Google
This is a digital copy of a book that was preserved for generations on library shelves before it was carefully scanned by Google as part of a project
to make the world's books discoverable online.
It has survived long enough for the copyright to expire and the book to enter the public domain. A public domain book is one that was never subject
to copyright or whose legal copyright term has expired. Whether a book is in the public domain may vary country to country. Public domain books
are our gateways to the past, representing a wealth of history, culture and knowledge that's often difficult to discover.
Marks, notations and other maiginalia present in the original volume will appear in this file - a reminder of this book's long journey from the
publisher to a library and finally to you.
Usage guidelines
Google is proud to partner with libraries to digitize public domain materials and make them widely accessible. Public domain books belong to the
public and we are merely their custodians. Nevertheless, this work is expensive, so in order to keep providing tliis resource, we liave taken steps to
prevent abuse by commercial parties, including placing technical restrictions on automated querying.
We also ask that you:
+ Make non-commercial use of the files We designed Google Book Search for use by individuals, and we request that you use these files for
personal, non-commercial purposes.
+ Refrain fivm automated querying Do not send automated queries of any sort to Google's system: If you are conducting research on machine
translation, optical character recognition or other areas where access to a large amount of text is helpful, please contact us. We encourage the
use of public domain materials for these purposes and may be able to help.
+ Maintain attributionTht GoogXt "watermark" you see on each file is essential for in forming people about this project and helping them find
additional materials through Google Book Search. Please do not remove it.
+ Keep it legal Whatever your use, remember that you are responsible for ensuring that what you are doing is legal. Do not assume that just
because we believe a book is in the public domain for users in the United States, that the work is also in the public domain for users in other
countries. Whether a book is still in copyright varies from country to country, and we can't offer guidance on whether any specific use of
any specific book is allowed. Please do not assume that a book's appearance in Google Book Search means it can be used in any manner
anywhere in the world. Copyright infringement liabili^ can be quite severe.
About Google Book Search
Google's mission is to organize the world's information and to make it universally accessible and useful. Google Book Search helps readers
discover the world's books while helping authors and publishers reach new audiences. You can search through the full text of this book on the web
at|http: //books .google .com/I
■I
\
I m
•J
I-
r
1
%
I
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,
0 . 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. dteidtd, 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 0 . 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 0 . 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 0 . 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,
0 . 1 c.c. of a serum from a horse frequently infected by the pneumococcus
will prevent the development in the body of a rabbit of many thousand
times the fatal dose of very virulent pneumococci, and a few times a
fatal dose of less virulent ones, the actual number as well as the virulence
of the bacteria affecting the protective value of the serum.
These protective substances are found also in other fluids of the
body than in the blood; they occur, indeed, in the substance of many
cells to a greater or less extent.
The immunity produced by these five methods affects the entire
body, as is natural, since the blood 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
0
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 0 .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
0
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 0 '* n 0 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
™. ...
■vDllMI 1
-1"
4
• •!■
« 1 » 1 IS 1 11 [ n
"
-_
H ^ ;
^P^/Mi
^-\=
LLv
.8
/yvt
^
^
-b.'p^yjW"
^^^
-=«2^^V^V
^^r^^\ \
-
1 1 1 1 1 1
1 1 1
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 0 . 6/£ to 0 . 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. The toxin is produced 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^ used for the inoculations. 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 with this twice the amount of anji-
toxin required to neutralize it. In'TivejIays this dose is don hied ^ and
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
contains the anjitoxin in suMcient__amount for thfixapeutic 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.
§5
at
I 'I
ill
si
its
i X-.- Hit .«is
I isisflrl 111
Bsn mn Kmrnnxmnn
llJifi illJi lilJili il°i
r-Ss-^1,5 ^^^11^5 l"t»^i^^ fd"
rf^slH llliJ:si ISgp-II II «
!lcJs^2 :5|,i£K'-3:» 'J'liisllg. ^'S'S
■[|]png pioqdXj-oo|00 jo dnojQ
THE COLON TYPHOID BACILLUS GROUP.
257
o
O
I
a
o
*o
OB
«
U
s.
CO
d
«
09
d
o
«3
•opog
5
a S
II
a
o
iCsfranfon
<n«»oj
p9j iBJinofif
IIIK
paanpoid 8v9 ^onomy
o^imrep^
osoivqaovg
e
au^xaci
»«ni«w
aso^arri
asai^XQQ
aoe^anpoxd lopaj
wi|a»B|j
^^nnoK
a
.5
a
•29
9
11
«
3
n
n
+++++++
++++++
++++++
+++'•■+'"'' ^++^
+ -H-H
I I xxxx
-H
-H^^ + + + -H-H+ I
+ + ++++++++
-H + + + I I I I I I i
+ + ++++++++X
+ + ++++++++
+ + + + XX+ + + + I I I I I I I
++++XX+ ++++++++++X
-H-H-H +
.1111
MIX
I I I I
XXXX
-H I
XXXX
X I X
XXXX
+ + I + I + I I +-H
I I I I I
+ +
<:..
<++"
+
+
+ -H I
+
+
1 1 +^ ++++;[| I +
M o ^
an
3
♦»
es
3
tt
a
<CQ
o
lis I
111 +
si
-^^^
X cc
S a o -
(u n n 3
• • • •
J4
1
= 3
O
5
a
1
1
0>
if
Is
d
o
9
M
O
o
a
eS
3i
-d-S S5
•S'S |5 5 s
+ X-H + -J
+ + «
'7
PATHOGENIC MICRO-ORGA.VISMS.
1° E
lifN .£2.2 °.l
s ill||l|| III
flllill Hi^Sfl QllJlllg. Illi
'!]||9«g ptoi(dX}-uo]03 JO dnojo
THE COLON TYPHOID BACILLUS GROUP.
257
o
O
I
a
OB
«
U
s.
CO
d
CO
d
•«•
o
a
<t3
•opog
5
a
art I
I
I !
X^sonion
<n«»oj
pai i«j^n9f«{
3nm
poanpoad 8v9 ^onoary
9)iinrep[
osoivqoov^
C
iiu)xaci
a«n|«K
aso^dTTi
d90J)X9Q
iioc)3npaid {opu]
«II»»»W
^^nnow
a
.5
S
o
+++++++
++++++
IS
5i
«
li
3
+ + + + -^1 +
+ -H-H
X+ + X I I XXXX I
+
+
-H
+ -h;[;^+++-h-h+ 1 1
+ + ++++++++
I
-H-H-H +
J I I I
MIX
I I I I
XXXX
-H + + + I I I I I I I I I I -H I
+ + ++++++++X
XXXX
+ + ++++++++ I XIX
+ + + + XX+ + + + II II II I I I III
++++XX+ ++++++++++X
XXXX
+ + I + I + II +-H
I I I I I
+ + I
<:..
<t+"
+
+
+ -H 1 I I
1 1 +;J; ++++^ I + 111 +
c
3
■*>
as
3
a
M g
^^ ^ ■ ^^ p*^ ^^ p* tJ "^
a a a d c a'^S S -.s'.? » -
cafe
98<
n
CCCC
edpQcdpd
5
a
1 %
o
= 3
O
1
73
Is
s
§
I
£
s
I
o
o
So ^ -i
•So ^mo a
+ X-H + -S
++«
17
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
0 . 4 per cent, of mineral acids, in from 0 . 3 to 0 . 45 per cent, of vetegable
acids, and in from 0.1 to 0 . 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
0 \ 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 0 0
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 rapid,
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
typhoid_bacilhis does not cause coagulation .
o. lEecoloii' bacillus is conspicuous for its power of causing fer-
mentation, with the production of gas in media containing glucose.
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 solutioiis.
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 0 . 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 0 . 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 0 . 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-
0 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 blood upon nutrient agar they may become con-
fluent. Transplantation of the original culture to ordinary agar or
serum cannot, as a rule, be successfully performed, owing to the want
of suflScient haemoglobin; but if sterile rabbit, pigeon, or human
blood be added to these media, transplantation may be indefinitely
performed, provided it is done every three or four days. Cultures
may remain alive up to seventeen days. By a series of beautifully
carried out experiments, Pfeiffer showed that not only were the red
blood cells the necessary part of the blood needed for the growth of the
influenza bacillus, but that it was the haemoglobin in the cells.
In bouillon in thin layers, to which blood is added, a good develop-
ment takes place if there is free excess of oxygen.
Resistaiice and Length of Life. — The influenza bacillus is very sensi-
tive to desiccation; a pure culture diluted with water and dried is
destroyed with certainty in twenty-four hours; in dried sputum the
vitality, according to the completeness of drying, is retained from
twelve to forty-eight hours. It does not grow, and soon dies in water.
In blood-bouillon cultures at 20° C. they retain their vitality for from
a few days to two or three weeks. In moist sputum it is difficult to
determine the duration of their life, since the other bacteria overgrow
and make it impossible to find them. It is probable that they can
remain alive for at least two weeks. The bacilli are very readily
killed by chemicals, disinfectants, and succumb to boiling within one
minute and to 60° C. within five minutes.
Detection of the Influenza Bacillus in Sputum.— The direct micro-
scopic examination of stained smears of sputum may give considerable
information as to the probable presence of influenza-like bacilli.
In patients suffering from influenza the bacilli are found chiefly in the
nasal and bronchial secretions. In acute uncomplicated cases they
may be observed microscopically in large masses, and often in abso-
lutely pure culture; the green, purulent sputum derived from the bron-
chial tubes is especially suitable for examination. The older the
INFLUENZA AND PSEUDOINFLUENZA BACILLI. 355
process is, the fewer free bacilli will be found and the more frequently
will they be seen lying within the pus cells, instead of being embedded
free in the secretion as at first. At the same time they stain less
readily and present more irregular and swollen forms. The frequent
presence of other infiuenza-like baciUi in the throat secretions leads to
so much doubt that it is advisable in important cases from the start to
make use of plate cultures, the best medium being nutrient agar
freshly smeared with a film of rabbit's blood.
Effect on Animals. — The bacillus of influenza, in so far as experi-
ments show, produces a disease at all similar to influenza only in
monkeys and to a less extent in rabbits. When a small quantity of
culture on blood agar, twenty-four hours old, suspended in 1 c.c. of
bouillon, was injected intravenously into rabbits, Pfeiffer found a char-
acteristic pathogenic effect was produced. The first symptoms were
developed in one and a half to two hours after the injection. The
animals became extremely feeble, lying flat upon the floor, with their
limbs extended, and suffered from extreme dyspnoea. The tempera-
ture rose to 41° C. or above. At the end of five or six hours they
were able to sit up on their haunches again, and in twenty-four hours
had recovered. Larger doses caused the death of the animals inocu-
lated. These results are attributed by Pfeiffer to toxic products
present in the cultures, and in none of his experiments was he ever
able to obtain effects resembling septicemic infection. Cultures killed
by moderate heat give much the same results. In some of the experi-
ments on monkeys, these animals, when cultures were rubbed into
the nasal mucous membrane, showed a febrile condition, lasting for a
few days; but in no instance has Pfeiffer observed a multiplication of
the bacilli introduced.
The cell bodied of the bacilli seem to possess considerable pyogenic
action.
Immnnity. — ^Possibly an immunity for a short period against the
influenza poison may be established after an attack. At least in three
experiments made by Pfeiffer on monkeys, these animals, after re-
covering from an inoculation with bacilli, seemed to be much less
susceptible to a second injection.
Pathogenesis for Man.— The invasion of the body by the influ-
enza bacillus is chiefly confined to the respiratory passages. Very fre-
quently the influenza process invades portions of the lung tissue. In
severe cases a form of pneumonia is the result, which is lobular and
purulent in character and accompanied by symptoms which may be
somewhat characteristic for influenza, or, again, almost identical
with bronchopneumonia due to the pneumococcus. The walls of the
bronchioles and alveolar septa become densely infiltrated with leuko-
cytes, and the spaces of the bronchial tubes and alveoli become filled.
The influenza bacilli are found crowded in between the epithelial
and pus cells and also penetrate the latter. There may be partial
softening of the tissues or even abscess formation. Bacilli are found
in fatal cases to have penetrated from the bronchial tubes not only
356 PATHOGENIC MICRO-ORGANISMS.
into the peribronchitic tissue, but even to the surface of the pleura,
and rarely they have been obtained in pure cultures in the pleuritic
exudation. The pleurisy which follows influenza, however, is
usually a secondary infection, due to the streptococcus or pneumo-
coccus.
Presence in Blood. — Bacilli that resemble influenza bacilli so
closely as to make their separation difficult or almost impossible
are found at times in the blood during the early days of an acute
infection; and sometimes in bad cases in young children a septi-
caemia develops before death. Whether the typical influenza bacillus
is found in the blood as supposed by Canon is still a matter of contro-
versy. It is found at times in otitis media accompanying influenza,
and has been found in the meninges in cases of meningitis. So far as
positive results have shown, influenza would seem to be almost always
a local infection confined chiefly to the air passages. The general, cere-
bral, gastric, and other symptoms produced are due to the absorption
of the toxic products of the specific organism, these poisons being
particularly active in their effects on the central nervous system.
Presence of Influenza Bacilli in Ghronic Influenza and in Tuber-
culosis.— Ordinarily influenza runs an acute or subacute course, and
not infrequently it is accompanied by mixed infections with the
pneumococcus and streptococcus. Pfeiffer was the first to draw
attention to certain chronic conditions depending ui>on the influenza
bacillus. Bacilli may be retained in the lung tissue for months at
a time, remaining latent a while, and then becoming active again,
with a resulting exacerbation of the disease. Consumptives are
liable to carry influenza bacilli for years and are particularly sus-
ceptible to attacks of influenza. Williams, in the examination of
sputa in cases of pulmonary tuberculosis, has found abundant in-
fluenza bacilli to be present in a large proportion of the samples of
sputum from consumptives, and this not only in winter but also in the
summer, when no influenza was known to be present in New York.
Taken together with results elsewhere, this indicates that at all times
of the year many consumptives carry about with them influenza ba-
cilli, and that very likely many healthy persons as well as persons
suffering from bronchitis also harbor a few. Given proper climatic
conditions, we have at all times the seed to start an epidemic.
Epidemiology. — The discovery of this bacillus enables us to explain
many things, previously unaccountable, in the cause of epidemic in-
fluenza. W^e now know, from the inability of the influenza bacillus
to exist for long periods in dust, that the disease is not transmissible to
great distances through the air. We also know that the infective ma-
terial is contained only in the catarrhal secretions. Sporadic cases or
the sudden eruption of epidemics in any localities from which the
disease has been absent for a long time, or where there has been no
new importation of infection, may possibly be explained by assuming
fh^^ ,th^ .b^c^lli^, as, already mentioned, often remain latent in the
l^i^gsjpfi brpinflbj^) js^r^tiops? qf^tte ibqdyjfQrnpi^nyiniontbs, .and per-
INFLUENZA AND PSEUDOINFLUENZA BACILLI. 357
haps years, and then become active again, when under favorable cir-
cumstances they may be communicated to others.
Bacteriological Diagnosis. — This is of importance for the identi-
fication of clinically doubtful cases, which, from their symptoms,
may be mistaken for bronchitis, pneumonia, or tuberculosis. Up to
the present time the diagnosis gives us little help in treatment.
In acute uncomplicated cases the probable diagnosis can be fre-
quently made by microscopic examinations of stained preparations of
the sputum. In chronic cases or those of mixed infection few or many
bacilli may be found and the culture method may be necessary to give
even a probable diagnosis. The bacillus of influenza is not readily
separated by its morphological, staining, and cultural peculiarities
from other bacteria belonging to the influenza group, and at present
it is almost impossible to identify it certainly.
The Pseudoinfluenza Bacillus.— This bacillus is culturally very
similar to the typical influenza bacillus, but may be distinguished
from it by its larger size and tendency to grow out into long threads.
It is not certain but that it is a form of the influenza bacillus.
Other Bacilli Resembling the Influenza Bacillus.— There are a
number of bacilli which differ slightly in morphology and growth in
culture from the characteristics of the typical influenza bacillus. One
of these strains is regulary found in whooping-cough. It produces,
when injected in animals, agglutinins which are specific for them,
but not for influenza bacilli. Both bacilli have in common group
agglutinins (Wollstein). The blood of those suffering from whooping-
cough usually agglutinates the whooping-cough bacilli, but not the in-
fluenza bacilli. Further investigation is required to establish their sig-
nificance in the disease (see, too, p. 484 for the bacillus of Bordet and
Gengou). The Koch- Weeks bacillus is also very .similar to the in-
fluenza bacillus.
Relation of the Olinical Sjnnptoms to the Bacterial Excitant.—
There is no doubt that other infections are also included under the
clinical forms of influenza, and during an epidemic of bronchopneu-
monias, irregular types of lobar pneumonias, and cases of bronchitis
frequently have symptoms so closely alike that the nature of the bac-
teria active in the case is very frequently different from that supposed
by the clinician. Thus in four consecutive autopsies examined by the
writer the influenza bacillus was found almost in pure culture in one
case believed, from the symptoms, to be due to the pneumococcus, and
entirely absent in two of the three believed to be due to it. Except for
these examinations the clinician would be of the opinion that he had
clearly diagnosed bacteriologically the cases, while in fact he had been
wrong in three of the four.
The striking symptoms in acute respiratory diseases are frequently
more due to the location of the lesions than to the special variety of
organisms producing them. In epidemics of influenza there are,
of course, many cases which, on account of their characteristic symp-
toms, can be fairly certainlv attributed to the influenza bacilli. Even
358 PATHOGENIC MICRO-ORGANISMS.
under these circumstances error may be made, as, for instance, two
cases of apparently typical influenza were re|>orted in a household
and both showed a total absence of influenza bacilli. The pneumo-
coccus was present in almost pure culture.
Examination of Sputum for Influenza Bacilli. — 1. Sputum coughed from
the deeper air passages and not from throat scraping should be used.
2. The sputum should be received in a sterile bottle, which should then
be placed immediately in cracked ice.
3. Blood-agar plates should be made by dropping a drop of fresh rabbit's
blood, obtained aseptically, on the centre of a hardened agar plate.
4. One of the more solid masses of the sputum should be tak6n from the
bottle with sterile forceps and placed on a plain agar plate. A small portion of
this mass should be separated with a sterile platinum needle and drawn through
the blood on the blood-agar plate from the centre out in different directions.
The larger part of what is left of this small portion is then placed in a similar
manner over a second blood agar^ and from this to a third, sterilizing the needle
between the transfers. The plate should be placed in the thermostat for
twenty-four hours.
5. After the plates are planted two smears should be made, one stained
by Gram and the other by weak carbol-fuchsin.
6. After twenty-four hours the plates are examined under low power.
The influenza colonies use up the hffimoglobin, and in parts of the blood-
agar plate where the blood is of right thickness such colonies show as almost
clear areas surrounded by the red blood. With a higher power (No. 6 or 7
objective), if such areas seem to be made up of fine indefinite granulations,
they are practically sure to be influenza colonies. Most influenza colonies are
more highly refractive than other light colonies, and they show this charac-
teristic best when they grow on the edge of a blood mass. Many influenza
colonies also show heapings in the centre. Influenza colonies growing away
from the blood cells are less characteristic in appearance and less easily differ-
entiated from other similar bacteria.
7. Fishings from the influenza-like colonies should be planted on blood-
agar tubes, and if, after twenty-four hours in the thermostat, the resulting
growth should consist of influenza-like organisms, plantings should be made
on plain agar. The first generation on plain a^ar may show slight growth
because of the blood carried over from the original tube, but the second
generation should show no growth if the organism is the influenza bacillus.
8. The agglutination characteristics of the cultures should be tested in the
serum from a rabbit injected with a single typical culture, and in the serum
from one injected with a number of cultures. The agglutination tests should
be carried out in order to gain information. The cultures tested in the Re-
search Laboratory have shown considerable variation.
For Testing the Agglutination of Influenza Bacilli in the Hanging
Drop. — Grow the cultures on slanted agar tubes to which, after cool-
ing to 40° C, \ c.c. of defibrinated blood has been added. When
twenty to twenty-four hours old, make a suspension of the bacilli in
normal salt solution, controlling the number of bacilli by examining
a hanging-drop preparation. The influenza bacilli seem to aggluti-
nate rather slowly, so it usually takes four to five hours to get a good
reaction.
Serum Therapeutics. — No protective serum has been produced
which has any value in treatment.
Vaccines. — These have not been proven to be of value.
IXFLUEXZA A.\D PSEUDOLVFLUEXZA BACILLI. 359
THE K00H-WEBK8 BA0ILLU8 OF OOWUHOTITITIS.
This bacillus was first observed by R. Koch in 1883 while making
certain investigations into inflammation of the eye occurring during
an epidemif of cholera in Alexandria. It was later, in 1887, more
specifically described by Weeks' in New York. Weeks obtained it in
pure culture in 1890.
The infective disease which is caused by this bacillus seems to be
very widely distributed, no land or clime probably being exempt
from it. In this country it occurs epidemically and with increasing
frequency during the spring and fait months. Weeks has found the
bacillus in over 1000 cases. This disease is known as pink eye,
Moiphology. — The bacilli from the purulent secretions are small
and slender, being not unlike the influenza bacilli but somewhat
longer. The shorter bacilli not infrequently have the appearance of
diplococci. Sometimes they exhibit slight polar staining. Their
width is very constant. The ends are rounded. They are rapidly
decolorized by Gram.
Staining. — They are best stained by very dilute solutions of car-
bol-fuchsin or Loeffler's methylene blue, but do not stain readily.
In smear preparations the Koch-Weeks bacilli are, as a rule, seen
alone or associated with isolated cocci and bacilli, especially xerosis
bacilli. They are not infrequently observed within the cells, and
are very rarely associated with gopococci and pneumococci, such
mixed infections being extremely uncommon.
Biological Oliaractors.— The Koch-Weeks bacillus grows only at
temperatures near to 37** C. Of the ordinary culture media none but
moist and slightly alkaline peptone agar can be employed. The best
results have been obtained with serum agar or a mixture of glycerin
agar and ascitic fluid, 2 to 1. Pure cultures are rarely obtained at first;
' Weeks. N. V. .Meii. Rpc., ISS7, xxxi,. page a'l.
360 PATHOGENIC MICRO-ORGANISMS.
they are usually associated with colonies of xerosis bacilli or staphy-
lococci. After twenty-four to forty-eight hours the colonies are
noticeable as moist, transparent, shining drops or points. Micro-
scopically examined under low magnifying power they appear like
small gas bubbles; by closer examination they are seen t6 be round,
lying loosely on the surface, and are readily removed. Under higher
magnification a number of fine points are observable. The colonies,
which resemble those of influenza, have a tendency to confluesce, but
are not so sharply defined as the latter and become more quickly in-
distinguishable. Isolated colonies, especially those in the neighbor-
hood or xerosis bacilli or staphylococci, grow larger and their con-
tour is slightly wavy; they are more opaque and granular than in-
fluenza colonies. In serum or blood bouillon a slight cloudiness is
produced which finally settles down.
Resistance. — In culture media the bacilli die rapidly, seldom liv-
ing more than five days. They resist a temperature of 50° for ten
minutes, but cannot withstand 60° for more than one or two minutes.
They cannot resist drying for any length of time.
Transmission. — This occurs only by contact either by direct or in-
direct conveyance of the moist infective material. Infection is not
communicated through the air by means of dust, as the bacilli soon
die when dried. It may, however, be conveyed by flies, etc.
Pathogenesis. — The Koch-Weeks bacillus is not pathogenic for
animals. Man, on the contrary, is extremely susceptible to infection
from this bactllus, which produces one of the most contagious diseases
known.
