
is pure filtrate. Now the increase in using these solutions is always 
by 1 decigram of each solution, which is convenient to measure and 
easy to remember. As 10 decigrams, or 1 c.c. of each solution is 
reached, the next solution, which is ten times stronger and in which 
1 decigram represents the same dose as 1 c.c. of the preceding solu- 
tion, is taken up and the increase is again by 0.1 of the new solution 
until 1 c.c. is given when the next solution is taken up iti the same 
way until the end of the treatment. Thus for ten doses the increase 
for each dose is by i jj^uir miUigram for ten doses; then by r^jVirfor 
ten doses; then by ^^ for ten doses; then by^V^^^ ten doses; then 
by 1 milligram for 10 doses; then by 10 milligrams for 10 doses; then 
by 100 milligrams, until 1 c.c. of the pure filtrate or old tuberculin 
is reached. The increase is by 0.1 of each solution, and as each 
solution is 10 times stronger than the preceeding, the progression in 
doses is ten times greater at the end of every ten doses. Approxi- 
mately the same plan may be followed by giving Bacillen-emulsion, 
provided it is remembered the doses referred to in the above schedule 
are liquid measure and not solid substance. 



332 PATHOGENIC MICRO-ORGANISMS. 

Dr. Brown has found that at the Adirondack Cottage Sanitarium 
reactions occur more freiijuently at the second or thirci injection of a 
new solution. This is not to be wondered at, as the increase in do.se 
is ten times larger when a new solution is taken up. To obviate this, 
instead of increasing 0.1, 0.2, 0.3, and so on to 10 decigrams, the in- 
crease may be 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, O.G, 0.8, 0.10. 

The intervals between the injections are three or four days; gen- 
erally two injections a week; but as the higher doses, such as 10 mil- 
ligrams, are reached, the intervals may be five days, and after 100 
milligrams six days, while the last three or four doses should be given 
a week or ten days apart. Lowenstein finds that for Bacillen-emul- 
sion longer intervals are necessary between the doses, especially when 
the large amounts are reached. 

If no intolerance is manifested the treatment will require six 
months; but in the majority of cases when any reactions occur it 
should be extended over ten months or a year, or even much longer, 
if necessary, to reach full doses. It is a mistake to try to shorten the 
time by increasing the doses too rapidly or decreasing the intervals. 
Whatever degree of immunity, antitoxic or otherwise, is produced 
by the treatment is produced only very gradually, anil besides the 
risk to the patient which is always involved by haste, the intolerance 
it may produce takes often so long to overcome that the duration of 
the treatment is lengthened rather than shortened in the end. 

Tolerance to tuberculin is an excellent prognostic sign and it bears 
a certain relation to the condition of the patient's general health; and 
the more this improves the less apt is he to develop symptoms of pro- 
longed intolerance, but the improvement in the general health is 
necessarily a slow process. 

How does intolerance show itself, and how are we to proceed when 
it does? The symptoms of intolerance may be divided into three 
groups; those of a general fever reaction, those which indicate local 
reaction, both at the site of disease and also the site of injection, and 
those which point to general constitutional disturbance, as mani- 
fested by malaise, headache, sleeplessness, wandering pains, anorexia, 
nausea, and loss of weight an<l strength. 

The fever reactions are of two kinds: the short and the prolonged 

reaction. The short reaction is identical with that produced by the 

tubercuhn test, and shows the classical fall and rise of temperature all 

ending in forty-eight hours; the prolonged reaction begins generally 

more gradually; the symptoms are mild; the fever rises less high but 

■ elf, with a morning remission, above the patient's normal 

range for several days, generally not more than a week. 

tion at the site of the lesion is a valuable guide to dosage; 

ugh and expectoration, pleuritic pains, aggravation of 

signs, hoarseness and aphonia if the larynx is involved 

I joint, ail point to local reaction, and are all indications 

1 increasing the dosage. 

tion at the .site of injection .shows it.self by more or less 



THE BACILLUS OF TUBERCULOSIS. 333 

extensive redness, cedema, and pain; when slight, it may be disre- 
garded, as it is somewhat influenced by the manner of injection or 
other causes; but if marked, it indicates commencing intolerance and 
should be considered in connection with the patient's other symptoms 
before increasing the dose. 

Most important, and most often disregarded because no rise in 
temperature may be present, is the group of symptoms which point 
to constitutional impairment resulting from overdosage. They are 
all the symptoms that chronic toxemia might be expected to produce, 
and all point to the supposition that the patient cannot respond by 
the formation of antitoxins and antibodies to the increasing doses of 
toxin which he is receiving. Even if no fever above the usual range 
be present, the patient who has been improving, and whose general 
condition has been satisfactory, may show marked arrest in his improve- 
ment; if the injections are persisted in and the dose steadily increased, 
he will complain of malaise, exhaustion, headache, sleeplessness, 
wandering pains, anorexia, nausea, and loss of weight. If these 
symptoms are disregarded, the injections continued, and the dose 
heedlessly increased, in time the patient's disease may take on an 
acute form. When intolerance manifests itself, whether by general 
fever reactions, by evidence of local reactions, or by some of the symp- 
toms of constitutional impairment, the rule is never to inject while 
any of these symptoms are still present, but to wait until the temperature 
has returned to its usual height, until the cough and increased expectora- 
tion have lessened, and all evidence of constitutional impairment, 
such as anorexia, malaise, debility, etc., have disappeared. Indeed 
all evidences of intolerance must have been absent for at least two 
days before the injections are again taken up, then start with lower 
doses. 

We have learned that **No reaction, no cure," has been a most 
misleading axiom, for we can have tuberculin immunity without 
reactions, and many reactions without any tuberculin immunity. 
Strong and frequent reactions are harmful, while patients who go 
through the entire treatment without appreciable fever reactions 
derive all the benefit that could be expected from the treatment. 

Trudeau has formed his favorable impression of the influence of 
tuberculin by noticing how rarely the disease seemed to progress by 
the usual exacerbations and relapses in patients who were tolerating 
progressively increasing doses of tuberculin well, and in watching 
chronic cases, who were running a slow but steadily downward course 
in spite of the climatic and open-air treatment, derive marked bene- 
fit and even gradually return to apparent health after a full course 
of injections. 

That tuberculin is not the vaunted and long-looked-for specific it 
was at first thought to be has been amply demonstrated by the bitter 
experience of the past. We have much to learn about tuberculosis, 
but even at the present state of our knowledge it seems established 
that the production of tuberculin immunity by the mild clinical 



334 PATHOGENIC MICRO-ORGANISMS. 

method is capable of favorably influencing the course of subacute 
and chronic tuberculosis, of prolonging life, and in many cases of 
aborting a commencing infection. 

As to the type of cases suitable for tuberculin treatment, Denys 
and some of the Germans claim that even in acute cases good results 
may be occasionally expected by a careful course of injections. 

Diagnostic Uses of Tnbercalin.— The chief use to which Koch's 
ori^nal tuberculin has been put is as an aid to diagnosis in human 
beings and cattle, and for this purpose it has proved to be of inesti- 
mable value. Numerous experiments made by veterinary surgeons 
show that the injection of tuberculin in tuberculous cows in doses of 
25 to 50 centigrams produces in at least 95 per cent, a rise of temper- 
ature of from 1° to 3° C. (2° to 5° F.). The febrile reaction occurs 
in from twelve to fifteen hours after the injection. Its intensity and 
duration do not entirely depend upon the extent of the tuberculous 
lesions, being even more marked when these are slight than in ad- 
vanced cases. In non-tuberculous animals no reaction occurs, or one 
much less than in tuberculous animals, and the results obtained on 
autopsy justify the suspicion that tuberculosis exists if an elevation 
of temperature of a degree or more centigrade occurs and remains 
for ten hours from the subcutaneous injection of the dose mentioned. 
It must always be remembered that cattle may have a rise of tempera- 
ture from other conditions, and it is only when due to tuberculin 
that infection is proved. When properly carried out, an error of 
more than 5 per cent, is impossible. For these injections, four- 
tenths c.c. of the original tuberculin is used, which for the conveni- 
ence of administration is diluted with water. 

United States OoTemment Directions for Inspecting Herds for 
Tabercolosis. — "Inspections should be carried on while the herd is 
stabled. If it is necessary to stable animals under unusual condi- 
tions or among surroundings that make them uneasy and excited, 
the tuberculin test should be postponed until the cattle have become 
accustomed to the conditions they are subjected to, and then begin 
with a careful physical examination of each animal. This is essen- 
tial, because in some severe cases of tuberculosis, on account of satu- 
ration with toxins, no reaction follows the injection of tuberculin, 
but experience has shown that these cases can be discovered by physi- 
cal examination. This should include a careful examination of the 
udder and of the superficial lymphatic glands, and auscultation of the 

liould be numbered or described in such a way that 
d without difficulty. It is well to number the stalls 
ansfer these numbers to the temperature-sheet, so 
jre of each animal can be recorded in its appro- 
ut danger of confusion. The following procedure 
msively and has given excellent results: 
temperature of each animal to be tested at least 
of three hours, before tuberculin is injected. 



JHE BACILLUS OF TUBERCULOSIS. 335 

** (6) Inject in the evening, preferably between the hours of six 
and nine, -^ c.c. of Koch's tuberculin previously diluted to 5 c.c. 
with sterile water. The injection should be made with a carefully 
sterilized hypodermic syringe. The most convenient point for injec- 
tion is back of the left scapula. Prior to the injection the skin should 
be washed carefully with a 5 per cent, solution of carbolic acid or 
other antiseptic. 

** (c) The temperature should be taken nine hours after the injec- 
tion, and temperature measurements repeated at regular intervals of 
two or three hours until the sixteenth (eighteenth)^ hour after the 
injection. 

"(d) When there is no elevation of temperature at this time the 
examination may be discontinued; but if the temperature shows an 
upward tendency, measurements must be continued until a distinct 
reaction is recognized or until the temperature begins to fall. 

** (e) If a cow is in a febrile condition tuberculin should not be 
used, because it would be impossible to determine whether, if a rise 
of temperature occurred, it was due to the tuberculin or to some transi- 
tory illness. 

"(/) Cows should not be tested within a few days before or after 
calving, for experience has shown that the result at these times may 
be misleading. 

** (g) In old, emaciated animals and in re-tests, use twice the usual 
dose of tuberculin, for these animals are less sensitive. 

"(A) Condemned cattle must be removed from the herd and kept 
away from those that are healthy. 

" (i) In making post-mortems the carcasses should be thoroughly in- 
spected, and all the organs should be examined." 

Diagnostic Use of Tuberculins in Man.— At first some believed 
that the irritation aroused in the tuberculous foci by the tuberculin 
sometimes caused a dissemination of the bacilli and an increase in 
the disease. When carefully used, however, in suitable cases there 
is probably no danger. A drawback to its usefulness is that it does 
not reveal the extent of the disease, nor whether the tuberculosis is 
active. It is, however, of great value in selected cases, both surgical 
and medical, where slight tuberculosis is suspected, and yet no de- 
cision can be reached. In the small first dose advised an absolutely 
latent infection would usually give no rise of temperature. I quote 
here Dr. Trudeau upon the use of the test: 

"The range of the patient's temperature is ascertained by taking 
it at 8 A. M., 3 P. M., and 8 p. m., for three or four days before making 
the test. The first injection should not exceed . 5 mg. in adults and 
0.3 in small children, and if any fever is habitually present should be 
even less, and is best given early in the morning or late at night, as the 

*The directions allow temperatures to be stopped the sixteenth hour, but even 
when there is no reaction at all it is much safer to always take temperatures for 
eighteen hours. We have found now and then a tuberculous cow that reacted 
on the eighteenth hour for the first time. 



336 PATHOGENIC MICRO-ORGANISMS. 

typical reaction usually begins, in my experience, within six or twelve 
hours. Such a small dose, while it will often be suflScient to produce 
the looked-for rise of temperature, has, under my observation, never 
produced unpleasant or violent symptoms. An interval of two or three 
days should be allowed between each of the two or three subsequent 
injections it may be necessary to give, as reaction in very rare eases 
may be delayed for twenty-four or even thirty-six hours. On the 
third day a second dose of 1 mg. is given, and if no effect is produced a 
third, of 2 mg., three days later. In the great majority of cases of latent 
tuberculosis an appreciable reaction will be produced by the time a 
dose of 2 mg. has been reached. If no effect has been caused by the 
tests applied as above I have usually gone no farther, and concluded 
that no tuberculous process was present, or at least not to a degree 
which need be taken into account in advising the patient, or which 
would warrant insisting on a radical change in his sourroundings and 
mode of life. If some slight symptoms, however, have been pro- 
duced by a dose of 2 mg., it may be necessary to give a fourth injec^ 
tion of 3 mg. in order to reach a positive conclusion. Nevertheless, 
it should be borne in mind that in a few cases the exhibition of even 
larger doses may cause reaction, when the smaller do not, and indicate 
the existence of some slight latent tuberculous lesion, and the nega- 
tive result should not, when applied within the moderate doses de- 
scribed, be considered absolutely infallible." 

**No evidence in connection with the tuberculin test as applied to 
man and animals has been forthcoming thus far from those who have 
made use of it, which would tend to sustain the general impression that 
this method is necessarily dangerous and tends invariably to ag- 
gravate the disease, and my own experience has developed nothing 
which would seem to confirm this impression. It is evident that the 
size of the doses given has much to do with the limitations of this 
method for usefulness, and the correctness of the conclusions reached 
by its application. The tuberculin used is also a matter of some im- 
portance in determining the dosage, as different samples vary con- 
siderably in their efficiency. If the test be pushed to the injection 
of such large amounts at 10 mg. or more, as advocated by Maragliano, 
such doses are by no means free from the objection of occasionally 
causing unpleasant and sometimes dangerous symptoms; and even 
if the amount given be not carried to the dose of 10 mg., which is 
known to produce fever in healthy subjects, it is likely that on account 
of individual susceptibility or the presence of some other morbid 
process in the body, reaction will be found to occur with the larger 
doses when no tuberculous process exists. The adoption of an 
initial dose so small as to guard against the absolute possibility of 
producing violent reactionary symptoms, and the graded increase 
of the subsequent doses within such quantities as are known never to 
produce reaction in healthy individuals, would seem to afford the best 
protection against unpleasant results and misleading evidence." 

Von Pirpuet's Cutaneous Tuberculin Test.— This has for manv 



THE BACILLUS OF TUBERCULOSIS. 337 

purposes supplanted the subcutaneous injections. It is perfectly 
harmless. This is carried out by placing a drop of a 25 or a 50 or a 
100 per cent, solution of tuberculin upon the skin of the forearm and 
then with a needle or instrument making through it a slight abrasion 
\%nthout drawing blood, as in vaccination. The skin is abraded at 
another point without the tuberculin as a control. Within 12 to 24 
hours a papule with a surrounding congested area forms about the 
inoculated point much as appears after the use of cowpox vaccine in a 
previously vaccinated person. The test is frequently carried out by 
making a scratch about an inch in length. This should if possible 
not cause bleeding. The tuberculin on a probe or slip of wood is 
rubbed into the scratch. Sometimes one spot is tested with the 10 
per cent, solution and a second with the 25 per cent. A reaction with 
the weaker solution is believed to indicate some activity in the process, 
while the stronger may give a reaction in a person having a recently 
healed lesion. 