Immunity. — Immunity is not produced to any extent by one at-
tack, but there does seem to be an individual susceptibility to the
disease.
Differential Diagnosis. — The only microorganisms from which
the Koch-Weeks bacillus would seem to require differentiation are
those of the influenza group. These latter bacilli, however, grow well
only on hsemoglobin media, which the Koch- Weeks bacillus does not
require. The colonies on serum agar are smaller than those of the
influenza bacilli and the edges more granular. While the influenza
bacillus is slightly pathogenic for certain animals, the Koch-Weeks
bacillus has so far given negative results with all animals.
CHAPTER XXVI.
THE PYOGENIC COCCI.
THE 8TAPHYL00000I.
Staphylococci were first obtained from pus by Pasteur in 1880.
In 1881 Ogston showed that they frequently occurred in abscesses,
and in 1884 Rosenbach fully demonstrated their etiological impor-
tance in circumscribed abscesses, osteomyelitis, etc. Of the staphy-
lococci those producing yellow and white pigments are by far the most
important since they are the pathogenic varieties.
The Staphylococcus Pyogenes Aureus.— The Staphylococcus au-
reus is one of the commonest pathogenic bacteria, being usually present
in the skin and mucous membranes, and is the organism most fre-
quently concerned in the production of acute, circumscribed, sup-
purative inflammations.
Morphology. — Small, spherical cells, having a diameter of 0.7/i
to 0.9ju, occurring solitary, in pairs as diplococci, in short rows of
three or four elements, or in groups of four, but most commonly in
irregular masses, simulating clusters of grapes; hence the name
staphylococcus, (See Fig. 116.)
Staining. — It staitis quickly in aqueous solutions of the basic ani^
line colors and with many other dyes. When previously stained
with aniline gentian violet it is not decolorized by Gram's method.
When slightly stained each sphere frequently is seen to be already
dividing into two semispherical bodies.
Biology. — The Staphylococcus pyogenes aureus is an aerobic, jac-
ultative anaerobic micrococcus, growing at a temperature from 8° to
43° C, but best at 25° to 35° C. The staphylococci grow readily
on all the common laboratory media, such as milk, bouillon, nutrient
gelatin, or agar. A slightly alkaline reaction to litmus is best for
the growth of the staphylococci, but they also grow in slightly acid
media.
Cultivation. — Growth in Nutrient Bouillon.— The growth of the
staphylococcus is rapid, reaching about 50,000,000 per c.c. at the end
of twenty-four hours at 30° C. The bouillon is cloudy and frequently
has a thin pellicle. Later a slimy sediment forms. The odor is dis-
agreeable. In peptone-water, growth occurs with indol production.
Growth on Gelatin. — Grown on gelatin plates it develops, at room-
temperature, within forty-eight hours, punctiform colonies, which when
examined under a low-power lens, appear as circular disks of a pale-
brown color, somewhat darker in the centre, and surrounded by
a smooth border. The colonies grow rapidly. The appearance of the
361
362 PA THOGESIC M/CRO-ORGA.MSMS.
growth is most characteristic. Immediately surrounding the col-
onies, which are of a pale yellow color, there is a deepening of the sur-
face of the gelatin, due to its liquefaction. By suitable light a num-
ber of these shallow depressions with sharply defined outlines may be
seen on the gelatin plate, having a diameter of from 5 to 10 mm,, in
the centres of which lie the yellow colonies. Later the liquefaction
becomes general, the colonies running together. In stab cultures in
gelatin a white confluent growth at first
^'°- "* appears along the line of puncture, fol-
lowed by liquefaction of the medium,
which rapidly extends to the sides of
the test-tube. At the end of two days
the yellow pigmentation begins to form,
and this increases in intensity for eight
days. Finally, the gelatin is completely
liquefied, and the staphylococci form a
golden-yellow or orange-colored deposit
at the bottom of the tube. Under un-
favorable conditions the staphylococcus
suphykwocciu. X iiuu dLwneieH. aureus gradually loses it ability to make
pigment and to liquefy gelatin.
Qrowtit on Agar, — In streak and stab cultures on agar a whitish
growth is at first produced, and this at the end of a few days becomes
a faint to a rich golden-yellow on the surface. The yellow pigmenta-
tion is produced only in the presence of oxygen ; colonies found at the
bottom of a stab culture or under a layer of oil remain white.
Hilk. — Milk is coagulated at the end of from one to eight days.
Potato. — The staphylococci grow readily on potato and produce
abundant pigment.
Growth on Loefflor's Solidified Blood Serom.^Growth vigorous, with
fairly good pigment production. Some varieties slowly liquefy the
Growth on Blood Agar. — If nutrient agar to which a little animal
blood has been added is streaked with staphylococci there appears, at
the end of twenty-four hours at 35° C, about the growth a clear zone,
owing to the hemolytic effect of the staphylococcus products.
Acids Prodnced.^In certain culture media, as a result of the growth
of the staphylococcus aureus, there is a production of acid in consider-
able quantities, these consisting chiefly of lactic, butyric, and valerianic
acids. These acids have been supposed to play a part in the production
'' ' '--'\, according to some observers, they are often present.
The staphylococcus is distinguished from most other
ing pathogenic bacteria by its greater power of resistance
lences, desiccation, etc., as well as to chemical disin-
ures of the staphylococcus pyogenes in gelatin or agar
ility for a year or more. Suspended in water its ther-
nt varies with different cultures and averages about
0° C, one-half hour at 60° C, ten minutes at 70" C,
THE PYOGENIC COCCI. 363
and five minutes at 80° C. Upon silk threads and in media rich
in organic matter its resistance is greater, but subjected to 80° C.
for thirty minutes or boiling for two minutes it is almost surely killed.
Cold has but little effect. Thirty per cent, of the organisms remained
alive after being subjected by us to freezing in liquid air for thirty
minutes. These are average figures. Some cultures are more
resistant than others.
They are quite resistant to direct sunlight and drying. Dried
pus contains living staphylococci for weeks and even months, and
they can be found alive in the fine dust of the air in living and in oper-
ating-rooms.
To most disinfectants the staphylococci are rather resistant. The
presence with staphylococci of organic substances, especially albumin,
increases their resistance. In watery solution dissolved mercuric
chloride, 1 : 1000, destroys the organisms in five to fifteen minutes, but
when in pus not for several hours. Hydrogen peroxide in 1 per cent,
solution kills in about one-half hour.
Products of Growth. — Besides the lipochrome and gelatin liquefying
enzyme, there are produced other enzymes. The specific haemolysin,
known as staphylolysin is destroyed by heating for twenty minutes at
56° C. An antibody for this is formed by inoculating animals with
culture filtrates. A substance called leukocidin is produced which
injures leukocytes. It also produces an antibody.
Toxic Substances. — Filtrates of cultures contain toxic substances.
Injected into the peritoneal cavity they excite peritonitis. Under
the skin they produce infiltration or abscess formation. In the blood
they injure both the red and white corpuscles.
Cultures of the staphylococcus, when sterilized by boiling and in-
jected subcutaneously, produce marked positive chemotaxis and often
local abscesses. Leber found also that sterilized cultures introduced
into the anterior chamber of the rabbit's eye would bring about a
fibro-purulent inflammation, the cornea becoming infiltrated, and
perforation alongside of the sclerotic ring finally taking place. This
was followed by the formation of pus in the anterior chamber and
recovery. These local changes follow the inoculation of small quan-
tities only of the dead cultures; but when large amounts are injected
into a vein or into the abdominal cavity, toxic effects are produced.
The hsemolytic effects of certain products of virulent staphylococci
have recently been studied. In cultures they can be detected about
the third or fourth day of incubation and reach their maximum on
the ninth to fourteenth day. Virulent staphylococci are more apt to
produce this substance than the non-virulent, but there is no definite
rule.
Pathogenesis. — The pathogenic effect of the Staphylococcus pyo^
genes aureus on test animals varies considerably, according to the
mode of application and the virulence of the special culture employed.
In man a simple rubbing of the surface of the unbroken skin with
pus from an acute abscess is, as a rule, sufficient to produce a purulent
364 PATHOGENIC MICRO-ORGAMSMS.
inBanimation, and the introduction of a few germs from a septic case
into a wound may lead to a fatal pyiemia. These conditions can only
be reproduced in lower animals with difficulty, and by the inoculation of
large quantities of the culture. Small subcutaneous injections, or the
inoculation of open wounds in mice, guinea-pigs, and rabbits, are
commonly without result; occasionally abscess formation may follow
at the point of inoculation, which usually ends in recovery. The pus-
producing property of the organism is exhibited in proportion to the
virulence of the culture employed. Slightly virulent cultures, which
constitute the majority of those obtained from pus taken from the
human subject, when injected subcutaneously in large quantities
(several c.c. of a fresh bouillon culture) in rabbits or guinea-pigs, give
rise to local pathological lesions — acute abscesses. \\'hen virulent
cultures are used — which are rarely obtainable — 0.5 c.c, of a fresh
bouillon culture is sufficient to produce similar results. The abscesses
generally heal without treatment; sometimes the animals die from
marasmus in consequence of the suppurative process. In intraperito-
neal inoculations the degree of virulence of the culture employed is
still more conspicuous in the effects produced. The animals usually
die in from two to nine days. The most characteristic pathological le-
sions are found in the kidneys, which contain numerous small collec-
tions of pus, and under the microscope present the appearances re-
sulting from embolic nephritis. Punctiform, whitish-yellow masses of
the size of a pea are found permeating the pyramids. Many of the
capillaries and .some of the smaller arteries of the cortex are plugged
up with thrombi, consisting of micrococci. Metastatic abscesses
may also be observed in the joints and muscles. The micrococci
may be recovered in pure cultures from the blood and the various
organs; but they are not numerous in the blood and are often difficult to
demonstrate microscopically. Intravenous inoculations of animals are
followed by similar pathological changes. Orth and Wyssokowitsch
first pointed out that injection of staphylococci into the circulation of
rabbits whose cardiac valves have previously been injured produced
ulcerative endocarditis. Subsequently, Weich.selbaum, Prudden, and
Fraenkel and Sanger obtained confirmatory results, thus establishing
the fact that when the valves are first injured, mechanically or chemic-
ally, the injection into a vein of a pure culture of staphylococcus aureus
gives rise to a genuine ulcerative endocarditis. It has been further
shown by Ribbert that the same result may be obtained without
previous injury to the valves by injecting into a vein the -staphylococcus
from a potato culture .suspendeil in water. In his experiments not only
'rom the surface, but the superficial layer of the potato
r with a .sterilized knife and mixed with distilled water
ul result is ascribed to the fact that the little agglome-
icocci and infected fragments of potato attach them-
argins of the valves more readily than isolated cocci
t infrequently, also, tn intravenous inoculations of
liere occurs a localization of the injected material in the
THE PYOGENIC COCCI. 365
marrow of the small bones. This may take place in full-grown animals
when the bones have been injured or fractured. The experimental
osteomyelitis thus produced has been demonstrated to be anatomically
analogous to this disease in man.
Occurrence in Man. — Practically all microorganisms have been
shown by experiment to produce, under certain conditions, the for-
mation of pus by their products when inoculated into the animal
body; but, while this has been demonstrated, the researches of bac-
teriologists show that only a few species are usually concerned in
the production of acute abscesses in man. Of these the two most
important, by reason of their frequent occurrence and pathogenic
power, are Staphylococcus pyogenes and Streptococcus pyogenes. These
two organisms are often found in the same abscess; thus, Passet, in
33 cases of acute abscess, found Staphylococcus aureus and alhus
associated in 11, aUms alone in 4, aUms and citreus in 2, Streptococcus
pyogenes alone in 8, albus and Streptococcus in 1, and albus, citreus , and
streptococcus in 1. The staphylococcus is liable to enter as a mixed
infection into most infections due to other bacteria, and is almost
always met with in all inflammations of the skin and mucous mem-
branes or in cavities connected with them.
The staphylococcus (staphylococcus aureus) has been demonstrated
not only in furuncles and carbuncles, but also in various pustular
affections of the skin and mucous membranes — impetigo, sycosis,
purulent conjunctivitis and inflammation of the lacrymal sac; in acute
abscesses formed in the lymphatic glands, the parotid gland, the tonsils,
the mammae, etc.; in metastatic abscesses and purulent collections in
the joints; in empyema, infectious osteomyelitis, ulcerative endocarditis,
pyelonephritis, abscess of the liver, phlebitis, etc. It is one of the chief
etiological factors in the production of pysemia in the various pathologi-
cal forms of that condition of disease. It is remarkable how many
staphylococci may be present in the blood without a fatal result, if the
original source of infection is removed. We met with one case in which
over 800 staphylococci were present in 1 c.c. of blood. A week later
only five were found. The patient finally died from pneumonia.
Not all persons are equally susceptible to infection by the staphy-
lococcus; those who are in a cachectic condition or suffering from
constitutional diseases, like diabetes, are especially predisposed to
infection. In healthy individuals certain parts of the body, as the
back of the neck and the buttocks, are more liable to be attacked than
others, with the production of furuncles, carbuncles, etc. In persons in
whom sores are readily caused, in consequence of disturbances of
nutrition, as in exhausting diseases, the micrococci settle at the points
of least resistance. Such conditions are present in the bones of debili-
tated young children, in fractures, and in injuries in general.
The pyogenic properties of the staphylococcus have been demon-
strated upon man by numerous experiments. Garr^ inoculated a
small wound at the edge of one of his finger-nails with a minute quan-
tity of a pure culture, and purulent inflammation, extending around
366 PATHOGENIC MICRO-ORGAXISMS,
the margin of the nail, resulted from the inoculation. Staphylococcus
aureus was recovered in cultures from the pus thus formed. The same
observer applied a considerable quantity of a pure culture obtained
from this pus — third generation — to the unbroken skin of his forearm,
rubbing it well into the skin. At the end of four days a large carbuncle,
surrounded by isolated furuncles, developed at the point where the
culture had been applied. This ran the usual course, and it was
several weeks in healing. No less than seventeen scars remained to
testify to the success of the experiment.
Tnvmiinity. — Rabbits have been rendered immune by means of
inoculations with both dead and living cultures. Unless the inoculations
are carefully done the animals frequently succumb. The staphylococci
injected into an immunized animal are more rapidly taken up by the
leukocytes than when injected into an untreated animal.
A serum having some protective power has also been elaborated.
Therapeutic Use of Vaccine. — The treatment of abscesses, boils,
and other localized staphylococcus infections by injections of repeated
doses of one hundred to three hundred million staphylococci has given
very successful results. Pyaemias have also been treated, but with
uncertain results. The serum has not been used with success.
Staph3^ococcas Pyogenes Albus.— It is morphologically identi-
cal with the Staphylococcus pyogenes aureus, and is probably the same
organism which has lost the property of producing pigment. On
the average it is somewhat less pathogenic and seldom produces pj'w-
mia or grave infections. The surface cultures upon nutrient agar
and potato have a milk-white color. Its biological characters are
not to be distinguished from the Staphylococcus aureu>s.
The majority of bacteriologists agree with Rosenbach, that the
aureus is found at least twice as frequently in human pathological
processes as the albus.
Staphylococcus Epidermidis Albus (Welch.)— Probably identical
with the Staphylococcus pyogenes albus. With reference to this micro-
coccus, Welch says: **So far as our observations extend — and already
they amount to a large number — this coccus may be regarded as
nearly, if not quite, a constant inhabitant of the epidermis. It is now
clear why I have proposed to call it the Staphylococcus epidermidis
albus. It possesses such feeble pyogenic capacity, as is shown by its
behavior in wounds as well as by experiments on rabbits, that the
designation Staphylococcus pyogenes albus does not seem appropriate.
Still, I am not inclined to insist too much upon this point, as very prob-
ably this coccus — which has hitherto been unquestionably identified
by others with the ordinary Staphylococcus pyogenes albus of Rosenbach
— is an attenuated or modified form of the latter organism, although,
as already mentioned, it presents some points of difference from the
classical description of the white pyogenic coccus/'
According to Welch, this coccus differs from the Staphylococcus
albus in the fact that it liquefies gelatin more slowly, does not so
quickly cause coagulation in milk, and is far less virulent when in-
THE PYOGENIC COCCI. 367
jected into the circulation of rabbits. It has been shown by the ex-
periments o[ Bossowski and of Welch that this microorganism is
very frequently present in aseptic wounds, and that usually it does
not materially interfere with the healing of wounds, although some-
times it appears to cause suppuration along the drainage-tube, and it
is the common cause of "stitch abscess."
Stapbylococcna Pyogenes Oitreus and other Staphylococci.—
Isolated by Passet (1885) from the pus of acute abscesses, in which
it is occasionally found in association with other pyogenic cocci. It
is distinguished from the other species p,g jjj
only by the formation of a lemon-yellow
pigment.
Many other varieties of staphylococci
have been occasionally met with which
differ- in some respects from the typical
varieties. This difference may be in the
fact that they liquefy gelatin more slowly
or not at all, or in pigment formation, or
in agglutination, or in still other respects.
None of these varieties are of great im-
portance.
The Micrococcns Tetragenus.— This Micnxwc^, tctn^m.
organism was di.scovered by GafFky (1881).
It is not infrequently present in the saliva of healthy individuals and
in the sputum of consumptive patients. In sputum it is sometimes
an evidence of mouth contamination rather than lung infection.
It has repeatedly been observed in the walls of cavities in
pulmonary tuberculosis associated with other pathogenic bacteria,
which, though playing no part in the etiology of the original disease,
contribute, doubtless, to the progressive destruction of the lung.
Its pyogenic character is shown by its occasional occurrence in the
pus of acute abscesses. Its presence has also been noted in the pus of
empyema following pneumonia.
Morphology. — Micrococci having a diameter of about l/<, which
divide in two planes, forming tetrads, and bound together by a trans-
parent, gelatinous substance, enclosing the cell like a capsule. In
cultures the cocci are seen in various stages of division as large, round,
cells, in pairs of oval elements, and in groups of three and
four {Figs. 117 and 118). When the division is complete they re-
mind one of sarcinie in appearance, except that they do not divide
in three directions and are not built up like diminutive cotton bales.
Staining. — This micrococcus stains readily with the ordinary ani-
line dyes; the transparent gelatinous envelope is only feebly stained.
It is not decolorized by Gram's method.
Biology. — The growth of this micrococcus is slow under all condi-
tions. It grows both in the presence and absence of oxygen; it grows
best from 35° to 38° C, but may be cultivated also at the ordinary
room-temperature — about 20° C.
368 PATHOGENIC MICRO-ORGANISMS.
Growth on Golatin- — On gelatin plates small, white colonies are
developed in from twenty-four to forty-eight hours, which, when ex-
amined under a low-power lens, are seen to be spherical or lemon-
shaped, grayiah-yellow disks, with a finely granular or mulberry-
like surface, and a uniform but somewhat roughly dentated border.
When the deep colonies push
^"'' "^ forward to the surface of the
gelatin they form white, ele-
vated, drop-like masses, hav-
ing a diameter of 1 to 2 mm.
In gelatin stick cultures the
gelatin is not licjuefied.
Orowtb on A^^ar and Blood
Senmi. — The colonies appear
as small transparent, round
points, which have a grajHsh-
yellow color and are slightly
elevated above the surface of
the medium.
Pathogenesis.— Subcu-
taneous injections of a culture
of this micrococcus in minute
.,,,,„._, quantity is usually fatal to
MiiTDCocPm tetroiepm inim periUmesl fluid. Stained ' , •'. ,™ "^ .
with fuchsin. (Freenkel.l XIOOO diBmetera. white TOICe. llie microCOCCl
are found in comparatively
small numbers in the blood of the vessels and heart, but are
more numerous in the spleen, lungs, liver, and kidneys. Intraperi-
toneal injections given to guinea-pigs and mice are followed by
purulent peritonitis, beautifully formed cocci in groups of four
being obtained in immense numbers from the exudate. Rabbits and
(logs are not affected by large doses of a culture subcutaneously or
intravenously administered.
The serum from immunized cases has not been used therapeutically
in human infection. Vaccines may be employed as with staphylococci.
THE 8TREPTO0O0OI.
Under this name must be included not only the streptococci which
excite inflammation in man, but all spherical bacteria which divide,
as a rule, in one plane only and remain attached in longer or
shorter chains. This name comprises by no means so many varieties
of bacteria as are grouped under the title bacilli. There are,
nevertheless, a considerable number of distinct groups of streptococci
which differ decidedly both in their cultural characteristics and their
pathogenic properties. The streptococci average about l/i in diam-
eter. None of them forms spores or is motile. They are rather
easily killed by disinfectants. Those that are pathogenic rarely re-
produce themselves outside the bodies of man and animals.
THE PYOGE.WIC COCCI. 369
Streptococcus Pyogenes. — The group of streptococci which in its
importance as related to human infections outweighs all other strepto-
cocci is that which comprises the streptococci which excite erysipelas,
many cases of cellulitis, abscess, septiciemia, pneumonia, etc., and
passes under the name ot Streptococcus pyogenes.
This organism was first discovered by Koch in stained sections of
tissue, attacked by septic processes, and by Ogston in the pus of acute
abscesses (1882). It was obtained by Fehleisen (1883) in pure cul-
tures from a case of erysipelas, its cultural and pathological char-
acters studied and demonstrated by him to be capable of producing
erysipelas in man. Rosenbach (1884) and Krause and Fasset (1885)
isolated the streptococcus from the pus of acute abscesses and gave
it the name of Streptococcus pyogenes. It has since been proved to be
one of the chief etiological factors in the production of many suppura-
tive inflammations. Formerly the streptococci of erysipelas, acute
abscesses, septiciemia, puerperal fever, etc., were thought to belong
to different species, because they were observed to possess apparent
differences in their biological and pathological characteristics, accord-
ing to the source from which they were obtained. Thus one species
of streptococcus was believed to be capable of causing erysipelas
only, another only acute abscesses; another sepsis, etc., but it is now
known that the slight differences between the majority of these strepto-
cocci are but acquired non-permanent variations of organisms derived
from the same species.
Morphology. — The cocci, when fully developed are spherical or
ovsl. They have no flagella or spores. They vary from o.4/( to 1/t
in diameter. They vary in dimensions in different cultures and
even in different parts of a singte colony. They multiply by binary
division in one direction only, forming chains of eight, ten, twenty,
and more elements, being, however, often associated distinctly in
pairs. On solid media the cocci occur frequently as diplococci, but
usually they grow in longer or shorter chains. Certain cocci fre-
quently exceed their fellows greatly in size, especially in old cul-
370 PATHOGENIC MICRO-ORGANISMS.
fures, when this may be considered to be the result of involution
forms. These were formerly called by Hueppe arthrospores. Some
varieties have distinct capsules when growing in the blood and in
blood-serum media (Hiss).
Staining.— -They slain readily by aniline colors and the pyogenic
varieties positively by Gram's method. Some varieties, mostly
saprophytic, growing in short chains are negative to Gram's stain.
Biology. — Streptococci grow readily in various liqui<l and solid
culture media. The most favorable temperature for their develop-
ment is from 30" to 37" C, but they multiply rather freely at ordi-
nary room temperature— 18° to 20° C. They are facultative anae-
robes, growing both in the presence and absence of oxygen.
Cultivation. — Growth on Oelatui. — Tubes of gelatin which have been
inoculated with streptococci by puncture with platinum needle show-
on the surface no growth beyond the point of entrance. In the depth
of the gelatin on the second or third day a distinct, tiny band appears,
with granular edges or made up of granules. These granules may
be very fine or fairly coarse. They are nearly translucent, with a
whitish, yellowish, or brownish tinge. With characteristic cultures
the gelatin is not liquefied.