Moro's Test. — Equal parts of tuberculin and lanolin are mixed 
together to make an ointment. A little of this is rubbed thoroughly 
upon a portion of the skin of the arm. Twelve to twenty-four hours 
afterward a crop of papules develops in cases in which the cutaneous 
tests proves effective. 

Directions for the Ophthahno-tuberculin Diagnostic Test.— Method 

of Application. — Two solutions in two strengths are employed in diag- 
nosis, one of the alcohol precipitate in 0.5 per cent, and 1 per cent, 
and the other of 1 and 2 per cent, of tuberculin (T. O.). The weaker 
and stronger may be used successively in each eye if time permits. 
In this way unnecessarily severe reactions may be avoided. 

The eyelid should be held down until the drop is distributed about 
the sac without overflowing on the cheek. The same eye should 
not be used for a second test as it usually becomes sensitized to some 
degree by one test The tested eye should be kept from external 
irritation due to rubbing, wind, dust, and smoke. 

Reactions. — The first symptoms of a reaction appear in from 3 to 12 
hours in most cases, but may be delayed 24 and even 48 hours, and 
continue for a week. The presence of a reaction is indicated by a 
scratchy feeling, secretion and redness of the inner canthus, caruncle 
or lower lid which may increase and include the entire conjunctiva 
with oedema of lids. 

Schema for Recording Reactions. — The following schema is propose 
for recording the degree of reaction. 

Negative: No difference in color when lower eyelids are pulled 
down. 

Doubtful: Slight difference with redness of caruncle. 
-f = Distinct palpebral redness with secretion. 
-h -f = Ocular and palpebral redness with secretion well marked. 

-\- -{--{- = Deep injection of entire conjunctiva with oedema of lids 

and photophobia, and secretion. 

Contraindications. — Any existing disease of either eye or lids. 

22 



338 PATHOGENIC MICRO-ORGANISMS. 

Interpretation of Reaction. — ^This is practically the same as with 
the cutaneous test. About 80 per cent, of latent or active tuberculosis 
react and about 40 per cent, of very advanced cases. Persons very 
ill from other diseases frequently do not react. Tuberculosis so slight 
that it is impossible to detect it during life may give a good reaction. 
A negative result in a person in fair general health indicates strongly 
that no tuberculosis is present. The location of the tuberculosis is 
of course not revealed by the test. 

Deleterious Effects. — Our personal experience accords with that pub- 
lished by others that about once in every four hundred tests a serious 
conjunctivitis, keratitis, or .iritis results. This possible injury has led 
largely to giving preference to the cutaneous test. 

Antituberculous Senixn. — Every conceivable way of obtaining the 
true products of the tubercle bacilli has been tried, so as to cause the 
injected animals to produce antibodies both antitoxic and bactericidal. 
At present Maragliano and Marmorek are presenting claims that their 
sera are truly curative. Although both these men have had a large 
experience in this field of investigation, it is probable that the final 
judgment will be that little good comes from the injection of their 
serum. Very few observers have succeeded in obtaining appreciable 
results with the serums prepared by other experimenters. In spite 
of much conflicting testimony, it is probably safe to assert that no sera 
now obtainable have any great value. Nor as we look at the progres- 
sive nature of tuberculosis can we see much ground to hope for the 
abundant development of curative substances in the blood of animals. 
This view, however, in no way lessens the necessity of continued en- 
deavor until every method conceivable has been tried. 

Prophylaxis. — Meanwhile all energies should be directed to the 
prevention of tuberculosis, not only by the enforcement of proper 
sanitary regulations as regards the care of sputum, milk, meat, dis- 
infection, etc., but also by continued experimental work and by the 
establishment of free consumptive hospitals, and by efforts to im- 
prove the character of the food, dwellings, and conditions of the 
people in general, we should endeavor to build up the indi\idual 
resistance to the disease. It may be years before the public are 
sufficiently educated to cooperate with the sanitary authorities in 
adopting the necessary hygienic measures to stamp out tuberculosis 
entirely; but, judging from the results which have already been ob- 
tained in reducing the mortality from this dread disease, we have 
reason to believe that in time it can be completely controlled. 

Among the numerous medical agents that have been tried without 
avail to protect animals against the action of the tubercle bacillus 
may be mentioned tannin, menthol, sulphuretted hydrogen, mercuric 
chloride, creosote, creolin, phenol, arsenic, eucalyptol, etc. 

Agglutination. — The results obtained by various observers have 
been very conflicting. Two methods are employed in making the 
test. In one a \igorous growth of bacilli is dried, ground up, and an 
emulsion made. In the other Arloing and Courmont grow the cul- 



THE BACILLUS OF TUBERCULOSIS. 339 

ture for a time on potato and then in bouillon. In this way a homo- 
geneous culture of separate bacilli is obtained which can be used for 
agglutination. The examination is usually made macroscopically, 
and requires twelve to twenty-four hours. At present the test 
cannot be advised as useful in diagnosis as the sera of cases suf- 
fering from tuberculosis frequently fail to give a reaction, while 
the sera from those having no detectable tuberculosis frequently cause 
a good reaction. A positive agglutination test is thought by some 
to be a favorable sign as indicating resistance to infection by the body. 
A reaction in dilutions of 1:10 or 1:15 is considered a positive test. 

The Tubercle Bacillus of Oattle, Pigs and Sheep, and its Relation 
to Human Tuberculosis. — Among the domestic animals tuberculosis 
is most common in cattle. On account of the milk which they pro- 
vide for our use, and which is liable to contain bacilli, the relation of 
these to human tuberculosis is a matter of extreme importance. 

The chief seat of the lesions is apt to be the lungs, and with them 
the pleura; less often the abdominal organs and the udder are af- 
fected. In pigs and horses the abdominal organs are most often 
involved, then the lungs and lymphatic glands. In sheep and goats 
tuberculosis is rare. 

Differences between Tubercle Bacilli of Human and Bovine 

Type. — As has been already noted in the tables given of the incidence 
of bovine and human infection, it is possible to tell in any case the type 
of infection. The essential differences are in cultural characteristics 
and in virulence for rabbits and calves. 

Oultaral Differences. — The bovine bacillus grows very poorly when 
isolated, the human bacillus very freely. This is noted on plain egg, 
but to a less extent than on glycerin egg. The glycerin restrains or 
adds little to the growth of bovine bacilli, but increases markedly 
the amount of growth of the human bacillus. In fact, primary cul- 
tures on glycerin egg of bovine material commonly fail. This dif- 
ference is very noticeable in the first few generations and is suflScient 
in the great majority of instances for differentiation to one who has 
had some experience with such cultures. Further, the majority of 
human strains can be transplanted to glycerin potato or glycerin 
broth and give vigorous growth in the first few generations, whereas 
the bovine bacillus fails or growth is very slight. After further culti- 
vation the bovine bacillus gradually increases its amount of growth 
until it is indistinguishable from the human type. This increase in 
luxuriance "toay be rapid or very slow. 