Orowth on Agar. — On agar plates the colonies are visible after
twelve to thirty hours' growth at 37° C, and present a beautiful ap-
pearance when magnified sufficiently to see the individual cocci In
the chain. The colonies are small, iiot averaging over 0 5 mm. in
diameter (pin head). From different sources they vary in size,
thickness, mottling, color, and in the appearance of their borders.
The streptococcus growing in short chains in bouillon shows but
hltte tendency to form true loops, but rather projecting rows at the
edges of the colonies, while those growing in long chains show beauti-
ful loops, which are characteristic of this organism.
Growth in Bouillon. — Most streptococci grow well in slightly alku-
THE PYOGENIC COCCI. 371
line bouillon at 37° C, reaching their full development within thirty-
six to forty-eight hours. Those which grow in long chains usually
give an abundant flocculent deposit and leave their liquid clear. The
deposit may be in grains, in tiny flocculi, in larger flakes, or in tough,
almost membranous masses, the differences depending on the strength
of union between the pairs of cocci in the chains. Some of the strep-
tococci growing in long chains, however, cause the broth to become
cloudy. This cloudiness may be only temporary or it may be lasting.
Those growing in short chains, as a rule, cloud the broth, this cloudiness
remaining for days or weeks. A granular deposit appears at the
bottom of the tube. An addition of 0 . 5 to 1 per cent, glucose aids the
development of streptococci, but the acid produced tends later to hasten
their death and make them lose virulence. A trace of calcium aids
the growth. This is best added as a pfece of marble, which has the
additional advantage of neutralizing some of the acids produced.
Growth in Ascitic or Semm Bouillon. — ^The development in this,
which is the best medium for the growth of the streptococcus, is more
abundant than in plain bouillon. The liquid is generally clouded,
and a precipitate occurs after some days, the fluid gradually clearing.
The addition of blood serum frequently causes streptococci, growing
in short chains in nutrient bouillon, to produce long chains. The
reverse is also true, and in the blood all forms are usually found,
partly, at least, as diplococci or in short chains.
Effect on Inlnin. — This is not fermented by most varieties.
Growth on Solidified Blood Senun. — This is also an excellent medium
for the streptococcus. Tiny, grayish colonies appear twelve to
eighteen hours after inoculation.
Growth in Milk. — All streptococci grow well in milk. As a rule,
when growth is luxuriant a marked production of lactic acid with
coagulation of the casein occurs.
Development of Hsmolytic Substances. — Most streptococci produce
these. This is especially true of those from human septic infections.
As the pneumococci and some types of streptococci produce them
in a much less degree, blood-agar plates are a very useful means for
a probable identification. If 1 c.c. of fresh or defibrinated blood
is added to 6 c.c. of melted agar at 40° to 45° C, well shaken, in-
oculated with characteristic streptococci and poured in a Petri dish
there will appear in twelve to twenty-four hours tiny colonies sur-
rounded by clear zones of about i to ^ inch in diameter. Pneu-
mococci and many varieties of streptococci, which occur together with
characteristic forms in the throat, lungs and elsewhere, on the other
hand produce only narrow zones of a green pigment. Anthony in
our laboratory has found that from a streptococcus producing abun-
dant hemolytic substances strains may be obtained by selecting certain
colonies which fail to make them. She has not been able to obtain
from strains producing in first cultures the green pigment only any
strains producing hsemoljiic substances.
Duration of Life Outside of the Body. — This is not, as a rule, very
372 PATHOGENIC MICRO-ORGANISMS.
great. When dried in blood or pus, however, they may live for
several months at room temperature, and longer in an ice-chest, and
in gelatin and agar cultures they live for from one week to three
months. In order to keep streptococci alive and virulent, it is best
to transplant them frequently and to keep them in serum or ascitic
fluid bouillon in small sealed glass tubes in the ice-chest.
Resistance to Heat and Ohemicals. — The thermal death point of the
streptococcus is between 52° and 54° C, the time of exposure being
ten to twenty minutes.
Mercuric chloride, 1 :5000; sulphate of copper, 1 : 200; trichloride
of iodine, 1 : 750; peroxide of hydrogen, 1 : 50; carbolic acid, 1 : 100;
cresol, 1 :250; lysol, 1 : 300'; creolin, 1 : 130, all kill streptococci
within a few minutes.
Pathogenesis. — The majority of test animals are not very sus-
ceptible to infection by the streptococcus, and hence it is difficult to
obtain any definite pathological alterations in their tissues through
the inoculation into them of cultures of this organism by any of the
methods ordinarily practised. White mice and rabbits, under simi-
lar conditions, are the most susceptible, and these animals are, there-
fore usually employed for experimentation. Streptococci, how-
ever, differ greatly in the effects which they produce in inoculated
animals, according to their animal virulence, which is very different
from human virulence. The most virulent when injected in the
tainutest quantity into the circulation or into the subcutaneous tissues
of a mouse or rabbit, produce death by septicaemia. Those of somewhat
less virulence produce the same result when injected in considerable
quantities. Those still less pathogenic produce septicaemia, which
is mild or severe, when injected into the circulation; but when injected
subcutaneously, they produce abscess or erysipelas. The remaining
streptococci, unless introduced in quantities of 20 c.c. or over, produce
only a slight redness, or no reaction at all, when injected subcutaneously,
and Uttle or no effect when injected directly into the circulation.
Many of the streptococci obtained from cases of cellulitis, abscess,
empyema, and septicaemia belong to this group.
A number of varieties of streptococci have thus been discovered,
differing in virulence and in their growth on artificial media; but
all attempts to separate them into various classes, even with the use
of specific serum, have largely failed, because the differences observed,
though often marked, are not constant, many varieties having been
found to lose their distinctive characteristics, and even to apparently
change from one class to another. A further objection to any of
the existing classifications of streptococci, which are based on the
manner of growth on artificial culture media, is that it has been
impossible to make any w hich would at the same time give even an
approximate idea of their virulence. Experiments have proved that
streptococci originally virulent may become non-virulent after long
cultivation on artificial media, and, again, that they may return to
their original properties after being passed through the bodies of
THE PYOGENIC COCCI. 373
susceptible animals. The peculiar type of virulence which they may
acquire tends to perpetuate itself, at least for a considerable time.
One important fact that experience teaches us is that those strepto-
cocci which are the most dangerous are those which have come imme-
diately from septic conditions, and the more virulent the case the
more virulent the streptococci are apt to be for animals of the same
species. There seems also to be a strong tendency for a Strepto-
coccus to produce the same inflammation, when inoculated, as the one
from which it was obtained; for example, streptococci from ery-
sipelas tend to produce erysipelas, from septicaemia to produce septi-
caemia, etc. Streptococci, however, obtained from different sources
(abscesses, puerperal fever, sepsis, erysipelas, etc.) are in many in-
stances capable, under favorable conditions, of producing erysipelas
when inoculated into the ear of a rabbit, as has been proved by experi-
ment, provided they possess sufficient virulence.
Occurrence in Man. — Streptococci have been found to be the pri-
mary cause of infection in the following diseases: Erysipelas,
circumscribed and extensive acute abscesses, impetigo, cellulitis (cir-
cumscribed as well as diffused), sepsis, puerperal infection, acute
peritonitis, angina, bronchopneumonia, periostitis, osteomyelitis,
synovitis, otitis media, mastoiditis, enteritis, irregular cases of rheu-
matic fever, meningitis, pleurisy, empyema, and endocarditis. Asso-
ciated with other bacteria in diseases of which they were the specific
cause, they have also been found as the secondary infection in many
diseases, such as in pulmonary tuberculosis, bronchopneumonia,
septic diphtheria, and diphtheritic scarlatina.
In cases of septic thrombus of the lateral sinus following mastoid-
itis there is almost certainly a streptococcus septicaemia. Libman has
shown that an examination of the blood may be useful in diagnosis.
In diphtheritic false membranes this micrococcus is very commonly
present, and is frequently the source of deeper infection, such as ab-
scesses and septicaemia; and in certain cases attended with a diphther-
itic exudation, in which the I^oeflBer bacillus has not been found by
competent bacteriologists, it seems probable that the Streptococcus
pyogenes, alone or with other pyogenic cocci, is responsible for the
local inflammation and its results. These forms of so-called diph-
theria, as first pointed out by Prudden, are most commonly associated
with scarlatina and measles, erysipelas, and phlegmonous inflammation,
or occur in individuals exposed to these or other infectious diseases.
So uniformly are long-chained streptococci present in the pseudomem-
branes of patients sick with scarlet fever, that many investigators have
suspected a special variety of them to be the cause of this disease.
The same is true for smallpox. Many varieties are regularly found,
however, in the throat secretion of healthy individuals (in 100 exami-
nations by us we found long-chained streptococci in 83, and probably
could have found them in some of the others by longer search). Their
abundance in scarlet fever and smallpox is most probably due to their
increase in the injured mucous membrane and entrance into the
374 PATHOGENIC MICRO-ORGANISMS.
circulation when the protective properties of the blood have been
lowered.
Occurrence in Animals. — Besides streptococci similar to those in
man, animals are infected by strains that are negative to Gram and
fluidify gelatin. Udder infections of the cow and glandular diseases
of the horse are frequently due to these. The streptococcic inflam-
mations in animals are almost as frequent and serious as they are
in man.
Effect on Tumors. — Fehleisen inoculated cultures, obtained in
the first instance from the skin of patients with erysipelas, into patients
in the hospital suffering from inoperable malignant growths —
lupus, carcinoma, and sarcoma — and has obtained positive results,
a typical erysipelatous inflammation having developed around the
point of inoculation after a period of incubation of from fifteen to
sixty hours. This was attended with chilly sensations and an eleva-
tion of temperature. Persons who had recently recovered from an
attack of erysipelas frequently proved to be immune. These experi-
ments were undertaken on the ground that malignant tumors had
previously been found to improve or entirely disappear in persons
who had recovered from accidental erysipelas. During the last few
years this fact has been therapeutically applied to the treatment of
malignant tumors by the artificial production of erysipelas by the
inoculation of pure cultures of streptococcus or of their toxic prod-
ucts. Lately the mixed toxins of the streptococcus and B. pro-
digiosus have been given, and it now appears as if the toxins of the
latter organism were much the more important.
Results from Injections in Tumors. — In some cases of sarcoma this
method has met with considerable success; in carcinoma, however,
the results have been very slight. In this country the experimental
work upon this subject and the actual treatment of cases have been
largely carried out by or under the direction of Coley. He has kindly
sent me the following notes on his results:
**The improvement and inhibitory action which the toxins have
upon carcinoma have proved to be, in nearly all cases, but temporary.
**On the other hand, in sarcoma, which is the only form of malig-
nant tumor in which I have advocated the treatment, sufficient time
has elapsed to enable us to draw the following conclusions:
**The toxins injected subcutaneously into the tissues, either into
the tumor substance or into parts remote from the tumor, exercise a
distinctly inhibitory action upon the growth of nearly all varieties
of sarcoma. This action is the least marked in melanotic sarcoma,
and thus far no cases of this form of tumor have disappeared under
the treatment. The influence of the toxins upon round-celled sarcoma
is much more powerful than it is upon melanotic, although distinctly
less than upon the spindle-celled variety. A number of cases of
round-celled sarcoma in which the diagnosis was questioned dis-
appeared, and the patients have remained well beyond three years.
Nearly half of the cases treated show no appreciable decrease in
THE PYOGENIC COCCI. 375
size; the majority of the others which did show marked improve-
ment at first, after decreasing in size for a few weeks, again began to
increase and were no longer influenced by the treatment.
"In half of the cases of spindle-celled sarcoma treated by the
toxins the disease had disappeared entirely, and the majority of the
successful cases have remained well suflBciently long to justify their
being regarded as cured. It should be distinctly stated that all of
the tumors under consideration were inoperable, as I have never ad-
vised treatment except in such cases.
**I have now a number of cases of spindle-celled sarcoma which
have remained well beyond three years; one case of mixed (round
and spindle) celled, after remaining well three years, had a return
in the abdomen, and died about eight months later. The result
here certainly establishes the correctness of the early diagnosis."
Some surgeons have not had nearly as favorable results as Coley. I
think there is no question that in a small percentage of cases good
results have been obtained.
Production of Toxic Substances. — ^There is no doubt that the strepto-
coccus causes fever, general symptoms of intoxication, and death by
means of toxic substances which it forms in its growth; but we know
very little about these substances or how they are produced. The
cell substance of streptococci possesses only slight toxicity. Ruediger*
has shown that a specific streptolysin is formed which produces a true
antibody. The poisons while partly extracellular are mostly contained
in the cell substance. Heat destroys a portion of them. They appear
to attack especially the red blood cells, and this hemolytic action seems
to be to some degree in proportion to the virulence of the organism.
Susceptibility to Streptococcus Infection. — As with the ever-present
staphylococci, whose virulence, as we have seen, is usually slight,
the streptococci are more likely to invade the tissues, forming abscesses
or erysipelatous and phlegmonous inflammation in man when the
standard of health is reduced from any cause, and especially when
by absorption or retention various toxic organic products are present
in the body in excess. It is thus that the liability to these local infec-
tions, as complications of operations or sequelae of various specific
infectious diseases, in the victims of chronic alcoholism, and consti-
tutional affections, etc., are to be explained. It seems established
that the absorption of toxic products formed in the alimentary canal
as a result of the ingestion of improper food, or in consequence of
abnormal fermentative changes in the contents of the intestine, or
from constipation predispose to infection.
Immunity. — In none of the streptococcus inflammations do we
notice much apparent tendency to the production of immunizing and
curative substances in the blood by a single infection.
Severe general infections usually progress to a fatal termination
after a few days, weeks, or months. It is true, however, that cases
of erysipelas, cellulitis, and abscess, after periods varying from a few
* Ruediger. Jour. Amer. Med. Assn., 1903, xli., page 962.
376
PATHOGENIC MICRO-ORGANISMS.
days to months, tend to recover, and to a certain extent, therefore, we
may assume that protective agents have been produced. In these
cases, however, we know from experience that faulty treatment, by
lessening the local or general resistance, would, as a rule, cause the
subsiding infection again to progress perhaps even to a more serious
extent than the original attack. Koch and Petruschky tried a most
interesting experiment. They inoculated cutaneously a man suf-
fering from a malignant tumor with a streptococcus obtained from
erysipelas. He developed a moderately severe attack, which lasted
about ten days. On its subsidence they reinoculated him; a new
attack developed, which ran the same course and over the same area.
This was repeated ten times with the same results.
This experiment proved that in this case, at least, little if any
lasting curative or immunizing substances were produced by repeated
attacks of erysipelas, and that the recovery from each attack was due
to local and transitory protective developments.
The severe forms of infection, such as septicaemia following in-
juries, operations, and puerperal infections, show little tendency to
be arrested after being well established. Having, then, in remem-
brance, the above facts, let us consider the results already obtained
in the experimental immunization and treatment of animals and
men suffering from or in danger of Infection with streptococci. Knorr
succeeded in producing a moderate immunity in rabbits against an
intensely virulent streptococcus by injections of very slightly virulent
cultures. Marmorek was the first to attempt the production of a
curative serum on a large scale.
Influence of Serum from Inmiunized Animals upon Streptococcus
Infections in Other Animals. — In the table are given the results fol-
lowing the injection of small amounts of a serum which represents
in immunizing value what about one-third of the horses are able to
produce when given in gradually increasing doses the living, virulent
streptococcus. In the following experiments the serum and culture
were injected subcutaneously in rabbits at the same time, but in
opposite sides of the body:
Table — Showing Strength of Average Grade of Antistreptococcic Serum given by
Selected Horses after Six Months of Injection of Suiiable Amounts of Living
Streptococci.
Weight
of
rabbit
Amounts
inoculated
Results I Autopsy
Serum and culture:
1. Inoculated together
2. Inoculated together
3. On opposite side*
4. On opposite sides
Controls:
1. Rabbits injected with culture only.
2. Rabbits injected with culture only .
Grms.
1430
1350
1770
1630
1750
1870
Serum
0.25 c.c.
0.125 c.c.
0.1 c.c.
0.1 c.c.
Cult.
0.01
0.01
0.01
0.01
c.c.
c.c.
c.c.
c.c.
0.001 c.c.
0.001 c.c.
Lived
Lived
Lived
Lived
Died in
4 days
Died in
24 hre.
Streptococci
infection.
Streptococcic
infection.
THE PYOGENIC COCCI. 377
•
The above results have been repeatedly obtained, and are abso-
lutely conclusive that the serum of properly selected animals, which
have been repeatedly injected with living streptococci in suitable
doses possesses bactericidal properties upon the same streptococcus
when it comes in contact with it within the bodies of animals.
Definite protection from the serum has been obtained by many
reliable observers since Marmorek's first reports.
Is Protection Afforded by the Same Serum against all Varieties of
Streptococci? — We have tested the protective value of one serum
against streptococci derived from five different sources. First, the
streptococcus given us by Marmorek, which was obtained from a case
of angina. Its virulence is now such, after having passed through
hundreds of rabbits, that 0.000001 c.c. is the average fatal dose.
Second, a streptococcus obtained from a case of erysipelas in Eng-
land. Its virulence is 0.00001 c.c. on the average. Third, a strep-
tococcus obtained from a case of cellulitis, its virulence being about
6 c.c. Fourth, a streptococcus sent me by Theobald Smith. Its
virulence is such that 0.1 c.c. is the average fatal dose. Fifth,
another culture sent me by Smith, which grew in short chains and
was obtained from milk; its virulence was similar to No. 4.
Against the first three streptococci derived from three different va-
rieties of infection existing in three different countries the serum
produced by the streptococcus from England had nearly the same
value. Against the latter two streptococci, as well as against a strep-
tococcus from a case of endocarditis, which resembled in some re-
spects the pneumococci, the serum had no effect.
The results of numerous investigators indicate that the majority
but not all of streptococci met with in cellulitis, erysipelas, and abscess
will be influenced by the same serum. Those obtained from cases
of pneumonia and endocarditis and other exceptional infections
are apt to have individual characteristics.
Polyyalent Serum. — In order that the serum may have specific
anti-bodies for the variety of streptococci causing each separate in-
fection each horse is now injected with a large number of different
varieties of streptococci. This serum will not be quite as eflBcient
as if made by the streptococcus infecting the treated case, but will
be fairly efficient for all cases.
Preparation of the Serum. — Antistreptococcus serum is obtained
from the horse after treatment by repeated injections of living or
dead streptococcus cultures derived from human sources. As a
rule, a number of varieties are given at the same time so that the
serum will be active against any variety causing the infection. If
the serum is to be used in scarlet fever, the streptococci used should
be from cases of scarlet fever. The procuring of a serum of the
highest potency requires a considerable number of animals, for some
produce with the same treatment a more protective serum than others.
The serum must be sterile from streptococcus as well as from other
contaminations.
378 PATHOGENIC MICRO-ORGANISMS.
Stability of tbe Semm. — It is fairly stable but, after several months,
the serum loses some of its protective value. It should be kept in a
cool and dark place.
Standardisation of the Valne of the Serum.— There is at present
no satisfactory way. The value of the serum is sometimes measured
by the amount required to protect against a multiple of a fatal dose
of a very virulent streptococcus of the same type as the one used to
inject the horses. The dose is usually a thousand times the average
fatal amount of a very virulent streptococcus.
Other methods of standardization, such as the estimation of the
amount of opsonins or agglutinins present, are also used.
Therapeutic Results. — To estimate the exact present and future
value of an ti streptococcus serum is a matter of the utmost difficulty.
Many of the cases reported are of little or no help, because, no cul-
tures having been made, we are in doubt as to the nature of the bac-
terial infection.
In the cases of puerperal fever, erysipelas, and wound infection
that we have seen, the apparent results under the treatment have not
been uniform. We have frequently observed favorable results which
appeared to be due to the serum when doses of 50 to 60 c.c. were
given intravenously. ,
In a number of cases of septicaemia where for days chills had oc-
curred daily they ceased absolutely or lessened under daily doses of
20 to 50 c.c. The temperature, though ceasing to rise to such heights,
did not average more than one or two degrees lower than before the
injections. In some cases the serum treatment was kept up for four
weeks. Some cases convalesced; others after a week or more grew
worse and died. In some cases the temperature fell immediately
upon giving the first injection of serum, and after subsequent injec-
tions remained normal, and the cases seemed greatly benefited. As
a rule, in these cases no streptococci or any other organisms were ob-
tained from the blood. In bronchopneumonia, laryngeal diphtheria,
tonsiUitis, smallpox, and phthisis, we have seen little effect.
The results obtained here in New York by both physicians and
surgeons have not, on the whole, been very encouraging.
In some of the cases where apparently favorable results were ob-
tained other bacteria than streptococci were found to be the cause of
the disease. We believe that the following conclusions will be found
fairly accurate:
The serum wilt tn animals limit an infection already started if it
has not progressed too far. The apparent therapeutic results in
cases of human streptococcus infection are variable. In some cases
the disease has undoubtedly advanced in spite of large injections, and
here it has not seemed to have ha<l any effect. In other cases good
observers rightly or wrongly believe they have noticed great improve-
ment from it. Except rashes, few have noticed deleterious results,
although very large do.ses have been followed in several instances,
for a short time, bv albuminous urine.
THE PYOGENIC COCCI. 379
In suitable cases we are warranted, we believe, in trying it, but we
should not expect very striking results.
For our own satisfaction, and to increase our knowledge, we should
always have satisfactory cultures made when possible, and the strep-
tococci, if obtained, tested with the serum used in the treatment. In
the cases where we want most to use the serum, such as puerperal
fever, septicaemia, ulcerative endocarditis, etc., we find that it is very
difficult to make a bacteriological diagnosis from the symptoms, and
in over one-half of the cases even the bacteriological examination
carried out in the most thorough way will fail to detect the special
variety of bacteria causing the infection. This is often a great hin-
drance to the proper use of curative antistreptococcic serum, for it, of
course, has no specific effect upon the course of any infection except
that due to the streptococcus and the full effect only on its own type.
Care should be taken to get the most reliable serum; much on the
market is worthless, and as it is weak, and the testing for strength is
difficult or impossible, full doses (30-50 c.c.) of serum should be given
if the case is at all serious, for the dose is limited only by the
amount of horse serum which we feel it safe to give, not because we
have given sufficient protective substance. Intravenous injections
give better results than those given subcutaneously. Studdiford has
obtained good results by adding to the intravenous injection the pack-
ing of the septic uterus with gauze impregnated with the serum.
Scarlet Fever. — In Vienna for some years the serum of horses treated
at each injection with a number of strains of streptococci derived
from scarlet fever cases has been used in this disease. The serum
given in large doses of 100 to 200 c.c. has apparently given good results
in about half of those treated. It is only used in severe cases. Moser
has chiefly advocated its use. One of us had the opportunity to look
over the histories of his cases. Although left in doubt as to its value,
it appears to us as worth a trial. Our own results in thirty cases have
been rather favorable.
Bacteriological Diagnosis. — Streptococci, using the name in a broad
sense, can often be demonstrated microscopically by simply making
a smear preparation of the suspected material and staining with
methylene-blue solution or diluted Ziehl's fluid. In order to demon-
strate them microscopically in the tissues, the sections are best stained
by Kiihne's methylene-blue method. In all cases, even when the
microscopic examination fails, the cocci may be found by the cul-
ture method on plate agar or slanted agar at 37° C. To obtain them
from a case of erysipelas it is best to excise a small piece of skin from
the margin of the erysipelatous area in which the cocci are most numer-
ous; this is crushed up and part of it transferred to ascitic or serum
bouillon, and part is streaked across freshly solidified agar in a Petri
dish on which a drop of sterile rabbit's blood had previously been
placed. Both are kept in the incubator at 37° C.
In septicaemia the culture method is always required to demon-
strate the presence of streptococci, as the microscopic examination of
380 PATHOGENIC MICRO-ORGANISMS.
specimens of blood is not sufficient. For this purpose from 10 to
15 c.c. of the blood should be drawn from the vein of the arm asepti-
cally by means of a hypodermic needle, and to each of three tubes
containing 10 c.c. of melted nutrient agar kept at about 43° C. 1 c.c.
of blood is added. After thoroughly mixing the contents are poured
into Petri dishes. The remainder is added to several flasks contain-
ing 250 c.c. of nutrient broth, in order to produce a development of
the cocci, which are found in small numbers in the blood. Petruschkv
is of the opinion that the cocci can be best shown in blood by animal
inoculation. Having withdrawn from the patient 10 c.c. of blood
by means of a hypodermic syringe, under aseptic precautions, he injects
a portion of this into the abdominal cavity of a mouse, while the other
portion is planted in bouillon. Mice thus inoculated die from septicae-
mia when virulent streptococci are present in only very small numbers
in the blood. If a successful inoculation takes place we can, through
the absence or presence of the development of capsules, often differen-
tiate between the pneumococcus and the streptococcus, which cul-
tures may fail to do. The development of a wide, clear zone about
the colonies (upon blood-agar), without a development of green pig-
ment, indicates that the streptococci belong to the pyogenes type.
The absence of a definite zone and the development of a green color
indicates that they are pneumococci,or streptococci which in these two
respects resemble pneumococci. The growth in the Hiss inulin serum
medium will generally differentiate between the two, as the pneumo-
cocci usually coagulate the serum, while the great majority of strepto-
cocci do not. The morphological and cultural characteristics of the
streptococcus give us, unfortunately, no absolute knowledge as to the
influence which the protecting serum will have. The actual test is here
our only method. The detection of the streptococcus in the blood is in
itself an unfavorable prognostic sign.
The blood cultures in many cases of supposed septicaemia give no
results, for many of these cases develop their symptoms and even die
from the absorption of toxins from the local infection, such as an
amputation wound or an infected uterus or peritoneum, and the bac-
teria never invade the blood. When we get negative results we are,
as a rule, utterly unable to test the case with curative serums with
any accuracy, for the sepsis may be due to either the streptococcus,
colon bacillus, staphylococcus, or a number of other pathogenic va-
rieties of bacteria.
CHAPTER XXVII.
THE DIPLOCOCCUS OF PNEUMONIA (PNEUMOCOCCUS, STREP-
TOCOCCUS PNEUMONIAE, MICROCOCCUS LANCEOLATUS).
THE DIPLOOOOOUS OF PNEUMONIA.
The diplococcus of pneumonia was observed in 1880 almost simul-
taneously by Sternberg and Pasteur in the blood of rabbits inocu-
lated with human saliva. In the next few years Talamon, Fried-
lander, A. Fraenkel, Weichselbaum, and others subjected this micro-
organism to an extended series of investigations and proved it to be
the chief etiological factor in the production of lobar or croupous
pneumonia in man.
The outcome of the various investigations proved that the acute
lung inflammations, especially when not of the frank lobar pneumonia
type, are not excited by a single variety of microorganism, and that
the bacteria involved in the production of pneumonias are also met
with in inflammations of other tissues.
In any individual pneumonic inflammation it is also found that
more than one variety of bacteria may be active, either from the
start or as a later addition to the original primary infection.
Among all the microorganisms active in exciting pneumonia, the
diplococcus of pneumonia is by far the most common, being almost
always present in primary lobar pneumonia and as frequently as any
other germ in acute bronchopneumonia and metastatic forms. Be-
sides the different varieties of pneumococci the following bacteria are
capable of exciting pneumonia: Streptococcus pyogeneSy Staphylo-
coccus pyogenes y Bacillvs pnevmcnioPy Bacillus inflvenzce, Bacillvs
pestis, Bacillus diphthericp, Bacillus typhi. Bacillus coli, and the Bacillvs
tuberculosis. Since the varieties of bacteria exciting acute pneumonia,
with the exception of the pneumococcus, are met with more fre-
quently in other inflammations and have been described elsewhere,
they will only be noticed in this chapter so far as their relation. to
pneumonia demand.
Morphology. — Typically, the pneumococcus occurs as spherical or
oval cocci, usually united in pairs, but sometimes in longer or shorter
chains consisting of from three to six or more elements and resem-
bling the streptococcus. The cells, as they commonly occur in pairs,
are somewhat oval in shape, being usually pointed at one end — hence
the name lanceolatus or lancet-shaped. When thus united the junc-
tion, as a rule, is between the broad ends of the oval, with the pointed
ends turned outward; but variation in form and arrangement of the
cells is characteristic of this organism, there being great differences
381
382 PATHOGESIC MICRO-ORGANISMS.
according to the source from which it is obtained. As obserxed
in the sputum and blood it is usually in pairs of lancet-shaped ele-
ments, which are surrounded by a capsule. (See Fig, 123.) When
grown in fluid culture media longer or shorter chains are frequently
formed, which can scarcely be distinguished from chains of certain
streptococci, except that, as a rule, the length of the chain is less and
the pairs of diplococci are farther apart. In cultures the individual
cells are almost spherical in shape, and except in certain varieties
are rarely surrounded by a capsule. (See Fig. 124,) The pneumo-
coccus is by some classed as a streptococcus.
The capsule is best seen in stained preparations from the blood and
exudates of fibrinous pneumonia or from the blood of an inoculated
animal, especially the mouse, in which it is commonly, though not
always, present. It is seldom seen in preparations from cultures
unless special media are employed. Flagella are not present.
Staining.^It gtains readily with ordinary aniline colors; it is not
decolorized after staining by Gram's method. The capsule may be
demonstrate<i in blood or sputum cither by Gram's or Welch's (glacial
acetic acid) method, or the copper sulphate method of Hiss.
Biology. — It grows equally well with or without oxygen; its parastic
nature is exhibited by the short range of temperature at which it usually
grows — viz., from 25° to 42° C. — best at 37° C. In the cultivation of
this organism neutral or slightly alkaline media should be empIoye<l.
The organism grows* feebly on the serum-free culture media ordinar-
ily employed for the cultivation of bacteria — viz., on nutrient agar
and gelatin, in bouillon. The best medium for its growth is a mi-xture
of one-third human or animal blood serum or ascitic or pleuritic
THE DIFLOCOCCUS OF P.VEUMOMA. 383
fluid and two-thirds bouillon, or nutrient agar streaked with human or
rabbit blood.
Growth on Agar. — Cultivated on plain nutrient agar, after twenty-
four to forty-eight hours at 37° C, the deep colonies are hardly visible
to the eye. Under the microscope they appear light yellow or brown
in color and finely granular. The surface colonies are large, equalling
in size those of streptococci, but are usually more transparent. If
blood serum or ascitic fluid be added to the agar the individual colonies
are larger and closer together, and the growth is more distinct in con-
sequence and of a graj-ish color. The surface colonies are almost circu-
lar in shape under a magnification of 60 diameters, finely granular in
structure, and may have a somewhat darker, more compact centre,
surrounded by a paler marginal zone. With high magnification
cocci in twos and short rows often distinctly separated are seen at the
edges.
Growth on Blood Serum. — The growth on Loeffler's blood-serum
mixture is very similar to that on agar, but somewhat more vigorous
and characteristic, appearing on the surface as a delicate layer of dew-
like drops.
Growth in BonlUon. — In bouillon, at the end of twelve to twenty-four
hours in the incubator, a alight cloudiness of the li<|uid will be found to
have been produced. On microscopic examination cocci can be seen to be
arranged in paira or longer or shorter chaina. After one or two transplanta-
tions the pneumocooci frefjuently fail to grow.
Growth in Hilk.^It grows readily in milk, causing coagulation with the
production of acid, though this is not constant with .some forms interme-
diate between the streptococcus and pneumococcus.
Growth on Gelatin. — The growth on gelatin is slow, if there is any devel-
opment at all, owing to the low temperature — viz., 24° to 27° C. — above
which even the most heat-resistant gelatin will melt. The^ gelatin is not
liquefied.
384 PATHOGENIC MICRO-ORGANISMS.
Special Media. — When cultures are grown on serum-free media the vitality
of some cultures may indeed be indefinitely prolonged; but after transplanta-
tion through several generations it is found that the cultures begin to lose in
virulence, and that they finally become non-virulent. In order to restore this
virulence, or to keep it from becoming attenuated, it is necessarj- to interrupt
the transplantation and pass the organism through the bodies of susceptible
animals.
With the view of overcoming these obstacles in the way of cultivating
this micrococcus, several special media have been proposed by various experi-
menters in the place of the ordinary culture media. The best fluid medium
for the growth of the pneumococcus is Marmorek^s mixture, consisting of
bouillon 2 parts and ascitic or pleuritic fluid 1 part. In this fluid pneumo-
cocci grow well, and cultures when preserved in a cool place and prevented
from drying retain their vitality and also their virulence for a number of
weeks. Lambert has found cultures in this medium alive and fully virulent
after eight months.
Loeraer's blood-serum mixture is a good, solid tube medium for making
cultures, and is very convenient and useful at autopsies. One and one-half
per cent, fluid nutrient agar mixed with one-third its quantity of warm ascitic
fluid makes an excellent plate medium.
Nutrient agar, with fresh rabbit blood smeared over it makes an excellent
medium for growth, but prevents the development of agglutinable substance.
On this medium, in a moist atmosphere at 36° C, the cultures retain their
viability and virulence for rabbits for months.^ ^
Hi88 Senun Media with and without Inulin. — These are verv useful.
The inulin is fermented by typical pneumoeocci with coagulation of
the serum, while most streptococci fail to ferment the inulin. This
medium is, therefore, of considerable diagnostic value.
Oaldmn Broth with or without Dextrose. — This medium has proven
of great value for the propagation of cultures where agglutination
tests are to be carried out. The addition of a small piece of marble
to each tube of broth is the most satisfactory way of preparing it.
Marble broth for this purpose was suggested independently by Bolduan
and Hiss.
Resistance to Light, Drjiag, and Oermicidal Agents. — On artificial
culture media the pneumoeocci tend to die rapidly. This is partially
due to the acid produced by their growth. In sputum they live much
longer.
Pneumonic sputum attached in masses to clothes, when dried in
the air and exposed to diffuse daylight, retains its virulence, as shown
by injection in rabbits, for a period of nineteen to fifty-five days.
Exposed to direct sunlight the same material retains its virulence
after but a few hours' . exposure. This retention of virulence for
so long a time under these circumstances is accounted for by the
protective influence afforded by the dried mucoid material in which
the micrococci were embedded. Guarnieri observed that the blood
of inoculated animals, when rapidly dried in a desiccator, retained
its virulence for months; and Fod found that fresh rabbit blood,
after inoculation and cultivation in the incubator for twenty-four
* The green color produced by all pneumoeocci in blood-a^ar media, and .showing
especially well m poured blood-agar plates is not diagnostic of this organism, as
some strains of streptococci produce just as intense a green.
THE DIPLOCOCCUS OF PNEUMONIA, 385
hours, when removed at once to a cool, dark place, retained its viru-
lence for sixty days. There are many conditions, therefore, in which
the virulence of the micrococcus is retained for a considerable length
of time. To germicidal agents pneumococci are very sensitive. The
fine spray expelled in coughing and loud speaking that remains sus-
pended in the air soon dries so completely that no pneumococci survive
after two hours.
Attennation of Virulence. — ^This may be produced in various ways.
The loss of virulence which occurs when the micrococcus is trans-
planted through several generations in culture fluid containing no
blood has already been referred to. An apparent attenuation of
virulence takes place also spontaneously in the course of pneumonia.
It has been shown by daily puncture of the lung in different stages
of the pneumonic process that the virulence of the organism dimin-
ishes as the disease progresses, and that the nearer the crisis is ap-
proached the more attenuated it becomes. This attenuation is prob-
ably only apparent. So many more microorganisms are living in
each cubic centimetre of fluid during the early stages of a pneumonia
that much smaller quantities kill. If a little sputum be taken) at
different periods in the disease and planted in ascitic bouillon the
resultant cultures will not vary greatly in virulence. The average
virulence for rabbits of cultures made from pneumonic sputum is
greater than in those from normal sputum.
Restoration and Increase of Virulence. — The simplest and perhaps
the most reliable method of restoring lost virulence for any sus-
ceptible animal is by passage through the bodies of highly susceptible
animals of the same species. Growth in fresh blood also increases it
for the homologous animal.
Toxin Production. — We have little exact knowledge upon the na-
ture of the substances produced by or through the growth of the
pneumococci in animal tissues or artificial media. Rosenow* showed
that the autolysis of virulent pneumococci in NaCl solution brings
into the solution a group of substances which inhibits the action of
the pneumococco-opsonin.
Occurrence in Man during Health.— It is probable that in crowded
communities the pneumococcus is present on the mucous membranes
of most persons. We have found it generally present not only in
the throats of persons living in New York City, but also in those of
persons living on farms and in the Adirondack Mountains. It is
commonly present only on the mucous membranes of the bronchi,
trachea, pharynx, and nostrils. The healthy lung seems to be gen-
erally free from it. Judging from animal tests it is very possible
that the virulence for man of the organisms present in health is much
less than the virulence of those in a pneumonic lung.
Pathogenicity in Man. — Characteristic or atypical pneumococci
are present in fully 95 per cent, of characteristic cases of lobar pneu-
monia. Usually no other bacteria are obtained from the lungs.
* Rosenow. Jour. Infect. Dis., 1907, iv., p. 285.
25
386 PATHOGENIC MICRO-ORGANISMS.
Atypical cases usually show the same conditions, but they may be
due to streptococci, influenza bacilli, etc. The more recent the in-
fet^ion the greater is the number of bacteria found in the disease^l lung
area. As the disease progresses these decrease in number until fin-
ally at the crisis they disappear from the tissues, though at this time
and long after convalescence they may be present in the sputum. In
atypical forms of pneumonia they may remain longer in the tissues,
and in walking pneumonia they may he absent in the original centres
of infection or present only as attenuated varieties, while the surround-
ing, newly formed foci may contain fully virulent cocci. It has been
shown by Netter that more than one-half of the caseS of broncho-
pneumonia, whether primary or secondary to some other disease, as
measles and diphtheria, both in children and ndults, are due to the
diplococcus of pneumonia. Others, such as Pearce, have found
other microorganisms, especially the streptococci, in the majority of
cases. These findings will be considered at the end of the chapter.
The pneuraococci are found partly in the alveoli and bronchioles
of the inflamed lung and partly in the lymph channels and blood
capillaries. Most of the organisms are found free, but a few are
found in the leukocytes. Through the lymph channels they find
their way to the pleura and to adjacent lymph glands. From the
capillaries they find their way to the general blood current, and thus
to distant parts of the body. In about 20 per cent, of cases the pneu-
raococci are so abundant that they can be found in cultures made
from 5 to 10 c.c. of blood. In a number of instances the foetus has
been found infected. The pneumococci are also responsible for:
Inflanmiatloiis Oomplicating Pneumonia.— In every case of lobar
pneumonia and in most cases of bronchopneumonia pleurisy is de-
veloped, which is excited by the same microorganism that was pre-
dominant in the pneumonia. With pneumococci the exudate is
usually moderate and of a fibrinous character, but may be more
abundant and of a serofibrinous or purulent character. When
the pleurisy is marked it is more apt to continue after the cessation of
the pneumonia. Pleurisy due to pneumococci is more apt to go on
to spontaneous recovery than that due to streptococci or staphylococci.
The most frequent pneumococcic infections next to pleurisy, fol-
lowing a pneumonia, are those of the middle ear, pericardium, en-
docardium, and meninges, and these not infrequently arise together.
Pneumococcic inflammations of the heart valves are apt to be fol-
lowed by extensive necrosis and growth of vegetations. In the.se
cases oneumococci can sometimes be found in the blood for many
ditis due to pneumococci is a frequent complication,
rery slightly developed. Meningitis due to pneumo-
ither fibrinous or purulent or both and is apt to be
)titis, mastoiditis, or pneumonia. Arthritis, periar-
'omvelitis are rarer complications of a pneumococcic
esides moderate parenchymatous inflammation of the
ccurs in most cases of pneumonia, well-marked inflam*
THE DIPLOCOCCUS OF PNEUMONIA. 387
mation may occur in which pneumococci exist in the kidney tissues in
large numbers.
How is the pneumococcus conveyed from its original seat in the
lungs to distant internal organs? Chiefly by means of the blood
vessels and lymphatics, in both of which it has been found in great
numbers. Proof enough of its conveyance through the lymphatics
is afforded by the frequent occurrence of inflammations of the serous
membranes complicating pneumonia; but two cases in particular
have been reported by Thue of pleurisy and pericarditis following
pneumonia in which the lymph capillaries have been found to be
filled with diplococci, as if injected. Their presence in the blood
after death has been amply proved by numerous investigations. In
many instances they have been recovered from the blood during life.
Lambert, as a rule, found them in all fatal cases twenty-four to forty-
eight hours before death. This examination has considerable prognos-
tic value, as nearly all cases in which the pneumococcus is found end
fatally. This micrococcus has been shown experimentally to be
capable of producing various forms of septicaemia — local phlegmonous
inflammations, peritonitis, pleuritis, and meningitis. A further proof
of the transmission of this organism by means of the blood is given
by Fod and Bordoni-Uffreduzzi in their investigations into intrauterine
infection in pneumonia and meningitis. These investigators have
demonstrated the presence of the micrococcus lanceolatus in fetal
and placental blood and in the uterine sinuses in maternal pneumonia.
There being no question, therefore, as to the possibility of the convey-
ance of the infective agent by means of the blood and the lymph to
all parts of the body, we need not wonder at the multiplicity of the
afiFections complicating a pneumonia, which are caused by this micro-
coccus; and not only the secondary, but also the primary diseases,
as of the brain and meninges, may be explained in the same way.
Knowing that the saliva and nasal secretions under normal conditions
so frequently afford a resting place for the micrococci, we have only
to assume the production of a suitable culture medium for these
parasites in the body, brought about by an abnormal condition of the
mucous membranes from exposure to cold, or a reduction of the vital
resisting power of the tissue cells in any of the internal organs, caused
by disease, traumatism, excess of various kinds, etc., to comprehend
readily how an individual may become infected with pneumococci,
either primarily affecting the lungs and secondarily other organs in
the body, or primarily attacking the middle ear, the pericardial sac,
the pleura, the serous cavities of the brain, etc.
Presence in Inflammatory Process Not Secondary to Pneumonia.
— It is now known that the pneumococcus may infect and excite
diseases in many tissues of the body independent of any preliminary
localization in the lung. As a rule, these processes are acute and
usually run a shorter and more favorable course than similar inflam-
mations due to the streptococci.
The most frequent primary lesions excited by the pneumococcus
388 PATHOGENIC MICRO-ORGANISMS,
after lobar pneumonia, bronchopneumonia, and bronchitis are prob-
ably meningitis, otitis media with its complicating mastoiditis, endo-
carditis, pericarditis, rhinitis, tonsillitis, conjunctivitis, and keratitis;
septicaemia, arthritis, and osteomyelitis; inflammations of the epi-
didymis, testicles, and Fallopian tubes; peritonitis, etc.
Pneumococcic peritonitis and appendicitis are not so very frequent.
The exudate is usually seropurulent.
Conjunctivitis due to pneumococci frequently occurs in epidemic
form and is frequently associated with rhinitis.
From statistics collected by Netter the following percentages of
diseases were caused by the pneuraococcus :
Pneumonia 65 . 9 per cent, in adults.
Bronchopneumonia 15.8 per cent, in adults.
Meningitis 13.0 per cent, in adults.
Empyema 8.5 per cent, in adults.
Otitis media 2.4 per cent, in adults.
Endocarditis 1.2 per cent, in adults.
In 46 consecutive pneumococcus infections in children there were:
Otitis media 29 cas^.
Bronchopneumonia 12 cases.
Meningitis 2 cases.
Pneumonia 1 case.
Pleurisy 1 case.
Pericarditis 1 case.
The pneumococcus and streptococcus are the two most frequent
organisms found in otitis media. The cases due to the pneumococcus
are apt to run the shorter course, but have a tendency to spread to
the meninges and cause a meningitis. The pneumococci may also
find their way into the blood current. This usually follows after
sinus thrombosis.
In bronchitis the pneumococcus is frequently met with alone or in
combination with the streptococcus, the influenza bacillus, or other
bacteria.
In certain epidemics pneumococcic bronchitis and pneumonia
simulate influenza very closely and cannot be differentiated except
by bacteriological examinations.
Primary pneumococcic pleurisy is frequent in children: it is very
often purulent, but may be serous or serofibrinous. Its prognosis
is better than that in cases due to other organisms. Frequently we
have streptococci and staphylococci associated with the pneumococci.
Pathogenesis in Lower Animals. — Most strains of the Micrococcus
lanceolatus are moderately pathogenic for numerous animals; mice and
rabbits are the most susceptible, indeed some stains are intensely
virulent for these animals, while guinea-pigs and rats are much less
susceptible. Pigeons and chickens are refractory. In mice ^nd
rabbits the subcutaneous injection of small or moderate quantities
of pneumonic sputum in the early stages of the disease, or of a twenty-
four-hour ascitic broth culture from such sputum, or of a pure, virulent
ascitic broth culture of the micrococcus, usuallv results in the death
THE DIPLOCOCCUS OF PNEUMONIA. 389
of these animals in from twenty-four to forty-eight hours. The course
of the disease produced and the post-mortem appearances indicate that
it is a form of septicsemia — what is known as sputum septicaemia.
After injection there is loss of appetite and great debility, and the ani-
mal usually dies some time during the second day after inoculation.
The post-mortem examination shows a local reaction, which may be
of a serous, fibrinous, hemorrhagic, necrotic, or purulent character;
or there may be combinations of all of these conditions. The blood
of "inoculated animals immediately after death often contains the
micrococci in very large numbers. For microscopic examination they
may be obtained from the blood, and usually from pleural and peri-
toneal exudations when these are present.
True localized pneumonia does not usually result from subcu-
taneous injections into susceptible animals, but injections made
through the thoracic walls into the substance of the lung may induce
a typical fibrinous pneumonia. This was first demonstrated by Tala-
mon, who injected the fibrinous exudate of croupous pneumonia,
obtained after death or drawn during Ufe from the hepatized por-
tions of the lung, into the lungs of rabbits. Wadsworth showed that
by injecting virulent pneumococci into the lungs of rabbits which
had been immunized, a typical lobar pneumonia was excited, the
bactericidal property of the blood being sufficient to prevent the
general invasion of the bacteria.
Varieties of the Pneumococcus. — As among all other microorgan-
isms minutely studied, difiFerent strains of pneumococci show quite
a wide range of variation in morphology and virulence. Some of the
variations are so marked and so constant that they make it necessary
to recognize several distinct varieties of the pneumococcus, and to
class as pneumococci certain varieties which have before this been
classed as streptococci — e. g., the so-called Streptococcus mucosus
capsidatus (Streptococcus mucosus Schottmiiller), when first isolated
from pneumonic exudate or elsewhere, and planted on artificial cul-
ture media containing serum, grows as a rounded coccus with a small
dense distinct capsule, principally in short or medium chains; it pro-
duces a large amount of mucus-like zooglia, forming very large spread-
ing colonies; it promptly coagulates fluid serum media containing
inulin. It is also very virulent for mice, but only moderately virulent
for rabbits. After a number of culture generations on ordinary nutrient
agar it apparently loses most of these characteristics. It then grows
in small colonies principally as naked diplococci which may be elon-
gated and pointed, produces no zooglia, and loses most of its virulence
for mice and rabbits. It still coagulates inulin serum media, and
when transferred to serum media regains its former morphological
characteristics. For these reasons we consider this organism a distinct
variety of the pneumococcus. This variety of pneumococcus has been
isolated by us from the lungs after death following lobar pneumonia,
out of twenty consecutive autopsies, as the only organism present twice,
and with another variety of pneumococcus once. Together with
390 PATHOGENIC MICRO-ORGANISMS,
other varieties it was isolated from four out of twenty specimens of
pneumonic sputum, and from sixty specimens of normal throat secre-
tion five times.