Rabbit Virulence. — The bovine bacillus is exceedingly virulent for 
rabbits by any method of inoculation; the hiiman bacillus only slightly 
so. The best method of diflFerentiation is by intravenous inoculation. 
A small amount of culture is weighed after the moisture has been ex- 
tracted with filter-paper and a suspension made in normal saline and 
diluted so that 1 c.c. =rJir ^g* ^^ culture; this amount is then 
injected into the ear vein of a rabbit. If the rabbit survives for 
from forty to fifty days, and on autopsy shows only lesions in the 



340 PA THOGENIC MICRO-ORGA NISMS. 

lungs or kidneys or both, the strain is of the human type. With the 
bovine type of bacillus the rabbit will die in the majority of instances 
before or about this time, if not it may be killed. On autopsy a pro- 
gressive generalized tuberculosis will be found. The lesions in the 
lungs will be very marked, the tubercles having become confl^ient 
with caseous centres. The liver or spleen or both will be peppered 
with tubercles. Tubercles will be present in the great majority of 
cases in the superficial lymph nodes and also in those of abdomen 
and thorax. There may be tubercles on the heart, in the rib marrow, 
or over the peritoneum. 

These two differences alone are sufficient to differentiate in every 
case the type of bacillus. It must be insisted upon again that the cul- 
tural characteristics be observed in the early generation and further 
that the virulence be tested in early generations. In case the bovine 
culture does not afford sufficient material for weighing, a suspension 
can be made and compared with a weighed suspension. 

Virulence for Calves. — In proving the non-identity of the two 
bacilli, calf experiments were resorted to. This was necessary as the 
supposed bovine cultures from children would have to be \drulent 
for calves to the same extent as cultures from bovine material. The 
commonly used method was the subcutaneous inoculation in the side 
of the neck with 50 mg. of culture. The human type of bacillus 
caused only a local lesion or at most a spreading to the nearest lymph 
node. The bovnne bacillus, on the other hand, caused a generalized 
tuberculosis which was or was not fatal. Sufficient data has been 
accumulated to make this test practically unnecessary for the deter- 
mination of type. 

Differences in Morphology. — The bovine bacillus tends to be shorter, 
thicker and solidly stained; the human type tends to be longer, slim- 
mer, usually bent, and shows beading and irregularities in staining. 
We have found this difference most marked on glycerin egg, slight or 
imperceptible on other media. 

Besides the above differences Theobald Smith made the interesting 
discovery that the production of acid differed with the two types when 
grown on glycerin broth. The bovine type renders the bouillon less 
and less acid; this may even progress till the medium becomes slightly 
alkaline to phenolphthalein. The human type causes a preliminary 
fall in the acidity; as growth progresses the acidity is then gradu- 
ally increased, and may exceed the original acidity of the broth used. 
This difference is evident in tuberculin made from the two types of 
bacilli. The bovine tuberculin is alkaline or very slightly acid while 
human tuberculin is markedly acid. The change is only noticed 
when glycerin is used in the media. Whether this diflFerence is specific 
is doubtful. The work of more recent investigators would seem to 
show that this difference, like all differences between the types, is 
purely quantitative, and that different strains vary in their reactions 
and give intermediate reactions between these two extremes. 

Bird (Avian) Tuberculosis. — Tuberculosis is very common and 



THE BACILLUS OF TUBERCULOSIS, 341 

infectious among fowl. The bacillus grows easily and freely on gly- 
cerin media. It tends to form a moist or even slimy growth, and com- 
monly produces an orange pigment. It is able to grow at a higher 
temperature than mammalian tubercle bacilli, the latter failing to grow 
above 41° C. ; the former growing at even higher temperatures. Guinea- 
pigs are less susceptible to inoculation with avian tubercle bacilli, and 
the \nrulence for these animals is usually quickly lost. Rabbits are 
somewhat more susceptible. Rats and mice are spontaneously infected 
with avian tubercle bacilli and are supposed to be an important factor 
in spreading the disease. Birds are refractory, ^ith few exceptions, 
to infection with the mammalian tubercle bacillus. Parrots, however, 
are susceptible to infection with all three types and commonly have 
spontaneous tuberculosis caused by the human type of bacillus. 

Stability of the Different Types of Bacilli.— The fact that the agglu- 
tination reactions and the tuberculin reactions of the different types is 
similar shows their close relationship. This has led to the endeavor 
to change one type into the other. This is usually done by passage 
through animals. The results have been peculiar. Some cultures 
have been passed through a series of calves without any change ex- 
cept for a moderate increase in virulence. Other cultures seem to 
have completely changed their type. We believe that this is not a 
change of type, but an additional bovine infection. Strong negative 
evidence is the fact that the bovine bacillus when infecting man loses 
none of its characteristics, though present in the human body for years. 

Tuberculosis in Fish. — In certain species of fish a tuberculous 
disease has been noted. The bacilli have the staining characteristics 
of the warm-blood types, but do not grow at body temperature and do 
not affect mammals. 

Methods of Examination for Tubercle Bacilli and Other Associated 

Bacteria. — One of the most important results of the discovery of the 
tubercle bacillus relates to the practical diagnosis of tuberculosis. 
The staining peculiarities of this bacillus renders it possible by the 
bacteriological examination of microscopic preparations to make an 
almost absolutely positive diagnosis in the majority of eases. A still 
more certain test in doubtful cases is the subcutaneous or intraperi- 
toneal injection of guinea-pigs, which permits of the determination 
of the presence of numbers of bacilli, so small as to escape detection 
by microscopic examination. For the animal test, however, time 
is required — at least three weeks, and, when the result is negative, 
at least six weeks — before any positive conclusion can be reached, 
for when only a few bacilli are present tuberculosis develops slowly 
in animals. In disinfection experiments where many dead bacilli are 
injected, care must be taken to exclude the local effect of dead bacilli. 
In doubtful cases a second guinea-pig should be injected from the first. 

Microscopic Examination of Sputum for the Presence of Tubercle 

Bacilli. 1. OoUection of Material. — The sputum should be collected 
in a clean bottle (two-ounce) with a wide mouth and a water-tight 
stopper, and the bottle labelled with the name of the patient or with 



342 PATHOGENIC MICRO-ORGANISMS. 

some other distinguishing mark. The expectoration discharged in 
the morning is to be preferred, especially in recent cases, and the 
material should be coughed up from the lungs. Care should be taken 
that the contents of the stomach, nasopharyngeal mucus, etc., are 
not discharged during the act of expectoration and collected instead 
of pulmonary sputum. If the expectoration be scanty the entire 
amount discharged in twenty-four hours should be collected. In 
pulmonary tuberculosis the purulent, cheesy, and mucopurulent spu- 
tum usually contains bacilli; while pure mucus, blood, and saliva, as 
a rule, do not. When hemorrhage has occunred, if possible, some 
purulent, cheesy, or mucopurulent sputum should be collected for 
examination. The sputum should not be kept any longer than neces- 
sary before examination, for, though a slight delay or even till putre- 
faction begins, does not vitiate the results so far as the examination 
for tubercle bacilli is concerned, it almost destroys any proper inves- 
tigation of the mixed infection present; it is best, therefore, to ex- 
amine it in as fresh a condition as possible, and it should be kept on ice 
until examined if cultures are to be made. 