Another group of pneumococci quite constantly produces large
forms and large capsules. Still another group produces principally
small forms and small capsules. Another group might be made of
morphologically typical pneumococci which do not coagulate inulin
serum media.
Immunity. — Following an attack of pneumonia some immunity is
established, but this lasts but a short time. Early in the history of
this organism experiments were begun for the production of immunity
in animals by means of preventive inoculations. Later it was found
that after successive injections of gradually increasing doses of virulent
pneumococci into certain animals (horse, sheep, goat, rabbit), a serum
of some protective and curative power in experimental animals was
obtained. The mode of action of this serum is still the subject of study.
According to Wright, Neufeld, and others, its activity is due to the
presence of certain substances called opsonins (Wright), or bacterio-
tropic substances (Neufeld), which act on the bacteria in such a way
as to prepare them for ingestion by the phagocytes. The extent to
which phagocytosis brings about the crisis and healing in pneumonia
is not known.
Agglutination Reactions. — Neufeld, Clairmont, and others demon-
strated agglutinating substances for the pneumococcus in the blood
of immunized animals; they concluded, from their observations, that
this test might be used as a means of diagnosis. The low index
obtained by them and the few strains used seemed to justify this
assumption.
We have found, however, in our laboratory (Collins) that when a
high index is reached or a large number of strains tested, the variability
of the reaction is so great as to render it impractical as a means of
diagnosis.
For instance, one strain may produce agglutinins common to itself
and four or five other strains, while 70 or 80 other strains (all being
typical pneumococci) will fail to react in the serum.
This diversity of reaction is confirmed also by the absorption tests.
In the case of the pneumococcus mucosus {Strejdococcus mucosus
Schottmuller) Collins found greater uniformity of reaction, all strains
tested reacting alike, and the agglutinins of one member of the group
were absorbed by the other members.
Therapeutic Experiments. — The number of cases reported in
which the blood serum of animals artificially immunized against
pneumonic infection has been used for the treatment of the disease
in human beings, although numerous, has not led to the formation
of a definite opinion as to the final value of this as a therapeutic agent.
In the cases we have observed there has been, in some a slight immediate
lowering of the temperature, in others no apparent change. As a rule,
the cases did rather better than was expected, but certainly no striking
THE DIPLOCOCCUS OF PNEUMONIA. 391
curative effects were apparent/ The cases did not'develop pneumo-
coccus blood infection, and it seems probable that the serum may be
able to prevent a general infection from taking place from the diseased
lung, even though it may fail to influence the local process. It has
also been shown that these injections of antipneumococcic serum are
practically harmless. In pneumococcus septicaemia no marked results
have been seen. The majority who received the injections, as well as
those not receiving them, died. Large intravenous injections of
50 c.c. of a polyvalent serum might be of value.
Vaccines. — The use of injections of dead pneumococci in pneu-
monia and other acute pneumococcic inflammations has not been
followed by appreciable beneficial results in those t'ases which have
come under our observation. In subacute cases the results appear
to be more favorable. With better understanding of the proper
dosage better results may become possible.
CHAPTER XXVIII.
MENINGOCOCCUS OR MICROCOCCUS (INTRACELLULARIS)
MENINGITIDIS, AND THE RELATION OF IT AND OF
OTHER BACTERIA TO MENINGITIS.
In the description of the diplococcus of pneumonia reference was
made to this organism as the most frequent cause of isolated cases of
meningitis, especially when it complicated pneumonia. In 1887
Weichselbaum* discovered another micrococcus in the exudate of
cerebrospinal meningitis in six cases, two of which were not compli-
cated by pneumonia. He obtained it in pure cultures, studied its
characteristics, and showed that this organism was clearly distin-
guishable from the pneumococcus and especially by its usual presence
in the interior pus-cells, on which account he called it Diplococctts
intracellularis meningitidis. In 1895 Jaeger and Schuerer drew
especial attention to the etiological relationship of the organism to
the epidemic form of cerebrospinal meningitis. They also believed
it to be very probable that in most cases of primary meningitis it is
from the mucous membrane of the nasal cavities and the sinuses
opening out from them that both the diplococcus of pneumonia and
the micrococcus intracellularis find their way through the blood or
perhaps directly through the lymph channels to the meninges. The
former we know to be almost constantly present in the nasal cavities,
and the latter we have reason to believe is not infrequently there.
The prevalence of epidemics in winter and spring, a time favorable
to influenza and pneumonia, also suggests the respiratory tract as the
place of the infection and where an increase in virulence takes place.
We do not as yet know why meningitis follows in some persons and
not in others after infection of the mucous membranes.
Infected persons as well as things recently soiled by their nasal
secretion are dangerous.
Morphology. — This organism occurs as biscuit-shaped micrococci,
usually united in pairs, but also in groups of four and in small masses;
sometimes solitary and small degenerated forms are found. It has no
well-defined capsule. Cultures resemble strongly those of gonococci
(see Fig. 127). In cultures more than twenty-four hours old larger and
smaller forms occur and some which stain poorly. These are involu-
tion forms. In the exudation, like the gonococcus, to which it bears
a close resemblance in form and arrangement, it is distinguished by its
presence, as a rule, within the polynuclear leukocytes. It never appears
within the nucleus and rarely within other cells (Fig. 126).
> Weichselbaum. Fortschr. d. Med., 1887, p. 573.
392
MICROCOCCUS MENINGITIDIS. 393
Staining. — It stains with all the ordinary aniline colors, but best
with Loeffler's methylene blue. It is readily decolorized by Gram's
solution. Some organisms in many cultures are more resistant than
others, but none are Gram-positive. Elser and Huntoon' carefully
tested this matter and never found a Gram-positive stain in 200 different
cultures. We have had a similar experience in a smaller number. It
is almost certain that the positive cocci which have been described by
Jaeger and others as meningococci P,,, ^^
are really contaminating organism.s.
Stained with loeffler's alkaline
solution of methylene blue the
cocci frequently show a central
metachromatic granule. The cells
have no definite capsule.
Biology.— It grows between 25'
and 40° C, best at about 37.5"
C, and its development is usually
scanty on the surface of nutrient
agar, but sometimes a few colonies
grow quite vigorously. Now and
then cultures grow at 23° C. or
slightly less. It grows scarcely at
all in bouilloD, and scantily in Dipioc«cu. iQirB«iiuijj™ menuwiticii* in
bouillon plus one-third blood- *"" " ' ""'
serum. It develops comparatively well on Loeffler's blood-serum
medium as used for diphtheria cultures, and on blood-serum or ascitic
fluid agar. The addition of 1 per cent, glucose favors growth.
Of the sugars the meningococcus ferments dextrose and maltose only,
and even these not sufficiently to coagulate the serum media.
When grown for some time, a tolerably good growth develops at the end of
forty-eight hours in the ineubator on nutrient agar or glucose agar. This
appears as a flat layer of colonies, about one-eightli of an inch Id diameter,
grayish-white in color, finely granular, rather vimid, and non-confiuent unless
very close together. On Loeffler's blood serum the growth forms round,
whitish, shining, viscid-looking colonies, with smooth and sharply-defined
outlines; these may attain diameters of one-eighth to one-sixteenth of an inch
in twenty-four hours. The colonies tend to l>ecome confluent and do not
liquefy the serum. From the spinal fluid in acute cases, where the organisms
are apt to be more abundant, a great many minute colonies may develop
instead of a few larger ones. On agar plates the deep-lying colonies are almost
invisible to the naked eye; somewhat magnified they ap|>ear (inely granular,
with a dentated border. On the surface they are larger, appcarmg as pale
disks, almost transparent at the edges, but more compact toward the centres,
which are yellowish-gray in color. On blood agar or scrum agar the growth
is much more luxuriant than on plain agar and larger than the gonococcus.
Not infrequently no growth is obtained when the cerebrospinal fluid contain-
ing the diplococei is jilaced on plain agar, and in rare instances no growth
appears when scrum agar is used. Cultivated in artificial media, while it
often lives for weeks, it may die within four days, and requires, therefore, to
be transplanted to fresh material at short intervals — at least every two days.
' The Jour, of Med. Research, Vol. tx,, No. 4, page 3!I0.
394 PATHOGEXIC MICRO-ORGANISMS.
I. — It IS readily killed by heat, disinfectants, sunlight,
and drying. A few cocci may remain alive for 1 to 3 days in the dried
state. To maintain cultures it is necessary to replant recently isolated
cultures daily, but after several weeks once a month will suflBce.
Some cultures are very difficult to keep alive.
Pathogenesis. — This organism does not show marked pathogenic
power for adult animals. It is most pathogenic for mice and guinea-
pigs, less so for rabbits and dogs. Subcutaneous injections in animals
when large cause death.
When mice are inoculated into the pleural or peritoneal cavities
they usually fall sick and die within thirty-six to forty-eight hours,
showing slight fibrinopurulent exudation.
Certain experiments made by Weichselbaum on dogs, though not
entirely successful, are interesting as showing the similarity of the
disease produced in them artificially with meningitis as occurring in
man. The three dogs, trephined and inoculated subdurally with
0.5 to 2 c.c. of a fresh culture, all died: No. 1 within twelve hours,
No. 2 in three days, and No. 3 in twelve days. In Nos. 1 and 2
there were found hypersemia of the meninges, with inflammatory
softening of the brain at the point of inoculation, which on nearer
inspection proved to be a true encephalitic process. In dog No. 2,
in which the disease was of longer duration, these changes were the
most pronounced. Numerous diplococci were observed in the sec-
tions removed, for the most part free, but some few within the pus
cells. In dog No. 3, in which the disease lasted twelve days, a thick,
reddish, purulent liquid was found between the dura mater and the
brain at the point of the inoculation; in the brain itself an abscess
had formed, about the size of a hazel-nut, filled with tough, yellow
pus, while the abscess walls consisted of softened brain substance
infiltrated with numerous hemorrhagic deposits. The ventricles on
that side contained a cloudy, reddish fluid, with flocks of pus; but
no diplococci could be demonstrated in the blood or exudations. In
our experience injection of a recent culture into the spinal canal of
very young puppies is regularly followed by the results noted by
Weichselbaum. Such effects are not observed in older dogs. In
monkeys Flexner,^ von Lingelsheim, and McDonald have been able
to produce rather characteristic symptoms and lesions by intraspinal
injections. Inoculated in other ways, the usual type of infection was
not produced.
Presence in the Nasal Oavity of the Sick and Those in Oontact with
Them. — In 1 of his 6 cases Weichselbaum succeeded in obtaining dip-
lococci from the nasal secretion. In 1901 Albrecht and Ghpn demon-
strated them in healthy individuals. Scheurer, in his 18 cases, found
the diplococci in the nasal secretions of all of them during life. In 50
healthv individuals examined they were found in the nasal secre-
tions of only two, one being a man suffering at the time from a severe
cold. This man, it is interesting to note, had been employed in a
' Flexner. Jour. Exp. Med., 11)07, ix., page 142.
MICROCOCCUS MENINGITIDIS. 395
room which had just previously been occupied by a patient with cere-
brospinal meningitis. Lately, there has been a tendency to throw
doubt on these findings, but from our experience in the 1906 epidemic
in New York, one can state that the meningococci are usually present
in great numbers in the nose and naso-pharynx in most cases of menin-
gitis during the first twelve days of illness. After the fourteenth day
they cannot usually be found. In one case Goodwin of our laboratory
obtained them on the sixty-seventh day. She also found them in
five persons out of sixty tested who had been in close contact with the
sick, and in two of fifty medical students.
Pathogenicity for Man. — The most marked lesions occur at the base
of the brain. The cord is always afiFected. This is not true to the
same extent in other bacterial infections. In some epidemics the
course of the disease is very rapid. The mortality varies from between
50 and 80 per cent.
Complicating Infections. — Occasionally we find secondary to the
cerebrospinal meningitis, and due to the micrococcus, inflamma-
tions of nasal cavities and their accessory sinuses, also catarrhal
inflammations of the middle ear, acute bronchitis, and pneumonia.
The absolute determination of the identity of the micrococcus found
in these conditions has not been established, so that the above com-
plications can only be considered as probably due to this organism.
Except in cases of meningitis the micrococcus has been absolutely
identified only in cases of rhinitis. Several observers believe they
have found it in the diseases mentioned above as occasionally com-
plicating meningitis.
Meningococci in the Blood.^Elser in forty cases examined during
the early days of the disease found them in ten.
Agglutination Characteristics. — A considerable percentage of cul-
tures of meningococci are relatively inagglutinable. Strains that
are agglutinable respond to the agglutinins developed in an animal
immunized with a true strain. Careful absorption tests are capable
in almost, if not all, instances of separating true meningococci from
other Gram-negative organisms. The serum reaction is rarely used
in diagnosis. The microscopic test is so much more definite.
Semm Treatment. — It is diflBcult to apportion the credit for the pro-
duction of the first protective serum. Bonhoff and Lepriere produced
in animals a serum which showed definite protection. The world-wide
epidemic beginning in 1904 stimulated a number of laboratories to pro-
duce sera in horses with the idea of treating human cases.
Thus Kolle* and Wasserman, Jochmann, Flexner, and ourselves
immunized horses. The usual method was to begin with cultures
recently obtained from human cases and grow them on ascitic agar
or plain nutrient agar in tubes. The growth was scraped off, added
to physiological salt solution, and heated to 55 ° to 60° for one hour.
Living cultures were often substituted later.
The original injections were quite small, being only one or two
• Deutsche Med. Woch.. 1906, xxxii., No. 16.
396 PATHOGENIC MICRO-ORGANISMS.
moderate-sized platinum loopfuls. Each succeeding injection was
doubled in size each time, until the maximum dose of the growth on
two Petri dishes was given, when the size of the injection was not
changed. The injections were given about every eight days. Horses
give the best serum after eight months to one year. Kolle and
Wasserman injected one horse with the watery extract of recent cultures.
They also used both the intravenous and subcutaneous methods.
The Therapeutic Use op Serum. — In 1905 there was inaugu-
rated in Hartford, the use of subcutaneous injections of diphtheria
antitoxic serum in meningitis. This influenced us to prepare and trj^
the subcutaneous injection of an antimeningococcus serum. The re-
sults reported by the physicians in some twenty cases did not seem to
establish that any beneficial effects were obtained, so no further serum
was issued. Later KoUe^ and Wasserman reported somewhat favor-
able results in a number of cases from the subcutaneous injection of a
serum prepared by them. Meanwhile a serum prepared by Jochmann
was employed by the intraspinal method in a series of cases. This
method soon supplanted the subcutaneous injections.
The first successful use of an immune serum in cases of human
cerebrospinal meningitis, by the intraspinal method, should, therefore,
so far as we know, be credited to Jochmann,' and the physicians who
used his serum in the winter of 1905 and 1906. He reported a series
of cases treated by the intraspinal method before the Congress for
Internal Medicine held in Munich in April, 1906, and published his
paper on May 17th, 1906.
The serum was prepared by injecting horses with increasing doses
of meningococcus killed at about 58° C. The doses were given every
eight days, beginning with a loopful and increasing until the growth on
the surface of ascitic agar covering two Petri dishes was used. After
this dose was reached living cultures were given.
Characteristics of Serum. — The serum was shown to possess
both bactericidal and opsonic power. He reported forty cases of
cerebrospinal meningitis which had been treated, but gave details
concerning 17 patients which all occurred in one hospital and were
treated by Kromer. Five of these patients died and twelve recovered,
a mortality of 29 per cent. He directed that after lumbar puncture,
20 to 50 c.c. of fluid should be removed and then 20 c.c. of immune
serum injected. These injections should be repeated once or twice
if the fever did not abate or returned. He noticed in general a better-
ing of the headache, stiffness of neck, and mental condition. Joch-
mann showed that in animals colored fluids injected into the spinal
canal in the lumbar region passed the full length of the canal.
Although the serum prepared in different laboratories in Europe
was regularly used after Jochmann's report, it did not receive much
attention in the country until Flexner, through his important experi-
ments on infected monkeys, which demonstrated the value of the intra-
' Ibid., 1907, xxxiii., p. 1585.
" Deutsche med. Wocnenschrift, Vol. xxii.. No. 20, page 788.
MICROCOCCUS MENINGITIDIS, 397
spinal injections of the serum, aroused medical interest and paved the
way for him to try out the serum on a large scale. All cases treated
by him were subjected to most careful bacterial tests and clinical
observation. Eighteen months later, Flexner and Jobling published
their report which fully corroborated the earlier results of Jochmann.
The serum prepared at the Rockefeller Institute for Medical Research
has been sent to many places, both in this country and in Europe.
The results obtained have been of the utmost value in arriving at the
value of the intraspinal treatment. The following is abstracted from
their latest report.^
There were 712 cases of the disease in which the bacteriologic diag-
nosis was made and the serum treatment used. In the first table
the cases are subdivided according to certain age periods, and in the
second the total cases of each age period are further subdivided accord-
ing as the serum was injected in the three arbitrarily chosen periods of
duration of the disease.
Table I.
Cases op Epidemic Cerebrospinal Meningitis Treated with the
Antimeningitis Serum.
Cases Analyzed According to Age Groups.
Age, years Total No. cases Recovered
1-2 104 60
2-5 112 82
5-10 113 95
10-15 101 73
15-20 107 72
20+ 175 106
Died
% Mortality
44
42.3
30
26.7
18
15.9
28
27.7.
35
32.7
69
39.4
Total, all ages 712 488 224 31.4
The highest mortality is shown to have occurred in the first two
years of life. But, contrary to the rule, under the older forms of treat-
ment in which the mortality was 90 per cent, or over, in this series it
was 42.3 per cent. The average mortality in all the age periods
was 31.4 per cent.
Table II.
Cases of Epidemic Cerebrospinal Meningitis Treated with the
Antimeningitis Serum.
Cases Analyzed According to Day of Injection.
-
lst-3rci
A tri iv»v
1 Ml
4th-7th
14 M^aj
•
Later than 7th
Age, years
Rec.
Died
%
Rec.
Died
%
Rec.
Died %
1-2
16
1
5.8
22
10
31.2
22
33 60.
2-5
24
6
20.
40
12
23.
18
12 40.
5-10
43
8
15.6
35
6
14.6
17
4 19.
10-15
36
8
19.
23
9
28.1
14
11 40.-
15-20
25
17
40.4
25
8
24.2
22
10 31.2
20 +
36
21
36 8
34
24
41.3
36
24 40.
Totals 180 61 25.3 179 69 27.8 129 94 42.1
* Journal of the American Med. Assn., Oct. 30, 1909, Vol. liii., p. 1443.
398 PATHOGENIC MICRO-ORGANISMS.
"Table II is instructive in bringing out the importance of early in-
jections of the serum. The results in the first two years of life are es-
pecially noteworthy. The extraordinary figures given under the first
period of injection, namely, in the first three days of the disease, can
hardly be maintained. But the influence of period of injection is
shown by the rapid rise in mortality in the subsequent two periods.
The rule of the effects of early injection is preserved in the age periods
up to the period of from 15 to 20 years, when it disappears. The dis-
crepancy occurring in the two highest age periods cannot be wholly
accounted for at present. The explanations which suggest themselves
are that among older individuals there tends to be a large number of
very severe, rapidly fatal or fulminating cases of the disease, or that
older persons are less subject to the beneficial action of the serum.
As regards the actual proposition, it may be stated that adults not
infrequently respond promptly to the serum injections by abrupt
termination of the disease or ameliorated symptoms and pathologic
conditions.
" The total figures do not, however, fail to indicate that the early
injections are more effective than the later ones, as is shown by the
percentage mortality in the first-to-third-day period of 25.3, in the
fourth-to-seventh-day period of 27.8, and the period later than the
seventh day of 42. 1.*'
Our own experience and our conversations with a number of physi-
cians both here and in Europe convince us that the intraspinal in-
jections of the serum are of great advantage in the majority of cases
and should always be given. We would not advise waiting for a
bacteriological examination of the spinal fluid before giving the first
injection of the serum. Later injections should be guided by the
result of the examination if that has been possible. We feel that the
results tabulated by Flexner are a little too favorable because the neces-
sity of having a bacterial examination tended to eliminate the most
rapidly fatal cases. It is also true that the comparison between the
treated cases in which meningococci were found and the untreated
cases in which no such bacterial tests were made gives a too favorable
contrast. ' It is well known that a considerable number of cases due to
pneumococci, streptococci, and tubercle bacilli are diagnosed as cere-
brospinal meningitis. These are almost invariably fatal. These
facts, however, do not lessen our conviction of the great usefulness
of the serum, but simply reduce somewhat the degree to which we
believe the mortalitv has been reduced.
Bacteriological Diagnosis. — By means of lumbar puncture, fluid
can readily be obtained from the spinal canal without danger. The
skin must be thoroughly cleansed and the needle aseptic. The
fluid should be placed in a sterile conical glass to settle. The sedi-
ment should be used to make smears to examine (1) for pus cells,
(2) for tubercle bacilli, and (3) for other organisms. By Gram's
stain we are able to separate the three Gram-positive organisms most
frequently met with in meningitis (pneumococcus, streptococcus, and
MICROCOCCUS MENINGITIDIS. 399
staphylococcus) from the others. Of importance also is the point
that the micrococcus intracellularis is usually inside the leukocytes in
the form of diplococci of varying size, of coffee-bean shape, or of tetra-
cocci, while the pneumococcus is frequently outside the cells and is
usually spherical or lancet-shaped and frequently occurs in short chains.
Sometimes the bacteria are present in very small numbers, and then
many smears must be looked through before a probable diagnosis can
be made. In all cases absolute certainty can only be obtained through
cultures. Here plain nutrient agar, serum agar, and blood-agar plates
should be made, and, if desired, tubes also. When considerable
quantities are inoculated upon these media and meningococci are
present, as a rule, a greater or less number of colonies having the
characteristics already described will develop. The value, clinically,
of the examination is that about 50 per cent, of the cases due to this
coccus recover, while almost all of those due to the pneumococcus and
streptococcus die.
In many cases there are very few diplococci present in the spinal
fluid, so that a failure to find them in a microscopic examination
should not be taken to prove that the disease was not due to this
organism. In two cases we could find no diplococci in the fluid with-
drawn on the first day of the disease, but found them on the second
day. In 210 cases examined Elser and Huntoon^ made a positive
microscopic or cultural diagnosis in 92.4 per cent. Of 171 examined
microscopically, 141 were positive. During the first week of the
disease of 120 examined, the films were positive in 100. Of 177 ex-
amined by cultures, 152 were pxositive.
They believe that if cultures could have been made immediately
upon the withdrawal of the fluid better results could have been at-
tained. The number of organisms tend to diminish as the disease
advances. In a total of 152 consecutive isolations of Gram-nega-
tive cocci from the spinal canal they all proved on the most rigid tests
to be meningococci. A finding of Gram-negative organisms in leuko-
cytes is sufficient for a diagnosis. Some observers have considered
that gonococci occasionally excited meningitis. This is possible,
but it seems more likely that it was a mistake in identification. A
few Gram-negative cocci have been reported by different observers,
but it seems likely that there was contamination. These cocci were
usually free in the fluid. For cultures a considerable amount of fluid
must be used, for we have found, as described by Councilman and
others, that there may be very few living diplococci even in 1 c.c.
of fluid.
To obtain the fluid the patient should lie on the right side with the
knees drawn up and the left shoulder depressed. The skin of the
patient's back, the hands of the operator, and the large antitoxin
syringe should be sterile. The needle should be 4 cm. in length,
with a diameter of 1 mm. for children, and longer for adults.
The puncture is generally made between the third and fourth
* Elser and Huntoon. Ibid., p. 400
400 PATHOGENIC MICRO-ORGANISMS.
lumbar vertebrae. The thumb of the left hand is pressed between
the spinous processes, and the point of the needle is entered in the
median line or a little to the right of it, and on a level with the thumb-
nail, and directed slightly upward and inward toward the median
line. At a depth of 3 or 4 cm. in children and 7 or 8 cm. in adults
the needle enters the subarachnoid space, and on withdrawing the
obturator the fluid flows out in drops or in a stream. If the needle
meets a bony obstruction withdraw and thrust again rather than
make lateral movements. Any blood obscures the microscopic
examination. The fluid is allowed to drop into absolutely sterile
test-tubes or vials with sterile stoppers. From 5 to 15 c.c. should
be withdrawn. No ill effects have been observed from the opera-
tions. On the contrary, the relief of -pressure frequently produces
beneficial results.
Differential Diagnosis from Oonococci.— As a rule, the portion of
the body from which the organisms are obtained reveals their source.
When this is insufficient careful culture and agglutination tests are
required.
Other Organisms Exciting Meningitis.— 1. The pneumococcu^.
This diplococcus is one of the most frequent exciters of meningitis,
both as a4)rimary and a secondary infection.
2. The Streptococcus pyogenes and the Staphylococcus pyogenes.
Meningitis due to these organisms is almost always secondary to
some other infection, such as otitis, tonsillitis, erysipelas, endocarditis,
suppurating wounds of scalp and skull, etc.
3. The Bacillus inpuenzcB, Numerous reports have been pub-
lished of the presence of influenza bacilli in the meningeal exudate.
Those that are reliable state in almost every instance that the menin-
gitis is secondary to infection of the lungs, bronchi, the nasal cavities
or their accessory sinuses.
4. The coUm bacillu^Sy the typhoid bacilluSy that of bubonic plague
and of glanders, all may cause a complicating purulent meningitis.
5. In isolated cases of meningitis complicating otitis media and
other infections, other bacteria, such as the Micrococcus tetrayenvs,
the Bacillus pyocyaneus, the gonococcus, etc., may be found.
6. The tubercle bacillus. This is a very frequent cause.
MIOROOOOOUS OATARRHALIS (R. PFEIFFER).
Micrococci somewhat resembling meningococci are found in the mucous
membranes of the respiratory tract. At times they excite catarrhal inflam-
mation of the mucous membranes and pneumonia. These are at present
included under the designation of Micrococcus catarrhaHs.
Bfticroscopic Appearance. — They usually occur in pairs, sometimes in
fours; never in chains. The cocci are coffee-bean in shape and slightly larger
than the gonococcus, and are negative to Gramas stain.
The micrococci are not motile and produce no spores.
Cultivation.— They grow between 20° and 40° C, best at 37° C. and less
rapidly at somewhat lower temperatures, developing on ordinary nutrient
agar as grayish-white or yellowish-white, circular colonies of the size of
meningococci. The borders of the colonies are irregular and abrupt as though
MICROCOCCUS CATARRHALIS. . 401
gouged out. They have a mortar-like consistency. On serum-agar media the
growth is more luxuriant. Gelatin is not liquefied. Bouillon is clouded, often
with the development of a pellicle. Milk is not coagulated, but dextrose
serum media may be. Gas is not produced.
Location of Organisms. — In the secretion of normal mucous mem-
branes they are occasionally present. In certain diseased conditions of the
mucous membranes they may be abundant.
Pathogenic Effects in Animals. — For white mice, guinea-pigs, and rabbits,
some cultures are as pathogenic as meningococci, while others are less so.
Differential Points Separating them from the Meningococci. — These
organisms have undoubtedly been at times confused. Some assert that the
meningococci grow only above 25° C. Many cord cultures of meningococci
grow below this point. Some assert that the meningococci will not grow
on 5 per cent, glycerin agar. Many undoubted cultures do. Careful agglu-
tinin absorption tests are of great differential value, but can only be carried
out safely by one accustomed to them. The meningococci tested by us have
removed all the agglutinins acting upon meningococci from a specific menin-
gococcus serum while the allied organisms have removed only about sixty
per cent, of them. The probability is that the organisms described by differ-
ent writers as Micrococcus catarrhalis were not all the same variety, and some
of them were meningococci.
Vaccine Therapy. — Good reports have been made of the results of inject-
ing the dead organisms in a number of infections due to this micrococcus.
OTHER ORAM -NEGATIVE OOOOI RE8EMBLIN0 MENINOOOOOOI.
Pseudomeningococcus. — This organism cannot be diflFerentrated
from meningococci except by serum reactions.
Micrococcus pharyngis siccus (Von Lingelsheim^). — Diplococcus
mucosas. — Ghromogenic Oram-negative cocci.
» Klin. Jahrb., 1906, xv., H. 2.
26
CHAPTER XXIX.
THE GONOCOCCUS OR MICROCOCCUS GONORRHCE^ — THE
DUCREY BACILLUS OF SOFT CHANCRE-
MICROCOCCUS MELITENSIS.
The period at which gonorrhoea began to inflict man is unknown.
The earliest records make mention of it. WTierever civilized man
has penetrated gonorrhoea is prevalent among the people. Except for
a period after the fifteenth century it was generally recognized as a
communicable disease and laws were made to control its spread.
The differentiation between the lighter forms of gonorrhoea and some
other inflammations of the mucous membranes was, however, almost
impossible until the discovery of the specific microorganism by Neisser,
in 1879.
The organism was first observed in gonorrhoeal discharges, and, de-
scribed by him under the name of "gonococcus;" but though several
attempted to discover a medium upon which it might be cultivated,
it was reserved for Bumm, in 1885, to obtain it in pure culture upon
coagulated human-blood serum, and then after cultivating it for many
generations to prove its infective virulence by inoculation into man.
The researches of Neisser and Bumm established beyond doubt that
this organism is the specific cause of gonorrhoea in man. Gonorrhoea
is in almost all cases among adults transmitted through sexual inter-
course. Gonorrhoeal ophthalmia is a frequent accidental infection
at birth and vaginitis in the young child is frequently produced by the
carelessness of the nurse or mother carrying infection.
Microscopic Appearance. — Micrococci, occurring mostly in the
form of diplococci. The bodies of the diplococci are elongated, and,
as shown in stained preparations, have an unstained division or inter-
space between two flattened surfaces facing one another, which give
them their characteristic ** coffee-bean" or "kidney" shape. The
older cocci lengthen, then become constricted in their middle portion,
and finally divide, making new pairs (Fig. 127). The diameter of an
associated pair of cells varies according to their stage of development
from 0 . 8/i to 1 . G/i in the long diameter — average about 1 . 2oti — by
0 . ()/( to 0 . 8/( in the cross diameter.
Extracellular and Intracellular Position of Oonococci. — In gonor-
rhoea, during the earliest stages before the discharge becomes purulent,
the gonococci are found mostly free in the serum or plastered upon the
epithelium cells, but later almost entirely in small, irregular groups
in or upon the pus cells, and always extranuclear. With the disappear-
ance of the pus formation more free gonococci appear. Discharge
expressed from the urethra usually contains more free organisms than
the natural flow, (ionococci are sometimes irregular in shape or
402
THE GONOCOCCVS OR MICROCOCCUS GOSORRH(E.€. 403
granular in appearance, involution forms, found particularly in older
cultures and in chronic urethritis of long standing. Single pus cells
sometimes contain as many as one hundred gonococci and seem to be
almost bursting and yet show but slight signs of injury. These diplo-
cocci are also found in or upon desquamated epithelial cells. There
is still discussion as to whether the gonococci actively invade the pus
cells or only are taken up by them. There is no evidence that the
gonococci are destroyed by the pus cells (Fig. 128).
Staining. — The gonococcus stains readily with the basic aniline
colors. Loeffler's solution of methylene blue is one of the best stain-
ing agents for demonstrating its presence in pus, for, while staining
the gonococci deeply, it leaves the cell protoplasm but faintly stained.
Fuchsin is apt to overstain the cell substance. Beautiful double-
stained preparations may be made from gonorrhoeal pus by treating
cover-glass smears with methylene blue and eosin. Numerous meth-
ods for double staining have been employed, with the object of mak-
ing a few gonococci more conspicuous. None of them have any
specific characteristics such as the Gram stain. It is now established
that gonococci from fresh cultures and from recent gonorrhceal in- ^^
fections are, when properly treated by Gram's method, quickly and
surely robbeil of their color and fake on the contrast stains. Thtr"'
removal of the stain from gonococci in old flakes and threads from
chronic cases is not so certain. This difference is mostly due to the'"
fact that equally uniform specimens cannot be prepared. The de- [
colorized gonococci are stained by dipping the films for a few seconds
into a 1 : 10 dilution of carbol-fuchsin or a solution of bismarck
brown. This' staining should be for as short a time as sufiices to
stain the decolorized organisms. This method of staining cannot
be depended upon alone ab.solutely to distinguish the gonococcus
from all other diplocoeci found in the urethra and vulvo-vaginal
404 PATHOGENIC MICRO-ORGANISMS.
tract, for, especially in the female, other diplococci are occasionally
found which are also not stained by Gram's method. It serves,
however, to distinguish this micrococcus from the common pyogenic
cocci, which retain their color when treated in the same way, and in
the male urethra it is practically certain, as no organism has been
found in that location which in morphology and staining is identical
with the gonococcus. It is certainly the most distinctive character-
istic of the staining properties of the gonococcus, and it is a test that
should never be neglected in differentiating this organism from others
which are morphologically similar.
Biology. — Grows best at blood temperature; the limits being
roughly 25° and 40® C. It is a facultative anaerobe. It is not motile
and produces no spores.
Cuiture Media. — The gonococcus requires for its best growth the
addition to nutrient agar of a small percentage of blood serum or
some equivalent. The following media have proven of value:
1. Human blood from the sterilized finger streaked on common
nutrient agar.
2. Human ascitic, pleuritic, or cystic fluid, 1 part added to and mixed
with 2 parts melted 5 per cent, glycerin nutrient, 1.5 per cent, agar
having a temperature of 55° to 60° C. The whole after mixing being
poured into a Petri dish or cooled slanted in a tube. "^The same pro-
portions of nutrient broth and ascitic fluid make a suitable fluid
medium. One per cent, glucose may be added.
3. Swine serum nutrose media. Wassermann strongly recom-
mends this mixture. In our hands it has given good results.
4. Nutrient or 5 per cent, glycerin agar. When considerable pus
is streaked on simple agar media a good growth of gonococci is usually
obtained. After continued cultivation gonococci cultures frequently
grow on media containing no serum. Some strains grow on ordinary
glycerin or glucose nutrient agar and even on plain nutrient agar
IFrom the start.
Viability. — Cultures frequently die in forty-eight to seventy-two
hours when kept at room temperature. In the ice-box they may live
for several weeks. They frequently live for one week in the ther-
mostat at 36° C. on plain nutrient agar.
Appearance of Colonies. — A delicate growth is characteristic. At
the end of twenty-four hours there will have developed translucent,
very finely granular colonies, with scalloped margin. The margin
is sometimes scarcely to be differentiated from the culture me-
diura. In color they are grayish-white, with a tinge of yellaw.
The texture is finely granular at the periphery, presenting punctated
spots of higher refraction in and around the centre of yellowish color
(Fig. 129).
Surface Streak Culture. — Translucent grayish-white growth, with
rather thick edges.
Resistance. — The gonococcus has but little resistant power against
THE GOS'OCOCCUS OR MICROCOCCUS GOSORRH(E£. 405
outside influences. It is killed by weak disinfecting solutions and
by desiccation in thin layers. In comparatively thick layers, how-
ever, as when gonorrhoeal pus is smeared on linen, it has lived for
forty-nine days, and dried on glass for twenty-nine days (Heiman).
It is killed at a temperature of 45'^ C. in six hours and of 60° in about
thirty minutes.
Occturence of Oonococci. — Outside of the human body or material
carried from it gonococci have not been found.
Toxins. — In the gonococcus cells substances are present which are
toxic after heating and contact with alcohol. Injected in considerable
amounts into rabbits, they cause p^^ i^^
infiltration and often necrosis. Ap-
plied to the urethral mucous mem-
brane there is produced an in-
flammation of short duration. In
gonorrhcea the secretion is believed
to be due to these intracellular
toxins. Repeated injections give
only slight immunity. The filtrate
of recent gonococcus cultures con-
tains little toxin.
Pathogenesis. — Non-transmissi-
ble to all animals. Both the living
and dead gonococci contain toxic
substances which cause death or
. . ■ ' ■ . J ■ 1 Calonlfs o[ gonococci on pleuritic fluid aaar.
mjury when mjected in large (Hein»n.)
quantities.
The etiological relation of the gonococcus to human gonorrhoea has
been demonstrated beyond question by the infection of a number of
healthy men with the disease by the inoculation of pure cultures of
the microorganism.
Disease Conditions Excited by Oonococci.— Affections due to this
organism are usually restricted to the mucous membranes of the
urethra, prostate, neck of bladder, cervix uteri, vagina, and con-
junctiva. The conjunctival, vaginal, and rectal mucous membranes
are much more sensitive in early childhood than in later life. The
usual course of the inflammation is as follows: Thegonococci first
increase upon the mucous membranes which show congestion, infil-
tration with serous exudate and accumulation of leukocytes. The
cocci then penetrate the epithelial layer down to the submucous con-
nective tissue. Recovery or a prolonged chronic inflammation may
then persist. The original infection of the urethra or vagina and
cervix may remain localized or spread to a<ljacent parts or through
blood and lymph he carried to all parts of the body. Gonococci
thus cause many cases of endometritis, metritis, salpingites, oopho-
ritis, peritonitis, prastitis, cy.stitis, epididymitis, and arthritis. Ab-
scesses of considerable size, periostitis, and otitis are occasionally due
to the gonococcus.
406 PATHOGENIC MICRO-ORGANISMS.
Endocarditis and Septicsemia. — Cases of gonococcus endocarditis
and septicaemia are not infrequent. Gonococcus septicaemia may
occur in connection with other localizations or alone. Nearly every
year one or two of these cases are met with in every general hospital.
In a considerable number of cases where gonococci are obtained from
the blood the patients recover. The fever is sometimes typhoid-like
in character.
Complications. — General infections with gonococci are often fol-
lowed or accompanied by neuralgic affections, muscle atrophies, and
neuritis. Urticaria occasionally occurs.
Immunity. — Immunity in man after recovery from infection
seems to be only slight in amount and for a short period if present
at all. It is known that the urethra in man or cervix uteri in woman
may contain gonococci which lie dormant and may be innocuous
in that person for years, but which may at any time excite an acute
gonorrhoea in another individual or, under stimulating conditions,
in the one carrying the infections. Animals may, however, be im-
munized, and their blood is both bactericidal and slightly antitoxic.
Therapeutic Use of Serum and Vaccine.— The use of sera in acute
gonorrhoeal joint inflammation has given in a considerable percentage
of cases good results and seems to be worth trying. It seems to be
useless in acute gonorrhoea of the mucous membranes. Vaccines
(heated cultures) have also been used with apparently real benefit
in joint inflammations and even in very localized chronic infections
of the urethra, bladder, and elsewhere. They have also been used in
acute vaginitis in young children. In our cases the symptoms abated
sooner than we expected, but the gonococci persisted. The dose
is from twenty to a thousand millions given every three to seven days.
The benefit of serum and vaccine in septicaemia is doubtful.
Agglutination. — Torrey has shown that gonococci resemble pneu-
mococci in that there are a number of different strains which have
different specific and but little common agglutinins. The agglutina-
tion test is of no practical value in diagnosis.
Duration of Infections and of Contagious Period. — There is no
limit to the time during which a man or woman may remain infected
with gonococci and infect others. We have had one case under obser-
vation where twenty years had elapsed since exposure to infection,
and yet the gonococci were still abundant. It is now well established
that most of the inflammations of the female genital tract are due
to gonococci, and the majority of such infections are produced in
innocent women by their husbands who are suffering from latent
gonorrhoea.
Bacteriological Diagnosis of Gonorrhoea. — In view of the fact
that several non-gonorrhoeal forms of urethritis occasionally exist,
and also that micrococci morphologically similar to the gonococcus
Neisser are at times found in the normal vulvo-vaginal tract of adults
it becomes a matter of importance to be able to detect gonococci
when present, and to differentiate these from the non-specific or-
THE GONOCOCCUS OR MICROCOCCUS GONORRHCE^. 407
ganisms. Besides this, the gonococci which occur in old cultures
and in chronic urethritis of long standing sometimes take on a very
diversified appearance. From a medicolegal and social standpoint,
therefore, the differential diagnosis of the gonococcus has in certain
cases a very practical significance.
There are two methods of differential diagnosis now available —
the microscopic and the cultural. Animal inoculations are of no
value, as animals are not susceptible, and, of course, human inocu-
lations are generally impossible. In the microscopic diagnosis it
should be born in mind that after the acute serous stage has passed,
the specific gonococci in carefully made preparations are always found
largely within the pus cells. Diplococci morphologically similar
to gonococci occuring in other portions of the field and outside of
the pus cells should not be considered specific by the test only. It
should also be remembered that the gonococci are decolorized by
Gram's method, while other similar micrococci which occur in the
urethra are, as a rule at least, not so decolorized. Organisms having
these characteristics can for all practical purposes be considered as
certainly gonococci if obtained from the urethra. From the vulvo-
vaginal tract the certainty is not so great, since other diplococci are
occasionally found in gonorrhoeal pus from this area, and very rarely,
also, from the urethra, which stain as gonococci; here cultures should
also be made.
Cover-glass preparations from subacute or chronic cases should
be examined, if possible, with a microscope provided with a mechanical
stage, and films should always be stained by both Loeffler's methylene-
blue solution and by Gram's method, and the examination repeated on
three consecutive days. Should these specimens prove negative, to
exclude any possible doubt in the matter, cultures should then be
made, if a thoroughly competent bacteriologist is available, on human
ascitic fluid or serum agar, poured in dishes; also, if with negative
results, on three consecutive days. Heiman, who has paid much atten-
tion to gonococcus examinations, obtains his material by the following
method: in chronic urethritis he allows the patient to void his urine
either immediately into two sterilized centrifugal tubes or first into
two sterile bottles. The first tube will contain threads of the an-
terior urethra; the second tube will be likely to contain secretion from
the posterior urethra and from the prostate gland if, while urinating,
the patient's prostate be pressed upon with the finger. Tubes contain-
ing such urine are placed in the centrifuge and whirled for three minutes
at twelve hundred or more revolutions per minute; the threads are
thrown down. The centrifuged sediment will be found to contain
most of the baccteria present, epithelial cells, and, at times, spermatozoa.
Normal urine on being centrifuged at this velocity will be found at
times to show a slight turbidity at the bottom of the tube. This will
be found, on microscopic examination, to consist of epithelial cells, a
few leukocytes, and some bacteria.
The careful examination of gonorrhoeal threads stained by Gram's
408 PATHOGENIC MICRO-ORGANISMS,
method is a very tedious affair, as in every instance no less than three
cover-glass preparations should be looked over before the absence
of the gonococcus is considered probable. It would require many
hours upon each and every specimen, especially if the gonococci
are present in very small number, before a reliable and conscientious
opinion could be rendered. If, after all, a negative opinion is ven-
tured, we still are under the necessity of proving that because the
threads which we fished out for the cover-glass examination were free
from gonococci the remaining ones were also. For this reason the
culture medium is more sensitive for bacteria than is the cover-glass,
for we are able to plant each and every thread of the sediment in the
centrifugal tube. Results on culture media are only reliable when
obtained by thoroughly trained bacteriologists with suitable media and
methods. Fiirbringer, in his work, mentions the fact that in certain
cases the absence of the gonococcus in many examinations of cover-
glass preparations is not a positive proof that the gonococcus is not
present. The culture methods, of course, presuppose that one has
the facilities and knowledge to carry them out successfully, otherwise
the microscopic methods are to be used alone.
When the examinations are negative and it is important to be certain,
either massage or injections of a solution of silver nitrate may be em-
ployed. The latter by causing a temporary irritation with increase
of secretion will almost surely causea discharge of gonococci if any
infection was present.
In acute cases where the pus is abundant the specimen for exami-
nation may be collected, when the patient is before one, by passing
a sterilized platinum-wire loop as far up into the urethra as possible
and withdrawing some of the secretion.
Occurrence in Cultures from Chronic Urethritis. — Goll examined
1046 cases of chronic urethritis varying in duration between four
weeks to six years or more, finding gonococci in 178 cases, the remainder
giving negative results. Neisser, out of 143 cases, varying in duration
between two months and eight years, found gonococci in 80 cases.
BACTERIA RESEMBLINO OONOGOGGI.
Baumm described a number of micrococci which resembled gono-
cocci in form and staining. These assume importance largely be-
cause they may be confused with the gonococcus. They occur on
the conjunctival and vaginal mucous membranes and cause confusion.
One of these microorganisms, the Micrococcus catarrhalis (see p. 400),
has an importance of its own. When absolute certainty is demanded
cultural tests must be applied.
MALTA FEVER.
The Micrococcus Melitensis. — This microorganism was first dis-
covered in the spleen in a case of Malta fever by Bruce in Malta in
1887. The disease is mostlv confined to the shores of the Mediter-
ranean, but cases of it have been observed in Porto Rico, China, Japan,
MICROCOCCUS MELITENSIS. 409
and the Philippines. The disease does not seem to be directly trans-
mitted from person to person.
Olinical Symptoms. — Prodromal symptoms follow an incubation
period of five to fourteen days. Headache, sleeplessness, loss of
appetite, or vomiting accompany a high fever. The fever lasts for
weeks, with intermissions and remissions. The fever periods of one
to three weeks may occur from time to time during a period of many
months. The spleen and liver are enlarged. Neuralgic pains are
severe. The fatal cases appear similar to severe cases of typhoid
fever.
Autopsy. — The spleen is large and very soft. The liver is also
large and congested. Both organs show parenchymatous degener-
ation.
Distribution of Bficrococd. — These are abundant in the blood and
all organs.
Morphology and Biology. — ^Very small rounded or slightly oval
organisms, about 0.30/i in their greatest diameter. It is usually single
or in pairs. In old cultures involution almost bacillary forms occur.
They are not motile.
Staining. — Thev stain readily with aniline dves and are negative^
to Gram. ' ^ . ! * ^ '* ~
Oultivation. — At 37° C. they grow rather feebly on nutrient agar
and in broth. The colonies are not usually visible until the third C' ^
day. They appear as small round disks, slightly raised, with a yel- .* ' ' *
lowish tint in the centre. The broth is slightly clouded after four 'I ,, 4
to six days. The culture remains aHve for several weeks or months.
In gelatin the growth is very slow. Gelatin is not liquefied.
Pathogenesis in Animals. — Monkeys only are infected. They U^^ ^
pass through the disease much like man. They can be infected by / ' ^''^<,
subcutaneous or mucous inoculation. In Malta it has been found
that about half of the goats pass the organisms in fseces and so contami-
nale^tligiL"!^^' This is believed to be a source of infection. By safe- ^'
guarSmgme^milk the disease has been largely eliminated.