2. M«tbod8 of Examination. — Examination for Tubercle Bacilli. — 
Pour the specimen into a clean, shallow vessel, having a blackened 
bottom — a Petri dish placed upon a sheet of dull black paper answers 
the purpose — and select from the sputum some of the true expectora- 
tion, containing, if possible, one of the small white or yellowish-white 
cheesy masses or "balls," From this make rather thick cover-glass 
or slide "smears" in the usual way. In doubtful cases a number 
of these coarse or fine particles should be placed on the slide. The 
material being thick, should be evenly spread and very thoroughly 
dried in the air before heating. Immerse this in a solution of Ehr- 
lich's aniline-water fuchsin or better in the Ziehl-Neelson carbol- 
fuchsin solution contained in a thin watch-glass or porcelain dish, 
or hold slide completely covered with solution in the Cornet forceps 
' ■ earn over a small flame for two minutes. Then remove and 

vith water. Now decolorize by immersing the stained prep- 
in a 3 per cent, hydrochloric acid solution in alcohol for from 

If up to one minute, removing at the time when all color is just 

gone from the smear. Wash thoroughly with water, and make 

ra.st stain by applying a cold solution of Ix>efller's alkaline 

ene blue — 

icentrsteil alcoholic solutioD of methylene blue 30 c.c. 

istic potash (1:10,000 3olutio ' 



m fifteen to thirty seconds. Wash with water; press between 
' filter-paper; dry in air; mount, and examine, 
tubercle bacilli are distinguished by the fact that they retain 
i color imparted to them in the fuchsin solution, while the 
bacteria present, having been decolorized in the acid solu- 
ike the contrast stain and appear blue. (See Plate I., Figs. 1 
) 
3US methods have been suggested for the staining of tubercle 



THE BACILLUS OF TUBERCULOSIS. 343 

bacilli, but the original method, as employed by Koch, or some slight 
modification of it, is so satisfactory in its results that it is still generally 
employed. The above is a slight modification of the Koch-Ehrlich 
method, differing from it chiefly in the use of a weak for a strong acid 
decolorizer. It has been found that the strong acid solution originally 
employed (5 per cent, sulphuric acid solution in alcohol) often decolor- 
izes some of the bacilli entirely by its too energetic action, and that a 
weaker decolorizer, such as the above, gives more uniform results. 

The Koch-Ehrlich aniline-water solution decomposes after having 
been made for a time, so that it must be freshly prepared as needed. 
Solutions older than fourteen days should not be used. The advan- 
tages in using Ziehl's carbol-fuchsin solution are that it keeps well 
and is more convenient for use in small quantities. 

Another method, which is often of value on account of its sim- 
plicity and rapidity of performance, is that of Frankel as modified 
by Gabbett. This consists in staining the "smear" with steaming 
Ziehl's carbol-fuchsin solution for from one to two minutes, and then, 
after washing in water, placing it from one-half to one minute 
directly in a second solution which contains both the acid for decol- 
orizing and the contrast stain. This second solution consists of 

Sulphuric acid 25 c.c. 

Methylene blue in substance 2 grm. 

Water 75 c.c. 

It is then washed with water and is ready for examination. The 
tubercle bacilli will remain red as stained by the fuchsin, while all 
the other bacteria will be tinted blue. 

When the number of tubercle bacilli in sputum is very small they 
may easily escape detection. Methods have, therefore, been sug- 
gested for finding them under these circumstances. Several stains 
have been advised in this case, the simplest and most satisfactory 
being that of Herman. The advantage is that more bacilli are 
stained than with carbol-fuchsin. The stain consists of A, crystal 
violet, 3 per cent, alcoholic solution, B, ammonium carbonate, 1 per 
cent, in distilled water. Mix 1 part of A with 3 parts of B just before 
using. Steam as with carbol-fuchsin, decolorize in 10 per cent, nitric 
acid, rinse for a few seconds in alcohol, wash, and counterstain with 
Bismark brown. The tubercle bacilli are stained violet. Where 
the slides stained with carbol fuchsin are negative, this stain will 
occasionally demonstrate bacilli. | 

Biedert advises the following method: Dilute 10 c.c. of sputum with 
90 c.c. of water. Heat over the flame and add a 10 per cent, solution of 
sodium hydroxide, stirring, till the mucus is dissolved. Allow the 
coarser particles to separate. Add a few drops of phenolphthalein 
solution and neutralize with dilute acetic acid. Pour into twice its 
bulk of alcohol and allow to sediment. The coagulum that forms 
collects all the tubercle bacilli. Concentrate the sediment by centrifug- 
ing and make smears. Fix to the slide if necessary with some of the 
patient's own sputum. 



344 PATHOGEXtC MWRO-ORGAXISMS. 

Uhlenhuth advises the use of antiformin. This is a patented 
preparation consisting of a mixture of sodium hydroxide and sodium 
hypochlorite solution. If this is mixed with sputum so that the 
total strength is about lo per cent, of antiformin, the sputum quickly 
becomes fluid. This should be thinned with water or alcohol to 
help reduce the specific gravity of the mixture and centrifuged. The 
sediment is then mixed with water and recentrifuged, and the washed 
sediment used for smears. Besides the di.ssolving action, antiformin 
kills most of the bacteria in the .sputum, but not the tubercle bacilli, 
though they are slowly affected, so that .tediment may be used for 
cultural purposes or injection into guinea-pigs. 

A comparison of the above methods made by us gave the follow- 
ing results. Of twenty-eight sputa negative with carbol-fuchsin, two 
showed bacilli after a few minutes search with the crystal violet stain. 
On restaining with carbol-fuchsin and giving only a light counterstain 
with methylene blue the negative slides were also positive. Of the 
remaining twenty-six, four (15 per cent.) were quickly positive in the 
antiformin sediment when stained with crystal violet, whereas only 
three were positive with carbol-fuchsin and only after restaining as 
above. It is advisable, therefore, in using carbol-fuchsin to have 
only a light counterstain to make the method most efficient, and control 
the results with cry.stal violet if negative. 

Detection of Tubercle Bacilli in Urine and Fseces, Etc.— The cath- 
eterized urine is centrifuged. If little sediment appears, the upper 
portion of the fluid is removed and more urine added and again centri- 
fuged. If the urine is rich in salts of uric acid, the same may be di- 
minished by carefully warming the urine before treating it. If too 
alkaline add a little acetic acid. 

The feeces are examined for any purulent or mucous particles. If 
none are found, lai^er masses of fseees are removed and then the 
rest diluted and centrifugalized. The examiner must remember that 
bacilli swallowed with the sputum may appear in the fteces. In 
examining cerebrospinal fluid for tubercle bacilli it must be remembered 
that the majority of the bacilli are entangle<l in the dehcate clot that 
forms. This is also the case in other .serous fluids, but in ascitic or 
pleuritic fluid they are usually very few in number, A method has 
been devised called inoscopy to render tubercle bacilli easier of detec- 
tion in serous fluids. The fibrinous clot which forms is freed from 
id and treated with about 30 c.c. of the following diges- 

lOcc. 

leid (sp. fcr, 1 . 18) 10 c.c. 

■.".''..'.'.'.'.'.'.'.'.'.'.'-'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'. lOOOcc. 

> incubated twenty-four hours, and when digested and 

ntrifuged and smears made from the sediments, using 

X it to the .slide, 

tor Other Bacteria (Mixed Infection).— ^Vith regard 



THE BACILLUS OF TUBERCULOSIS. 345 

to the bacteriological diagnosis of pulmonary phthisis, many con- 
sider that it is not enough to show only the presence of tubercle ba- 
cilli; it is held to be of importance, both for purposes of prognosis 
and treatment, that the presence of other microorganisms which may 
be associated with the tubercle bacillus should also be determined. 
It is now usual to distinguish pure tuberculosis of the lungs from a 
mixed infection. Phthisis due to the tubercle bacillus alone, which 
constitutes but a small percentage of all cases, may occur almost 
without febrile reaction; or when fever occurs the prognosis is un- 
favorable, thus indicating that the disease is already advanced. It 
is in the uncomplicated forms of phthisis, moreover, where one must 
expect if anywhere the best results from treatment with tuberculin 
or antituberculous serum. The majority of cases, however, of pul- 
monary tuberculosis show a mixed infection, especially with varieties 
of the streptococcus and pneumococcus. These cases may be active, 
with fever, or passive, without fever, according, perhaps, as the 
parenchyma of the lung is invaded by the bacteria; or they are only 
superficially located in cavities, bronchi, etc. Mixed infection with 
the staphylococcus, other micrococci, and with the influenza bacilli 
have also been frequently met with by us. The tetragenus has not 
been often detected by us in thoroughly washed fresh sputum. At 
present the facts seem to prove that the tubercle bacilli have in the 
great majority of cases, at least shortly before death, a much more 
important r6le than the associated bacteria. 