Therapeutic Results. — Injections of heated cultures have been
thought to give good results.
Methods of Diagnosis. — The diagnosis of Malta fever can frequently
only be made with the help of bacteriological examination ..^MaJaigc,
typhoid fever, and sepsis are the three diseases most apt to be con-
founded with^t. "'
CM\t\)Tf^R itrp mfldp hy ^^prcgdlTiL PXer Jthc . surface j)f a number of
a^ar plates freshlv drawn blood. JFrequently no organisnaS-ilfi-yelop.
The agglutination test, is then required. Many bloods of persons
suffering from other infections agglutinate the micrococcus of M<a
fever_iILJow~dilutions so tliat 1:500 or over is required for. a positive
diagnosis.
Animals injected with the coccus produce a serum agglutinating in
high dilutions. Under suitable precautions this can be used to identify
suspected cultures.
♦ -«fc
^
410 PATHOGENIC MICRO-ORGANISMS.
Laboratory Infection. — A number of workers have infected them-
selves with more or less serious results.
MIGROGOGGUS ZYMOGENS.
MacCallum and Hastings^ observed this micrococcus in a case of
acute endocarditis. It has since been found in a few other pathologic
processes. It occurs in pairs and short chains. It grows well on agar,
ferments lactose and glucose, and slowly liquefies gelatin.
THE BAGILLUS OF SOFT GHANGRE.
This bacillus was first specifically described and obtained in pure
culture by Ducrey in 1889. An experimental inoculation is followed
in one to two davs bv a^sinall pustule. This ^oon ruptures and a_
small round depressed ulcer is left. About this other pustujes and
ulcers develop which tend to become confluent. The base of the ulcer
IS covered with a gray exudate and its edges Are undermined. TJiere
is no induration such as in the syphilitic chancre. The secretion is
seropurulent and very infectious.
Morphology. — About 1.5// long and 0.4/i thick, growing often in
chains and in cultures, sometimes twisted together in dense masses.
It stains best wilk carbol-fuchsin. and shows po^y s^aiping.
Cultural Characteristics.— The following" method of cultivation
has given the best results: Two parts agar are liquefied at 5CP C.
and mixed with one part human, dog, or rabbit blood. The blood
from the cut carotid of a rabbit may be allowed to run directly into
the agar tube, to which the pus from the ulcerated bubo is then added
in proper proportion, and the whole placed in the incubator at 35°
C. The pus may be obtained by puncture and aspiration from the
unbroken ulcer, or if the ulcer is already open it is first painted with
tincture of iodine and covered with collodion or sterile gauze. After
twenty-four to forty-eight hours, some pus having collected under the
bandage, inoculations are made from it. The bacillus grows well
also in uncoagulated rabbit-blood serum or in condensation water of
blood agar. In twenty-four to forty-eight hours, on the surface
of the media, well-developed, shining, grayish colonies, about 1 mm.
in diameter, may be observed. The colonies remain separate, but
only become numerous after further transplantation. The best re-
sults are obtained when the pus is taken close to the walls of the abscess.
Glass smears show isolated bacilli or short parallel chains ^ith
distinct polar staining.
After the eleventh generation of the culture, and from all old cul-
tures, on inoculation the characteristic soft chancre is produced in
man. Animals in general cannot be infected, but positive results have
been obtained with monkeys and cats.
The organisms are especially characteristic in the water of con-
' Jour. Exp. Med., 1899, iv., p. 521.
THE BACILLUS OF SOFT CHANCRE. 411
densation from blood agar, the bacilli being thinner and shorter,
with rounded ends; sometimes long, wavy chains are found. In
rabbit-blood serum at 37® C. a slight clouding of the medium is pro-
duced and small flakes are formed, consisting of short bacilli or mod-
erately long, curved chains, showing polar staining.
The bacillus lives several weeks on blood agar at 37® C, but it
soon dies in cultures on coagulated serum. All other ordinary cul-
ture media so far tried have given negative results, and even with the
media described development is difficult and often fails entirely.
The chancre bacillus possesses but little resistance to deleterious
outside influences. Hence, the various antiseptic bandages, etc.,
used in treatment of the affection soon bring about recovery by pre-
venting the spread of inoculation chancre.
CHAPTER XXX.
BACILLUS PYOCYANEUS (BACILLUS OF GREEN AND OF BLUE
PUS)— BACILLUS PROTEUS (VULGARIS).
BACILLUS PTOOTANEUS.
The blue and green coloration whicli is occasionally found to ac-
company the purulent discharges from open wounds is usually due
to the action of the BacUlua pyocyaneus. According to recent in-
vestigations, this bacillus appears to be very widely distributed and
not infrequently the cause of infection. It was first obtained in
pure culture and its significance noted by Gessard.
Morphology. — Slender rods from 0.3^ to l/i broad and from 2u
to &fi long; frequently united in pairs or in chains of four to six ele-
ments; occasionally growing out into long filaments and twisted
spirals. The bacillus is actively motile, a single flagellum being at-
tached to one end. Does not form
spores. Stains with the ordinary*
aniline colors; does not stain with
Gram's solution.
Biology. — Aerobic, liquef jing,
motile bacillus. Capable also of
an anaerobic existence, but then
produces no pigment. Grows
readily on all artificial culture
media at the room temperature,
though best at 37° C, and gives
to some of them a bright green
color in the presence of oxygen.
In gelatin-ptale cultures the col-
onies are rapidly developed, im-
BaciiiiH pyoc^^u^^^Frnm KoUb ftnci parting to the medium a fluores-
cent green color; liquefaction
begins at the end of two or three days, and by the fifth day the
gelatin is usually all liquefied. The deep colonies, before lique-
faction sets in, appear as round, granular masses with scalloped
margins, having a yellowish-green color; the surface colonies have
a darker green centre, surrounded by a delicate, radiating zone.
In slick cultures in gelatin liquefaction occurs at first near the surface,
in the form of a small funnel, and gradually extends downward;
later the liquefied gelatin is separated from the solid part of the medium
by a horizontal plane, a greenish-yellow color being imparted to
BACILLUS PYOCYANEUS, 413
that portion which is in contact with the air. On agar a wrinkled,
moist, greenish-white layer is developed, while the surrounding medium
is bright green; this subsequently becomes darker in color, changing to
blue-green or almost black. In bouillon the green color is produced,
and the growth appears as a delicate, flocculent sediment. Milk
is coagulated and assumes a yellowish-green color.
Pigment. — Two pigments are produced by this bacillus — one of a
fluorescent CTeeo. which is common to many bacteria. This is sol-
t^ie m :!'wat|'r put not in chloroform. The othpr (pynryaninl afir
blue coTqi; is soluble jn chloroform, and may be obtained from pure
solution in long, blue needles. 1 his pigment distinguishes the Bacilr
l^La jyii^jff^*t^io fy^ny nthpF fluor^^^ng bactefiaT
Ferment. — Besides the ferment causing liquefaction of gelatin
there is one which acts on albumin. It resists heat. This ferment
called pyocyanase has the power to dissolve bacteria, and it has been
stated to have some protective power when injected into animals. It
has been used locally in diphtheria in a number of cases. We do not
think it has any advantage over the cleansing preparations.
Distribution. — This bacillus is very widely distributed in nature;
it is found on the healthy skin of man, in the faeces of many animals,
in water contaminated by animal or human material, in purulent
discharges, and in serous wound secretions.
Pathogenesis. — Its pathogenic effects on animals have been care-
fully studied. It is pathogenic for guinea-pigs and rabbits. Sub-
cutaneous or intraperitoneal injections of 1 c.c. or more of a bouillon
culture usually cause the death of the animal in from twenty-four
to thirty-six hours. Subcutaneous inoculations produce an extensive
inflammatory oedema and purulent infiltration of the tissues; a sero-
fibrinous or purulent peritonitis is induced by the introduction of the
bacillus into the peritoneal cavity. The bacilli multiply in the body,
and may be found in the serous or purulent fluid in the subcutaneous
tissues or abdominal cavity, as well as in the blood and various organs.
When smaller quantities are injected subcutaneously the animal
usually recovers, only a local inflammatory reaction being set up
(abscess), and it is subsequently immune against a second inoculation
with doses which would prove fatal to an unprotected animal. It
is interesting to note that Bouchard, Charrin, and Guignard have
shown that in rabbits which have been inoculated with a culture of
the bacillus anthracis a fatal result may be prevented by inoculating
the same animal soon after with a pure culture of the bacillus pyo-
cyaneus. I^oew and Emmerich have shown that the enzymes produced
in the pyocyaneus cultures are capable of destroying many forms
of bacteria in the test-tube, and have a slight protecting value in the
body. The pyocyaneus bacillus produces these effects not only
through ferments, but by intracellular toxins.
Our knowledge of the pathogenic importance of the Bacillus pyo-
cyaneus in human diseases has been much increased by recent inves-
tigations. Its presence in wounds greatly delays the process of re-
414 PATHOGENIC MICRO-ORGANISMS.
pair, and may give rise to a general depression of the vital powers
from the absorption of its toxic products. This bacillus has been
obtained in pure culture from pus derived from the tympanic cavity
in disease of the middle ear, from cases of ophthalmia, and broncho-
pneumonia. Kruse and Pasquale have found the organism in three
cases of idiopathic abscess of the liver, in two of them in immense
numbers and in pure culture. Ernst and Schiirmayer report the
presence of the bacillus pyocyaneous in serous inflammations of the
pericardial sac and of the knee-joint. Ehlers gives the history of a
disease in two sisters who were attacked simultaneously with fever,
albuminuria, and paralysis. It was thought that they would prove
to be typhoid fever or meningitis, but on the twelfth day there was
an eruption of blisters, from the contents of which the bacillus pyo-
cyaneus was isolated. Krambals refers to seven cases in which a
general pyocyaneus infection occurred, and adds an eighth from his
own experience. In this the bacillus pyocyaneus was obtained post-
mortem from green pus in the pleural cavity, from serum in the
pericardial sac, and from the spleen in pure culture. Schimmel-
busch states that a physician injected 0.5 c.c. of sterilized (by heat)
culture into his forearm. As a result of this injection, after a few
hours he had a slight chill, followed by fever, which at the end of
twelve hours reached 38.8° C; an erysipelatous-like swelling of the
forearm occurred, and the glands in the axilla were swollen and
painful. Wassermann reports an epidemic of septic infection of the
newborn, starting in the umbilicus. In all there were eleven deaths.
Lartigau found it in well-water, and in great abundance in the intesti-
nal discharges of a number of cases made ill by drinking the water.
It has also been found in a certain number of cases of gastroenteritis
where no special cause of infection could be noted.
We may therefore conclude from these facts that the BaciUus pyo-
cyaneuSj although ordinarily but slightly pathogenic for man, may
under certain conditions, as in general debility, become a danger-
ous source of infection. Children would seem to be particularly
susceptible.
The differential diagnosis of the pyocyaneus from other fluorescing
bacteria is easy enough as long as it retains its pigment-producing
property. When an agar culture is agitated with chloroform a blue
coloration demonstrates the presence of this bacillus. When the
pyocyanin is no longer formed, however, the diagnosis is by no means
easy, particularly when the pathogenic properties are also gone.
Immunity. — Animal infection is followed by the production of
antitoxic and bactericidal substances. No practical use has been
made of this knowledge.
BACILLUS PROTEUS (VULGARIS).
This bacillus, which is one of the most common and widely dis-
tributed putrefactive bacteria, was discovered by Hauser (1885)
along with other species of proteus in putrefying substances. These
BACILLUS PROTEUS. 415
bacteria were formerly included under the name ^'Bacterium iermo"
by previous observers, who applied this name to any minute motile
bacilli found in putrefying infusions.
Morphology. — Bacilli varying greatly in size; most commonly
occurring 0 . 6/£ broad and 1 . 2/£ long, but shorter and longer forms
may also be seen, even growing out into flexible filaments which are
sometimes more or less wavy or twisted like braids of hair.
The bacillus does not form spores, and stains readily with fuchsin
or gentian violet.
Biology. — An aerobic, facultative anaerobic, liquefying, motile
bacillus. Grows rapidly in the usual culture media at the room
temperature.
Growth on Oelatin. — The growth upon gelatin plates containing 5
per cent, of gelatin is very characteristic. At the end of ten or twelve
hours at room temperature small, round depressions in the gelatin
are observed, which contain liquefied gelatin and a whitish mass
consisting of bacilli in the centre. Under a low-power lens these de-
pressions are seen to be surrounded by a radiating zone composed
of two or more layers, outside of which is a zone of a single layer,
from which amoeba-like processes extend upon the surface of the gelatin.
These processes are constantly undergoing changes in their form and
position. The young colonies deep down in the gelatin are somewhat
more compact, and rounded or humpbacked; later they are covered
with soft down; then they form irregular, radiating masses, and simu-
late the superficial colonies. But it is difficult to describe all the forms
which the proteus vulgaris takes on in all the stages of its growth on
gelatin plates. When the consistency of the medium is more solid,
as in 10 per cent, gelatin the liquefaction and migration of surface
colonies are more or less retarded. In gelatin-stick cultures the growth
is less characteristic — liquefaction takes place rapidly along the line
of puncture, and soon the entire contents of the tube are liquefied.
Upon NtUrient agar a rapidly spreading, moist, thin, grayish-white
layer appears, and migration of the colonies also occurs. Milk is
coagulated, with the production of acid.
Cultures in media containing albumin or gelatin have a disagree-
' able, putrefactive odor, and become alkaline in reaction. Growth
is most luxuriant at a temperature of 24° C, but is plentiful also at
37® C. It is a facultative anaerobe and grows also in the absence of
oxygen, but the proteus then loses its power of liquefying gelatin.
It produces indol and phenol from peptone solutions. The proteus
develops fairly well in urine, and decomposes urea into carbonate of
ammonia.
Pathogenesis. — This bacillus is pathogenic for rabbits and guinea-
pigs when injected in large quantities into the circulation, the ab-
dominal cavity, or subcutaneously, producing death of the animals
with symptoms of poisoning. Hauser has obtained the Bacillus
jyroteus {vulgaris) from a case of purulent peritonitis, from purulent
puerperal endometritis, and from a phlegmonous inflammation of
416 PATHOGENIC MICRO-ORGANISMS.
the hand. Brunner also reports similar infections in which this
organism was found associated with pus cocci, and Charrin describes
a case of pleuritis during pregnancy, in which the proteus was present
, and a foul-smelling secretion was produced. Death in this case,
which ensued without further complication, is said to have been due
probably to the poisonous products of the proteus.
An interesting example of 'pure toxsemia resulting from the toxin
of the proteus is reported by Levy: While conducting some experi-
ments on this organism he had an opportunity of making a bacterio-
logical examination in the case of a man who died after a short attack
of cholera morbus. From the vomited material and the stools he
obtained a pure culture of the proteus; but the blood, collected at
the autopsy, was sterile. In the meantime seventeen other persons
who had eaten at the same restaurant were taken sick in the same
way. Upon examination at the restaurant it was found that the
bottom of the ice-chest in which the meat was kept was covered with
a slimy, brown layer, which gave off a disagreeable odor. Cultures
from this gave the proteus as the principal organism present. In-
jections into animals of the pure cultures produced similar symp-
toms as occurred in the human subjects.
Levy concludes that in so-called "flesh poisoning" bacteria of
this group are chiefly concerned, and the pathogenic effects are due
to toxic products evolved during their development.
Booker, from his extended researches into this subject, concludes
that the proteus plays an important part in the production of the
morbid symptoms which characterize cholera infantum. Proteus
vulgaris was found in the alvine discharge in a large proportion of
the cases examined by him, but was not found in the faeces of healthy
infants. "The prominent symptoms in the cases of cholera infantum
in which the proteus bacteria were found were drowsiness, stupor,
and great reduction in flesh, more or less collapse, frequent vomiting
and purging, with watery and generally offensive stools."
Next to the Bacillus coli communis the Proteus vulgaris appears
to be the microorganism most frequently concerned in the etiology
of pyelonephritis. In cases of cystitis and of pyelonephritis this
bacillus is often found in pure cultures or associated with other bac-
teria. It probably gets into the bladder chiefly through catheteri-
zation. From the animal experiments of the authors above men-
tioned, simple injection of pure cultures of proteus into the bladder,
without artificial suppression of urine, invariably produces severe
cystitis. The fact that this organism grows in urine is sufficient to
account for the extension of the purulent process finally to the kidneys.
The Proteu^s vulgaris is usually a harmless parasite when located
in the mucous membrane of the nasal cavities. Here it only decom-
poses the secretions, with the production of a putrefactive odor. It is
found occasionally in the discharge from cases of otitis media in con-
nection with other bacteria.
CHAPTER XXXI.
GLANDERS BACILLUS (BACILLUS MALLEI).
This bacillus was discovered and proved to be the cause of gland-
ers, by isolation in pure culture and inoculation into animals, by
several bacteriologists almost at the same time (1882). The bacilli
were first obtained in impure cultures by Bouchard, Capitan, and
Charrin, and first accurately studied in pure culture by Loeffler and
Schtitz. They are present in the recent nodules in animals alTected
with glanders, and in the discharge from the nostrils, pus from the
specific ulcers, etc., and occasionally in the blood.
Morphology. — Small bacilli with rounded or pointed ends, from
nutrient agar cultures, 0.2ofi to 0.5/i broad and from 1 .5/i to 5/t long;
Pm I3J usually single, but sometimes united
in pairs, or growing out to long
filaments, especially in potato
cultures. The bacilli frequently
break up into short, almost coccus-
hke elements (Fig. 131).
Stainmg.— The bacillus mallei
ataiTtj with difficulty with the ani-
line colors, best when the aqueous
solutions of these dyes are made
feebly alkaline; it is decolorized by
Gram's method. This bacillus
presents the peculiarity of losing
very quickly in decolorizing solu-
tions the color imparted to it by
,., , . .„, . ,. the anilinestainingsolutions. For
tjlamle™ bmilL. Agar rulture. . , . ",._„ ,
X 1 100 diBBietera. this reasuu It IS difncult to stam
in sections. Ixjeffler recommends
his alkaline methylene-blue solution for staining .sections, and for
decolorizing, a mixture containing 10 c.c. of distilled water, 2 drops
of strong sulphuric acid, and 1 drop of a 5 per cent, solution of oxalic
acid; thin sections to be left in this acid solution for five seconds.
Biology. — An aerobic, non-motile bacillus, whose molecular move-
ments are so active that they have often been taken for motility. It
grows on various culture media at 37° C. Development takesplace
.slowly at 22° C. and ceases at 43° C. The bacillus does not form
spores. Exposure for ten minutes to a temperature of 5^° C, or for
five minutes to a 3 to 3 per cent, solution of carbolic acid, or for two
minutes to a 1 : 5000 solution of mercuric chloride, destroys its vitalitv.
As a rule, the bacilli do not grow after having been preserved in a desic-
J7 417
418 PA T HOG E NIC MICRO-ORGA NISMS.
cated condition for a week or two: in distilled water thev are also
quickly destroyed. It is doubtful whether the glanders bacillus findb
conditions in nature favorable to a saprophytic existence.
Cultivation. — (For obtaining pure cultures see page 420.) — It
grows well in the incubating oven on glycerin agar. Upon this meilium
at the end of twenty-four to forty-eight hours, whitish, transparent
colonies are developed, which in six or seven days may attain a diameter
of 7 or 8 mm. On blood serum a moist, opaque, slimy layer develops,
which is of a yellowish-brown tinge. The growth on cooked potato
is especially characteristic. At the end of twenty-four to thirty-six
hours at 37° C. a moist, yellow, transparent layer develops; this later
becomes deeper in color, and finally takes on a reddish-bro^^Ti color,
while the potato about it acquires a greenish-yellow tint. In bouillon
the bacillus causes diffuse clouding, ultimately with the formation of
a more or less ropy, tenacious sediment. It grows on media possessing
a slightly acid reaction, and both with and without oxygen. Milk is
coagulated with the production of acid.
Pathogenesis. — The bacillus of glanders is pathogenic for a num-
ber of animals. Among those which are most susceptible are horsej^.
asses, guinea-pigs, cats, dogs, ferrets, moles, and field mice; sheep,
goats, swine, rabbits, white mice, and house mice are much less sus-
ceptible; cattle are immune. Man is susceptible, and infection not
infrequently terminates fatally.
When pure cultures of Bacillus mallei are injected into horses or
other susceptible animals true glanders is produced. The disease is
characterized in the horse by the formation of ulcers upon the nasal
mucous membrane, which have irregular, thickened margins, and
secrete a thin, virulent mucous; the submaxillary lymphatic glands
become enlarged and form a tumor which is often lobulated; other
lymphatic glands become inflamed, and some of them suppurate and
open externally, leaving deep, open ulcers; the lungs are also involved,
and the breathing becomes rapid and irregular. In farcy, which is a
more chronic form of the disease, circumscribed swelling^, varying in
size from a pea to a hazel-nut, appear on different parts of the body,
especially where the skin is thinnest ; these suppurate and leave angry-
looking ulcers with ragged edges, from which there is an abun<iant
purulent discharge. The bacillus of glanders can easily be obtained
in pure cultures from the interior of suppurating nodules and glands
which have not yet opened to the surface, and the same material will
give successful results when inoculated into susceptible animals. The
discharge from the nostrils or from an open ulcer may contain com-
paratively few bacilli, and these being associated with other bacteria
which grow more readily on the culture media than the bacillus mallei,
make it difficult to obtain pure cultures from such material by the plate
method. In that case, however, guinea-pig inoculations are useful.
Of test animals guinea-pigs and field mice are the most susceptible.
In guinea-pigs subcutaneous injections are followed in four or five
days by swelling at the point of inoculation, and a tumor with ca>e-
GLANDERS BACILLUS. 419
ous contents soon develops; then ulceration of the skin takes place,
and a chronic purulent ulcer is formed. The essential lesion is the
granulomatous tumor, characterized by the presence of numerous
lymphoid and epithelioid cells, among and in which are seen the
glanders bacilli. The lymphatic glands become inflamed and gen-
eral symptoms of infection are developed in from two to four weeks;
the glands suppurate, and in males the testicles are involved; finally
purulent inflammation of the joints occur, and death ensues from
exhaustion. The formation of the specific ulcers upon the nasal
mucous membrane, which characterizes the disease in the horse, is
rarely seen when guinea-pigs are inoculated. In these the process
is often prolonged or remains localized on the skin. They succumb
more rapidly to intraperitoneal injection, usually in from eight to ten
days, and in males the testicles are invariably affected.
Mode of Spread. — Glanders occurs as a natural infection only in
horses and asses; the disease is occasionally communicated to man by
contact with affected animals, usually bv inoculation on an abraded sur-
face of the skin. The contagion may also be received on the mucous
membrane. Infection has sometimes been produced in bacteriological
laboratories. In man, as in horses, an acute and chronic form of
glanders may usually be recognized. The disease in human beings is
fatal in about 60 per cent, of the cases. It is transmissible also from
man to man. Washerwomen have been infected from the clothes of
a patient. The infective material exists in the secretions of the nose,
in the pus of glanders nodules, and frequently in the blood; it may
occasionally be found in the secretions of glands not yet affected, as in
the urine, milk, and saliva, and also in the foetus of diseased animals
(Bonome). From recent observations it appears that glanders is by
no means an uncommon disease among horses, particularly in southern
countries, sometimes taking a mild course and remaining latent for a
considerable time. Horses apparently healthy, therefore, may possibly
spread the disease.