Sputum Washing. — Some of the associated bacteria found in the 
expectoration come from the diseased areas of the lungs, while others 
are merely added to the sputa as it passes through the mouth or 
are developed after gathering. To endeavor to separate the one 
from the other we wash the sputa. The first essential is that the 
material is to be washed within a few minutes, and certainly within 
an hour after being expectorated. If a longer time is allowed to inter- 
vene, the bacteria from the mouth will penetrate into the interior 
of the mucus, and thus appear as if they came from the lungs. Spu- 
tum treated twenty-four hours after its expectoration is useless for 
examining for anything except the tubercle bacillus. A rough method 
is to pour some of the specimen of sputum to be examined into 
a convenient receptacle containing sterile water, and withdraw, by 
means of a sterilized platinum wire, one of the cheesy masses or 
thick "balls'' of mucus. Pass this loop five times through sterile 
water in a dish; repeat the operation in fresh water in a second and 
third dish. Spread what remains of the mass on cover-glasses and 
make smear preparations; stain and examine. With another mass 
inoculate ascitic bouillon in tubes and agar in plates. 

When we wish thoroughly to exclude mouth bacteria, a lump of the 
sputum raised by a natural cough is seized by the forceps and trans- 
ferred to a bottle of sterile water and thoroughly shaken; it is then 
removed to a second bottle of bouillon and again thoroughly shaken. 
From this it is passed in the same way through four other bottles of 



346 PATHOGENIC MICRO-ORGANISMS. 

bouillon. A portion of the mass is now smeared over cover-glasses, 
and the rest inoculated in suitable media, such as agar in Petri dishes, 
and ascitic fluid bouillon in tubes. If desired, the bacteria washed 
off in the different washings are allowed to develop. 

Practical Notes on tbe Examination for Mixed Infection. — I. The 
difficulties to be overcome, in order to obtain sputum consisting pre- 
sumably of exudate from the deeper portions of the lungs, are so 
great that the collection of the specimens should be supervised by the 
bacteriologist in charge of the work of examination. 

2. Specimens of sputum collected even with the greatest precaution 
may give evidence of decided mouth infection unless immediately 
washed. 

3. The sputum must be examined very soon after collection. 

4. The culture medium used for the final cultures must be suitable 
for the growth of the microorganisms, 

5. At least two successive examinations of sputum should be made 
in each case. 

6. The results, especially as to the number of colonies, vary accord- 
ing to the size and tenacity of the ball of sputum washed — e. g., a 
small bail of sputum which becomes more or less broken up upon 
thorough shaking may contain very few or no bacteria. 

Williams, in the examination of the sputum in some 40 cases, came 
to the following conclusions: 1. The presence of a large number of 
bacteria in a satisfactory and thoroughly washed specimen of sputum 
indicates that these bacteria probably play an active part in the dis- 
ease. 2. The presence of a small number of bacteria in such sputum 
does not necessarily indicate that they are not active in that case, for 
they may penetrate more or less deeply into the lung tissue, and pro- 
duce pathological changes without being thrown off in large numbers 
with the exudate. It is probable, however, that, as a rule, the smaller 
the number found the less the degree of mixed infection. 

3. Cases of clinically secondary infection frequently give pure cul- 
tures of some one organism (pneumococcus, influenza bacillus, or 
streptococcus), which are capable of causing the symptoms. 

4. In the majority of severe cases of clinically mixed infection 
many organisms have been found which usually have belonged to 
several different species or varieties. 

5. In the majority of cases of clinically non-mixed infection very 
few organisms have been found. 

6. Only bacteria which might cause pathological changes were 

of the organisms found were markedly virulent in 
ough coming from severe cases of mixed infection. 
■ for laboratory animals of bacteria obtained from the 
efore, no indication of their virulence for man he- 
ipossibility of reproducing in such animals the exact 
ceptibiMty present in human infection. 
i8 in Microscopic Examination of Spatmn. — Always 



THE BACILLUS OF TUBERCULOSIS. 347 

make two smear preparations from each specimen. Report no result 
as negative until at least two preparations have been subjected to a 
thorough search with a yV oil-immersion or 2 mm. apochromatic 
lens by means of a mechanical stage. From a very large experience in 
the examination of sputum for tubercle bacilH, the New York Health 
Department bacteriologists have concluded that the examination of 
two preparations of each specimen, in the careful manner described 
above, is usually suflScient to demonstrate the presence of the bacilli 
when they are present in the sputa, and they are usually found to be 
present to this extent in fairly well-developed cases of pulmonary 
tuberculosis, and in many cases which are in the incipient stage. 
There are, however, undoubted cases of incipient pulmonary tuber- 
culosis which require the examination of many preparations before 
the tubercle bacillus can be found ; and cases also occur in which the 
sputum for a time does not contain the bacilli, which were, neverthe- 
less, present at an earlier period, and which again appear later. 
Therefore, if cases occur w^hich may be still regarded as possibly 
tuberculosis, further examinations of the sputum should be made. 
It should also be constantly borne in mind that the demonstration of 
the presence of tubercle bacilli in the sputum prove about as con- 
clusively as anything can the existence of some degree of tubercu- 
losis; but that the absence of tubercle bacilli or the failure to find 
them microscopically does not positively exclude the existence of the 
disease. Here tuberculin can be made use of. 

Staining of Tubercle Bacilli in Tissues. — Thin sections of tuberculous tis- 
sues may be stained by the same methods recommended for cover-glass prep- 
arations, except that it is best not to employ heat to any extent. Fixation 
in bichloride of mercury is better than in alcohol. Formalin is a bad fixa- 
tive, as it makes the tissues hold the fuchsin with as much tenacity as the 
bacilli. Both paraffin and celloidin may be used for embedding; but the for- 
mer is better. 

EraiLiCH's Method. — Place the paraffin sections in aniline fuchsin apd 
leave at 37*^ C. for from six to twelve hours, or at about 80° C. for three to 
five minutes, the sections are then washed in water; then decolorize by placing 
them for about half a minute in dilute nitric acid (10 per cent.), or in 3 per 
cent, hydrochloric acid in alcohol; wash in 60 per cent, alcohol until no 
more color is given off; counterstain for two or three minutes in a saturated 
aqueous solution of methylene blue, or, better, with haematoxylin ; wash in 
water; dehydrate with absolute alcohol; clear in oil of cedar or xylol, and 
mount in xylol balsam. 

Method of Ziehl-Neelson. — Stain the section in warmed carbol-fuchsin 
solution for one hour; the temperature to be not over 45° to 50° C. Decol- 
orize for a few seconds in 5 per cent, sulphuric acid, then in 70 per cent, 
alcohol, and from this on as in the Ehrlich method. 

Inoculation of Animals. — The inoculation of suspected material 
into guinea-pigs sometimes produces tuberculosis when no bacilli 
could be detected by microscopic examination. The material should 
be injected subcutaneously as already described (p. 20). 

Cultivation. — This is so difficult and requires so much time that 
it is not used except in important investigations upon the nature of 
the tubercle bacilli. The special methods have already been given. 



CHAPTER XXIV. 

BACILLI SHOWING STAINING REACTIONS SIMILAR TO THOSE 
OF THE TUBERCLE BACILLI— LUSTG ARTE NS BACILLUS— 
SMEGMA BACILLUS— LEPROSY BACILLUS- 
GRASS BACILLI. 

LUSTQARTEN'S BACILLUS— SHEQ HA BAOILLnS. 

Bacilli were discovered by Lustgarten in syphilitic lesions or 
syphilitic ulcers (1884), and believed by him to be the specific cause 
of this disease. It has since been shown that in normal smegma 
from the prepuce or the vulva bacilli are found in great abundance, 
simitar in their morphology to the bacillus of Lustgarten, but dilTer- 
ing, as a rule, slightly in certain staining peculiarities. (See Fig. 

Motphology. — Straight or curved bacilli, which bear considerable 
resemblance to tubercle bacilli, but differ from them in staining 

" e not usually found free in the tissues, but 

ometimes in groups within the interior of 
I, or polygonal form, and apparently some- 

i of Lustgarten stains with almost as much 
bacillus, but is much less resistant to the 
ling agents, such as mineral acids, particu- 

joic Properties. — Xumerous attempts have 
e bacillus of Lustgarten on artificial media, 
but with doubtful success. The inocula- 
tion of animals has also given only negative 
re-sults. 

Lustgarten's bacillus has been found in 
various syphilitic tissues and lesions, in 
beginning sclerosis, in the papules, in con- 
dylomata anil gummatu, ami not only in 
the vicinity of the genitals, but also in the 
mouth, throat, heart, and brain. No satis- 
factory experimental evidence has Iteen 
given of its causative relation to syphili.s. 
The finding of saproph\'tic bacilli — the .so- 
called smegma bacilli — (Fig. Ill and Plate 
I., Fig. 4) almost identical morphologically 
with the bacillus of Lustgarten, under the 
ns, does not prove the identity of the two 
'nee of cultures and inoculation experiments, 
348 



BACILLI SIMILAR TO TUBERCLE BACILLI. 349 

we have not the means of establishing their relationship to one 
another. The smegma bacilli have never been identified in other 
parts of the body, except in the neighborhood of the genitals. While 
the bacillus of Lustgarten cannot resist the prolonged decolorizing 
action of acids, but is resistant to the action of alcohol, the smegma 
bacillus, when stained, is rather quickly decolorized by alcohol, but 
quite resistant to 5 per cent, sulphuric acid solution. Besides, 
Lustgarten's bacillus has been found in papules, in gummata, and 
other syphilomata, where there seems no probability whatever of the 
smegma bacillus having emigrated. 

The differential diagnosis of Lustgarten's bacillus must be made 
from the tubercle bacillus, the smegma bacillus, and the leprosy ba- 
cillus. According to Hueppe, the differential diagnosis between 
these four organisms depends upon the following reactions: When 
stained by the carbol-fuchsin method, commonly employed in stain- 
ing the tubercle bacillus, the syphilis bacillus becomes almost in- 
stantly decolorized by treatment with mineral acids, particularly 
sulphuric acid; whereas, the smegma bacillus resists such treatment 
for a much longer time, and the lepra and tubercle bacilli for a 
still longer time. On the other hand, if decolorization is practised 
with alcohol instead of acids, the smegma bacillus is the first to lose 
its color. The bacillus tuberculosis and the bacillus of leprosy are 
both verv retentive of their color, even after treatment with acids and 
alcohol. If, then, we treat the preparation, stained with carbol-fuchsin, 
with sulphuric acid, the syphilis bacillus becomes almost at once decol- 
orized. If it is not immediately decolorized, treat with alcohol; if 
it is then decolorized, it is probably the smegma bacillus. If it is still 
not decolorized, and it lies between these four bacilli only, it is either 
the leprosy or the tubercle bacillus. 

By these methods the differential diagnosis can usually be made. 
In all investigations of importance, however, animal inoculations 
should also be made, as by this means alone can a positive diagnosis 
from tuberculosis be established. Especial care should be observed 
in the examination of syphilitic ulcers of the genital region, as in 
this situation the smegma bacilli are almost always present. 

LEPR08T BA0ILLU8-B. LEPRJS. 

The bacillus of leprosy was discovered by Hansen and Neisser 
(1879) in the leprous tubercles of persons afflicted with the disease. 
This discovery was confirmed by many subsequent observers. 

Morphology. — Small, slender rods resembling the tubercle bacilli 
in form, but somewhat shorter and not so frequently curved. The 
rods have pointed ends, and in stained preparations unstained spaces, 
similar to those observed in the tubercle bacillus, are seen. They 
stain readily with the aniline colors and also by Gram's method. Al- 
though differing slightly from the tubercle bacillus in the ease with 
which they take up the ordinary aniline dyes, they behave like tubercle 



350 PATHOGENIC MICRO-ORGANISMS. 

bacilli in retaining their color when subsequently treated with strong 
solutions of the mineral acids and alcohol. The slight difference in 
staining characteristics is too little to be relied upon for diagnostic 
purposes. 

Biological Characters. — Clegg' reported in 190S that he had been 
able to cultivate an acid-fast bacillus from cases of leprosy in sym- 
biosis with amoeba? and cholera vibria. By heating a symbiotic cul- 
ture the leprosy bacillus was obtained in pure culture. From the 
first cultures different media were successfully inoculated. On nu- 
trient agar the surface colonies are small and brownish. Blood 
serum is liquefied after ten days. Lactose is not fermented. 



Leprosy bBCilli in doiIuIe. (KoLle aad WaHermHoii.) 

Pathogenesis. — Numerous inoculation experiments have been 
made on animals with portions of leprous tubercles, but there is no 
conclusive evidence that leprosy can 1)e transmitted to the lower ani- 
mals by inoculation. The inference that this bacillus bears an etio- 
logical relation to the disease with which it is associated is based chiefly 
upon the demonstration of its constant presence in leprous tissues 
(Fig. 1 12). Subcutaneous inoculations of cultures in guinea-pigs have 
produced local lesions which resemble leprous lesions in man. This 
has been repeated by Duval who states that he was able to continue 
the growth on later transfers. 

The bacilli are found in all the diseased parts, and usually in large 
numbers, especially in tubercles on the skin, in the conjunctiva and 
cornea, the mucous membranes of the mouth, gums, and larjTix, 
and in the interstitial processes of the ner\es, testicles, spleen, liver, 
and kidneys. The rods lie almost exclusively within the peculiar 
round or oval cells of the granulation tissue which composes the lep- 
rous tubercles, either irregularly scattered or arranged parallel to 
one another. In old centres of infection the leprosy cells containing 

'Tlie Philippine, Jour, of Scipnce, Vol. iv, No. (i. 



BACILLI SIMILAR TO TUBERCLE BACILLI. 351 

the bacilli are larger and often polynuclear. Giant cells, such as are 
found in tuberculosis, are claimed to have been observed by a few 
investigators (Boinet and Borrel). In the interior of the skin tuber- 
cles, the hair follicles, sebaceous and sweat-glands are often attacked, 
and bacilli have sometimes been found in these (Unna, etc.). Quite 
young eruptions often contain a few bacilli. A true caseation of the 
tubercles does not occur, but ulceration results. During acute exacer- 
bations with development of new lesions bacilli have been observed 
in the blood. 

In the anaesthetic forms of leprosy the bacilli are found most com- 
monly in the nerves and less frequently in the skin. They have been 
demonstrated in the sympathetic nervous system, in the spinal cord, 
and in the brain. The Bacillus leprce occurs also in fhe blood, partly 
free and partly within the leukocytes, especially during the febrile 
stage which precedes the breaking out of fresh tubercles (Walters 
and Doutrelepont). The bacilli have also been found in the intestines, 
in the lungs, and in the sputum, but not in the urine. 

With regard to the question of the direct inheritance of the disease 
from the mother to the unborn child there is considerable difference 
of opinion. Some cases have been reported, however, in which a 
direct transmission of the bacillus during intrauterine life seems to 
be the only or most plausible explanation of the infection. At the 
same time, we have no positive experimental evidence to prove that 
such an infection does take place. Although many attempts have 
been made to infect healthy individuals with material containing the 
bacilli of leprosy, the results are not conclusive. Even the experi- 
ments made by Arning, who successfully infected a condemned crim- 
inal in the Sandwich Islands with fresh leprous tubercles, and which 
have been regarded as positive evidence of the transmissibility of the 
disease in this way, are by no means conclusive; for, according to 
Swift, the man had other opportunities for becoming infected. These 
negative results, together with the fact that infection does not more 
frequently occur in persons exposed to the disease, may possibly be 
explained by the assumption that the bacilli contained in the tuber- 
culous tissue are mostly dead, or much more probably that an in- 
dividual susceptibility to the disease is requisite for its productions. 

The widespread idea, before the discovery of the leprosy bacillus, 
that the disease was associated with the constant eating of dried fish or 
a certain kind of food, has now been entirely abandoned. 

The relation of leprosy to tuberculosis is sufficiently evident from 
their great similarity in many respects. This is rendered still more 
remarkable by the fact that leprosy reacts, both locally and generally, 
to an injection of tuberculin in the same manner as tuberculosis, but to 
a somewhat less extent. 

Differential Diagnosis. — The differential diagnosis between lep- 
rosy and tuberculosis is not difficult in typical cases. The large num- 
bers of bacilli found in the interior of the cells would point with great 
probability to leprosy. Too much importance should not be placed 



352 PA THOGENIC MICRO-ORGASISMS. 

upon the staining peculiarities, as these are not c-onstant. Moreover, 
the two diseases not infrequently occur together in the same individual. 
In making the diagnosis, therefore, all the signs, histological and 
pathogenic, must be considered and animal inoculations made. 

TIMOTHY AND OTHEE ORASS BAOILU. 

On various grasses, in cow's manure, in butter, and in milk there 
have been discovered a number of varieties of bacteria which have 
more or less of the characteristics of the tubercle bacillus. Some of 
them are as difficult to stain and as resistant to the decolorizing action 
of mineral acids and alcohol as the tubercle bacillus found in man. 
Many of them are of the same general .size and shape as the tubercle 
bacillus, and, strangely enough, produce in animals small diseased 
areas which not only macroscopically but also microscopically re- 
semble miliary tubercles due to the tubercle bacillus. They are, 
however, entirely different in their culture characteristics, produc- 
ing in twenty-four to forty-eight hours, on ordinary culture media, 
moist, round colonies of an eighth to a quarter of an inch in diam- 
eter, and of a more or less intense pink color. In animals they pro- 
duce only localized lesions, causing death only when injected in large 
numher.s. The inoculated animals are unaffected by tuberculin in- 
hief interest which these bacilli have for us is the 
infusing them with tubercle bacilli. This danger 
t in milk, for grass bacilli find so many means of 
to it. In the examination of dust, healthy throat 
>ns, etc., the simple microscopic examination might 

separated from tul)ercle bacilli by inoculating ani- 

progressive lesions will develop. If there is any 
nature of the infection, inject 2 c.c. of a standardized 

if infected with tuberculosis they will die, but if by 
will show little or no reaction. If a second group 
re inoculated with a small amount of the infected 

1 develop progressive tuberculosis, if the doubtful 
:rcle bacilli, and practically no lesions if they were 
ultures from the lesions will also show, on ordinary 
inies if grass bacilli are present, and no growth if 



CHAPTER XXV. 



THE INrLTTENZA BAOILLVS. 



Infli'enza as a distinct entity can be traced back to the fifteeiHb 
century and probably existed at a much earlier date. . ■ ,; 

At times but few endemic cases occur, and then a great, ^idemic 
spreads over the civilized world. The last great epidemic reftohed 
Russia from the East in the fall of 1889 and gradually ispj'ead.ov^i 
Europe and to America, reaching the latter country ini De^i^inlMf pf 
that year. Since then we have had more or less of i^icgpeciatl^idur* 
ing the winter months. Many acute inf1ammationS|C(f;the,'^f»pi;t*^rtt 
mucous membranes, due to pncumocooci and streptococoi ^ , give 
symptoms similar to those due to the influenza bafiJlusK. ! : i ;!ii i.. 

The rapidity of the spread 
of epidemics of influenza sug- 
gested that persons were tlie 
carriers of the infection, while 
the location of the disease 
pointed to the respiratory tract 
as the location of, and to the 
expectoration as the chief source i 
of infection by, the microorgan-: | 
isms. ,i!,l 

After numerous un.successful'; 
attempts, during the epidemic of'r 
1889 and succeeding years, toii 
discover the specific ,t}au»t' /oji 
influenza, Pfeiffer (lSS'2)i. a«q-l 
ceeded in isolating aild i jcmwi^it^ 
upon blood agar a bhcjl|it$u(tiicbi: 
abounded in the pwruk-nt.hf'on-, . 

chial secretion (>f,pKtientSjmUIerIng from epidianjfc.iitfliienaa, whit^tlliij 
showed was the probable cause of t>^;di^at«;i! - ; '■. r imi;! 

Morpholflgy.'TVefiy;:flw||),ntn«dfim«l3c:;tiiJElt(6«)ffli'liO'.3l:tO!i^^ 
in thickn^s, to Q.^^tp' 3/1 .in length), uaually.«ccurnngisiogly or united 
in pairs,. , but ^hr^dlj opi. chains nl. thtee, if««r,,<>r more eWui«ntM<Ard 
occasjonallj' f()Mnd.'; .NoicdpaftkihaabwiidipmopSlraleilj - ,■ i: ■.■ nl 

Staininff.tr-.Tht^. baoillus »(a*M3i!t*-ithrdiffkuUyi with »hei ordioarji 

aniline,. t*Soii!Sr-fl»est, with : ;di hit d ZSdll's.iaolutioii <\*aittT ft.paittsto 

i^hl's ^(utitfp: l.jjaWlt .OE^Loefflrf's.imiHhj^eft^tblueitiolutton./fritih 

heat,, Wiiea' riiin%i!ttHihttsl/theitWoitfid«(i«f't[leib)teilli aire si}n)etjii)(>» 

73 3.53 



354 PATHOGENIC MICRO-ORGANISMS. 

more deeply stained than the middle portion. They are not stained 
by Gram's method. 

Biology. — An aerobic, non-motile bacillus; does not form spores; 
no growth occurs with most cultures below 26° C, or above 41° C, 
or in the entire absence of oxygen. 

Gnltivation. — ^This bacillus is best cultivated at 37° C, and on the 
surface of ordinary nutrient culture media containing haemoglobin. 
Plain or glycerin agar, or blood serum thinly streaked with rabbit, 
guinea-pig, or human blood, make the best media for its growth. At 
the end of eighteen hours in the incubator very small circular colonies 
are developed, which, under a low magnification (100 diameters), 
appear as shining, transparent, homogeneous masses, and even under 
a No. 7 lens scarcely show at all the individual organisms. Older 
colonies are sometimes colored yellowish-brown in the centre. A 
characteristic feature of the influenza bacillus is that the colonies tend 
to remain separate from each other, although when they are thickly 
sown in a film of moist