Attenuation of virulence occurs in cultures which have been kept
for some time, and inoculations with such cultures may give a nega-
tive result, or, when considerable quantities are injected, may pro-
duce a fatal result at a later date than is usual when small amounts
of a recent culture are injected.
Immanity. — Attempts have been made to produce artificial immu-
nity against glanders, but so far with unsatisfactory results. Ac-
cording to Strauss, by intravenous inoculations of small quantities
of living bacilli, dogs may be protected against an injection of quanti-
ties which usually kill them. Fenger has found that animals inoculated
with glanders bacilli react less powerfully to fresh injections; and that
rabbits which have recovered from an injection of glanders are sub-
sequently immune, the immunity lasting for from three to six weeks.
Ladowski has obtained positive results also in rabbits and cats by in-
travenous injections of sterilized cultures. Other observers have
reported not only the production of immunity, but also cures by the
420 PATHOGENIC MICRO-ORGANISMS.
use of mallein. This is prepared in the same way as tuberculin. It
consists of the glycerinated bouillon in which the glanders bacilli have
grown and which contains the products of their growth and activity.
Concentrated mallein is produced by evaporating a six-weeks-old
culture of the glanders bacillus in 5 per cent, glycerin nutrient veal
bouillon to 10 per cent, of its original bulk. Some evaporate the culture
fluid only to 50 per cent. The dose for diagnostic purposes in horses
is 2^ c.c. of the unevaporated preparation.
Use of Quinea-pigs for Cultures and Diagnosis.— It is often diffi-
cult to demonstrate microscopically the presence of the bacillus of
glanders in the nodules which have undergone purulent degenera-
tion, in the secretions from the nostrils, or in the pus from the specific
ulcers and suppurating glands. It is then necessary to make im-
mediate cultures and also animal tests of these discharges by inocu-
lating susceptible animals, as guinea-pigs and mice, and then from
those to obtain a pure culture; but this requires time, and in clinical
work it is of great importance for the diagnosis to be established as
quickly as possible. With this view Strauss has prepared a method
which is prompt and which has given very satisfactory results. This
consists in introducing into the peritoneal cavity of a male guinea-
pig some material or a culture from the suspected products. If it be
a case of glanders, the diagnosis may usually be made within two
to five days from the tumefaction of the testicles, which become swollen,
and show evidences of pus formation. One objection to this method,
however, is that occasionally from the injection of impure material,
as in the nasal secretion, the animal may die of septicaemia; but if pure
matter can be obtained, as from the lymphatic glands of the horse,
this method is generally satisfactory. Sometimes the reaction is delayed
or develops only to a moderate extent. Further tests must then be
carried out with the cultures obtained from the tissues.
Diagnostic Use of Mallein. — The diagnosis of glanders in horses
in which the usual symptoms of the disease have not yet manifested
themselves, or in which it is suspected, may often be made by the
use of mallein. Following an injection of mallein in a glanderous
horse (best made about midnight) there will be a local reaction, and
a general reaction with a rise of temperature. The temperature usu-
ally begins to rise three or four hours after the injection, and reaches
its maximum between the tenth and twelfth hour. Sometimes, how^-
ever, the highest point is not reached until fifteen or eighteen hours after
the injection. This elevation of temperature is from 1.5® to 2® C
{2? to 3.5° F.), above the normal mean temperature. In a healthy
animal the rise of temperature, as a rule, amounts to only a few tenths
of a degree, but it may reach 1° C. The rise of temperature, however,
should be considered always in connection with the general and local
reactions. In a glanderous animal, after an injection of mallein, the
general condition is more or less profoundly modified. The animal
has a dejected appearance; the countenance is pinched and anxious,
the hair is rough, the flank is retracted, the respirations are rapid, there
GLANDERS BACILLUS. 421
are often rigors, and the appetite is gone. In healthy animals the gen-
eral symptoms do not occur. The local reaction around the point of in-
jection in a glanderous animal is usually very marked. A few hours
after the injection there appears a large, warm, tense, and very painful
swelling, and running from this will be seen hot, sensitive lines of sin-
uous lymphatics, directed toward the neighboring lymphatic nodes.
This oedema increases for twenty-four to thirty-six hours and per-
sists for several days, not disappearing entirely for eight or ten days.
In healthy animals, at the point of injection, mallein produces only
a small oedematous tumor, and the oedema, instead of increasing,
diminishes rapidly and disappears within twenty-four hours. The
value of this test has been demonstrated by numerous experiments.
There are some exceptions to the rule as described above, but they
are infrequent, and mallein has been used with considerable success
as a diagnostic aid in detecting the existence or absence of glanders
in doubtful or obscure cases.
Agglatination Test for Qlanders.— The test may be carried out
according to the macroscopic or microscopic method.
Collection of the Blood. — In obtaining blood from horses a large-
sized hypodermic needle which has been sterilized is inserted into
the jugular vein which has been brought into view by pressing the
thumb upon it from below; the blood is allowed to flow through the
needle into a sterile tube or flask, 8 to 10 c.c. being suflScient.
In the case of human beings it is obtained by pricking the lobe of
the ear or finger and collected in small capillary pipettes sealed at
both ends. Care must be taken to keep the blood sterile.
Macroscopic Method. — ^The procedure of Meissner and Schiitz with
slight modifications is as follows: A forty-eight-hour glycerin agar
culture of Bacillus mallei is washed off with normal salt solution, to
which sufficient carbolic acid has been added to make a 5 per cent,
solution. This is incubated for two hours at 60° C. then filtered
and enough of the carbolized normal salt solution is added to give a
slight milky appearance. This emulsion will keep for two or three
weeks in the ice-box.
The serum is then made up into the required dilutions, such as
1 : 50, 1 : 100, etc. One c.c. of each dilution is pipetted into stoppered
sterile serum tubes and an equal amount of the epiulsion is added
to each tube. The tubes are incubated at 37° C. for twenty-four to
forty-eight hours.
If a reaction occurs the upper part of the fluid will be clear and a
fine granular sediment will be found at the bottom or fine clumps
clinging to the sides of the tube.
Meissner and Schiitz use a culture of Bacillus mallei that has been
recently passed through a guinea-pig, claiming that it agglutinates
better than a culture grown for some time on artificial media. This
is not in accordance with our experience. We have found that the
more recently isolated culture, as in the case of Bacillus ti/phi and
Bacillus dysenterice, shows much less agglutinability.
422 PATHOGENIC MICRO-ORGANISMS.
The Microscopic or Hanging Drop Method. — In this case a twenty-
four-hour glycerin broth culture is used which has been heated to
60° C. for one minute, and the test is made as in the Widal for typhoid.
The cover-glasses and slides must be sterilized and the hanging
drops made carefully and quickly to avoid contamination. The
slides are left at room temperature or at 22° C. for eighteen to twenty-
four hours and then examined microscopically.
In this method the reaction can be observed earlier than in the tubes,
that is as soon as agglutination occurs, and it is not necessary to wait
for precipitation which at times takes place slowly.
The microscopic method gives a higher reading than the macro-
scopic method. This will include more horses which are doubtful,
while on the other hand horses showing other symptoms of glanders
will sometimes give a negative reaction with the gross method.
The limit of agglutination of the normal horse is 1 : 500, but many
apparently healthy horses will agglutinate the BaciUus mallei in
dilutions as high as 1:5000 and 1:10,000. The cause of this has
not been fully decided. Such horses should be subjected to the mal-
lein test from time to time, the possibility of a slight infection which
may manifest itself at any time being kept in view. Very rarely
a horse in the last stages of glanders will fail to give a reaction, but
the clinical symptoms will be well-defined in such cases. So far,
we have found the agglutination reaction valuable as a guide to
the use of mallein, the facility with which it can be carried out admit-
ting of the testing of a large number of horses suspected of having
glanders or those having been exposed to the disease.
In human cases the reaction of 1 : 100 and above is considered posi-
tive, the normal blood not reacting above 1 : 50.
In very acute cases that run their course in a few days, the reaction
may be entirely absent.
CHAPTER XXXII.
A number of bacilli of similar characteristics have been described
as causing certain infectious diseases of lower animals, marked by the
appearance of hemorrhagic areas throughout the body (hemorrhagic
septicemia of Hueppe). The bacilli are short, bipolar-staining, non-
motile, n on -spore bearing organisms. They are Gram-negative and
do not liquefy gelatin. They are found in rabbit septicemia, fowl
cholera, swine plague, and a similar disease in cattle. The bacillus
of bubonic plague seems to be closely related to the bacteria of this
group, and Ricketts, who recently reported finding organisms similar
to these in Rocky Mountain spotted fever and in typhus fever of
Texas suggested that these three diseases be considered a group of
human hemorrhagic septicemias.
BACILLUS or BUBONIC FLAOUE (BACILLUS PE8TI8).
HisTORiCAU-Y we can trace the bubonic plague back to the third
century. In Justinian's reign a great epidemic spread over the
Roman empire ami before it terminated destroyed in many portion.s
of the country nearly .50 per cent, of the people. The fourteenth
century saw the whole of Europe stricken. Except for occasional
cases, Europe and America have of late been free, but in India the
disease has recently broken out in all its horrors so that at the present
423
424 PATHOGENIC MICRO-ORGANISMS.
time over 500,000 persons die annually from it. Among the most
fetal forms of infection is that of the lungs. Pneumonic ca^es are
not alone very serious, but they readily spread the infection. The
bacillus exciting the disease was discovered simultaneously by Kitasalo
and Yersin (1894) during an epidemic of the bubonic plague in China.
It is found in large numbers in the seropurulent fluid from the recent
buboes characteristic of this disease and in the lymphatic glands;
more rarely in the internal organs except in pneumonic cases when
the lungs and sputum contain immense numbers, it occurs in the bloo<l
in acute hemorrhagic cases and shortly before death. It also occurs
in malignant cases in the f»ces
of men and animals. The
bacillus, as we have stated, is
closely allied to the hemor-
rhagic septicemia group.
Morphology.— The bacilli
in smears from acute abscesses
or infected tissues are, as a
rule, short, thick rods with
rounded ends. The central
portion of the bacillus is
slightly convex. When lightly
stained the two ends are more
colored than the middle por-
tion. The bacilli are mostly
single or in pairs. Bacilli in
short chains occur at times.
Invomtion form^n Mk^gnr (Kolle and Xhc length of the bacilU
varies, but on the average is
about 1.6/1 (l.oju to 1.7/i), breadth 0.5/1 to 0.7/1. Besides the
usual oval form, the plague bacillus has many exceptional variations
which are characteristic of it. In smears, especially from old buboes,
one looks for long bacilli with clubbed ends (similar to involution forms
(Fig. 134), yeast-like forms, and bladder shapes. Some of these stain
with difficulty. When obtained from cultures the bacilli present
not only the forms already mentioned, but also long chains.
Staining. — They stain readily with the ordinary aniline dyes, and
e:*pecially well with methylene blue, the ends being usually more
deeply colored than the central portion; they do not stain by Gram's
method.
Biology. ^An aerobic, non-motile bacillus. Grows best at 30° to
3.'»° C. Does not form spores. Grows on the usual culture media,
which should have a shghtly alkaline reaction." Does not liquefy
gelatin. Grows well on bhod-aerum media. It grows rapidly on
(lli/cerin agar, forming a grayish-white surface growth. The bacilli
appear, as a rule, as short, plump, oval bacilli, but a few present
elongated thread forms which are very characteristic. In bouillon
which is kept still a very characteristic appearance is produced, the
MICRO-ORGANISMS— HEMORRHAGIC SEPTICEMIA
423
culture medium remaining clear while a pellicle forms on the sur-
face from which projections sprout downward (stalactite forma-
tion) toward a granular or grumous deposit which forms on the
walls and on the bottom of the tube. In bouillon and most fluid
media the growth is in the form of short or medium chains of very
short, oval bacilli, which look almost like streptococci.
Pathogenesis. — This bacillus is pathogenic for rats, mice, guinea-
pigs, monkeys, rabbits, fleas, flies, and other insects, which usually die
within two or three days after inoculation. Then at the point of
inoculation is found a somewhat hemorrhagic infiltration and oedema,
with enlargements of the neighboring lymph glands, hemorrhages
into the peritoneal cavity, and ^^ ^^^
parenchymatous congestion of the
organs. The spleen sometimes
shows minute nodules resembling
miliary tubercles. Microscopically
the bacilli are found in all the
organs and in the blood. The
disease is rapidly cojnmunicated
from one animal to another
through the bites of infected fleas,
and thus its extension is facilitated.
During epidemics, rats, mice, and
flies, in large numbers, become in-
fected and die, and the disease is
frequently transmitted through
them to man. The organism is
found at times in the faeces of sick
animals, in the dust of infected houses, and in the soil.
Ground squirrels in California have been shown to be susceptible
to infection and they are supposed to help spread the disease.
The virulence of the bacilU in cultures and in nature seems to varv
considerably, and rapidly diminishes when grown on artificial media.
The growth in cultures becomes more abundant after frequent trans-
plantation. The virulence of the organism is increased by successive
inoculation in certain animal species, and then its pathogenic prop-
erties for other species are less marked.
In man there are often subcutaneous hemorrhages in severe cases
which gave the disease its name of ** black death."
Immunity. — Yersin, Calmette, and Borrel have succeeded in im-
munizing animals against the bacillus of bubonic plague by the
intravenous or intraperitoneal injection of dead cultures, or by re-
peated subcutaneous inoculation. They also succeeded in immuniz-
ing rabbits and horses, so that the serum afforded protection to small
animals, after subcutaneous injection of virulent cultures, and even
cured those which had been inoculated, if administered within twelve
hours after injection. The serum has considerable antitoxic as well
as bactericidal properties. More recently this serum has been applied
Bacilli in smear from acutely inflamed gland.
426 PATHOGENIC MICRO-ORGANISMS.
to the treatment of bubonic plague in man, with promising results.
Experience has shown that the treatment is more efficacious the
earlier the stage of the disease. When treatment is begun in the first
day of the attack, fever and all alarming symptoms sometimes dis-
appear with astonishing rapidity. In cases treated at a later stage
larger doses of the serum are required, and even in the favorable cases
suppuration of the buboes is not always prevented. In some of the
early cases and in many of the rather late ones the serum fails. WTien
the disease is far advanced the serum is powerless. For immunizing
purposes the serum should be valuable, and a single injection would
probably give protection for several weeks.
Vaccines. — Haffkine, in India, has appUed his method of preventive
inoculation to the bubonic plague, as he previously did with cholera,
and apparently with equally good results. This method consists in
an inoculation of dead cultures, and is essentially a protective rather
than a curative treatment. It gives after six to ten days a consider-
able immunity, lasting a month or more. By means of these two
methods of inoculation, along with strict quarantine regulations and
the destruction of rats and fleas, it is to be hoped that this disease
which, under the name of Black Death, once decimated the populations
of the earth and which in the East still causes a great mortality
may finally be greatly restricted.
Duration of Life Outside of the Body.— In cultures protected
from the air and light the plague bacilli may live one year or more.
In the bodies of dead rats they may live for two months. In sputum
from pneumonic cases the bacilli lived ten days. Upon sugar sacks,
food, etc., they may live six to fifteen days.
Resistance to Deleterious Influences. — The bacilli resemble the
colon bacilli in their reaction to heat and disinfectants. Boiling for
one to two minutes kills them. Carbolic acid, 5 per cent, solution,
kills cultures in one minute, in 2^ per cent, in two minutes, etc.
Bacteriological Diagnosis. — When the lymph glands are acutely
inflamed but not yet suppurated, cut down on one and make cultures
on nutrient agar slanted in tubes. If pus has formed withdraw a
little by means of the hypodermic needle. There should also be
made smears from the suspected bubo, or in case of pneumonia from
the sputum. If the patient is dead, cultures from the spleen and
heart's blood are also taken when possible. Suspected animals, such
as rats and mice, when freshlv killed, are examined as in man; when
decomposed, rats and guinea-pigs should be inoculated.
ROOKT MOUNTAIN SPOTTED FEVER.
Rocky Mountain spotted fever and typhus fever have this in com-
mon with bubonic plague, they are acute infectious diseases charac*
terized by fever and a more or less hemorrhagic eruption. These
two diseases have been especially studied for the last few years by
Ricketts of Chicago and his associates. They began with Rocky
Mountain spotted feve^^ In this disease, some years ago, Wilson and
» Ricketts, H. T. Jour. Am. Med. Assoc, 1909, Hi, 379.
MICRO-ORGANISMS— HEMORRHAGIC SEPTICEMIA. 427
•
Chowning thought they found protozoa similar to Babesia of Texas
fever. Anderson was inclined to agree with them, but nobody else
could find these bodies even in the original slides. These investi-
gators proved, however, that rabbits are susceptible to the disease and
that a tick of the genus Dermacentor carries the infection. Then
Ricketts and Gomez made some very interesting studies on the
disease. They found that guinea-pigs and monkeys are susceptible as
well as rabbits, and they further found that in guinea-pigs and monkeys
an attack of spotted fever produces a strong active inherited immunity
characterized by a serum with high protective but low curative
power, and that the production of the serum in the horse with the use
of sero-vaccination in man may give practical results.
They found a moderate number of diplococcoid bodies in the blood of
infected guinea-pigs and monkeys, and fever in man. They described
these bodies as two small, lanceolate, chromatin-staining (Giemsa
stain) bodies separated by a small amount of eosin-staining substance.
They did not find the bodies in normal blood, but they state that,
considering the complex morphology of the blood and the fact that they
could get no culture, it could not yet be stated that these are micro-
organisms. They found that the virus is transmitted by the infected
female tick to her young through the eggs. If the lar\8e from these eggs
are allowed to feed upon normal guinea-pigs, these animals come down
with the disease. Immense numbers of these apparent organisms
are found in infected eggs and none were found at first in normal eggs.
Afterward Ricketts found a few, but he thought these might be an
avirulent species of the same organism.
The salivary glands, alimentary, sac and ovaries of infected female
ticks are swarming with these bodies, while normal ticks seem to have
none. Lastly, Ricketts found that these bodies agglutinate with speci-
fic serum, 1 to 300 dilution. These bodies resemble the bacilli belong-
ing to the hemorrhagic septicaemia group of organisms.
TTPHUS FEVER.
Nicolle (July, 1909)^ had showed that old world typhus can be
transmitted to the chimpanzee and from this to the macacus with
typical eruption in each case. He also showed that the disease is
transmitted by the ordinary body louse (Pediculus yesiimenti). He
was not able to transmit from monkey to monkey.
• Anderson and Goldberger (December, 1909)^ were the first to trans-
mit the typhus fever of Mexico (tabardillo) to monkeys. They were
able to transmit directly from human beings to the macacus and
capuchin.
Ricketts and Walker (February, 1910)' also found that the macacus
was directly susceptible to the disease. They based their diagnosis
*Compt. rend. Acad. Sci., cxlix.
* Public Health Reports (U. S.), xxiv, Nos. 50 and 52.
*Jour. of Am. Med. Assoc, liv, 463, 1304, 1373.
428 PATHOGENIC MICRO-ORGASISMS.
chiefly upon a rather indeSnite fever, and, in most cases, somewhat
distinct symptoms of illness.
TTieir conclusions are as follows:
1. It seems that M. rhesus can be infected with tabardillo invariably
by the injection of virulent blood from man taken on eighth to tenth
days of fever. The blood should be diluted with salt solution.
2. Attempts to maintain typhus in the monkey by passage through
other monkeys were not successful.
3. The monkey may pass through an attack of typhus so mild that
it cannot be recognized clinically. Vaccination results.
4. The immunity test is a reliable proof of the previous occurrence or
no n -occurrence of typhus at least within a period of one month,
5. Typhus was transmitted to the monkey by the bite of the louse
{Pediculus veatimenti) in second experiment, the lice in one instance
deriving their infection from man and in another from the monkey.
6. Another monkey was infected by typhus through the introduction
of the faeces and abdominal contents of infected lice into small incisions.
7. In stained (Giemsa) preparations of blood of patients taken from
seventh to twelfth days of disease we invariably found a few short ba-
cilli (300 to 2,000 bacilli to 0.01 c.c. of blood) which have roughly the
morphology of those which belong to the hemorrhage septicaemia
group.
8. In moist preparation similar forms have been seen in all cases.
No motility observed. No cultures could be obtained.
9. Dejecta and organisms of many lice were examined and similar
stained bodies have been found in large numbers in infected lice, occa-
sionally in non-infected ones.
CHAPTER XXXIII.
THE ANTHRAX BACILLUS AND THE PATHOGENIC ANAEROBES.
BACILLUS ANTHBAOIS.
Anthrax is an acute infectious disease which is very prevalent
among animals, particularly sheep and cattle. Geographically and
zoologically it is the most widespread of all infectious disorders. It
is much more common in Europe and in Asia than in America. The
ravages among herds of cattle in Russia and Siberia and among
sheep in certain parts of France, Hungary, Germany, Persia, and
India are not equalled by any other animal plague. Local epidemics
have occasionally occurred in England, where it is known as splenic
fever. In this country the disease is rare. In infected districts
the greatest losses are incurred during the hot months of summer.
The disease also occurs in man as the result of infection, either
through the skin, the intestines, or in rare instances through the
lungs. It is found in persons whose occupations bring them into
contact with animals or animal products, as stablemen, shepherds,
tanners, butchers, and those who work in wool and hair. Two forms
of the disease have been described — the external anthrax, or malig-
nant pustules, and the internal anthrax, of which there are intestinal
and pulmonary forms, the latter being known as "wool-sorters*
disease."
Owing to the fact that anthrax was the first infectious disease
which was shown to be caused by a specific microorganism, and to
the close study which it received in consequence, this disease has
probably contributed more to our general knowledge of bacteriology
than any other infectious malady.
Pollender in 1849 observed that the blood of animals suffering
from splenic fever always contained minute rod-shaped bacteria.
Davaine in 1863 announced to the French Academy of Sciences the
results of his inoculation experiments, and asserted the etiological
relations of the microorganism to the disease, with which his investi-
gation showed it to be constantly associated. For a long time this
conclusion was energetically opposed until, in 1877, Koch, Pasteur,
and others established its truth by obtaining the bacillus in pure
cultures, and showing that the inoculation of these cultures produced
anthrax in susceptible animals as certainly as did the blood of an
animal recently dead from the disease.
Morphology. — Slender, cylindrical, non-motile rods, having a
breadth of 1// to 1 . 25;<, and ranging from 2/jt or 3/£ to 20fi or 25// in
length. Sometimes short, isolated rods are seen, and, again, shorter
429
430 PA THOGEMC MICRO-ORGA MSMS.
or longer chains or threads made up of several rods joined end to end.
In suitable culture media very long, flexible filaments may be ob-
served, which are frequently united in twisted or plaited cord-like
bundles. (See Fig. 136 and Fig. 137 and Fig. 5, p. 11.) These
filaments in hanging-drop cultures, before ihe development of spores,
appear to be homogeneous or nearly so; but in stained prepara-
tions they are seen to be composed of a series of rectangular, deeply
stained segments. When obtained directly from the bloo<l of an
infected animal the free ends of the rods are slightly rounded, but
those coming in contact with one another are quite square. In cultures
the ends are seen to be a trifle thicker than the body of the cell and
somewhat concave, giving the appearance of joints of bamboo. At
one time much stress was laid upon these peculiarities as distinguished
marks of the anthrax bacillus; but it has been found that they are the
effects of artificial cultivation and not necessarily characteristic of the
organism under all conditions. Another peculiarity of this bacillus
is that it is enclosed in a transparent envelope or capsule, which in
stained preparations (from albuminous material) may be distinguished
by its taking on a lighter stain than the deeply stained ro<U which il
surrounds.
Under favorable conditions in cultures spores are developed in
the bacilli. These spores are elliptical in shape and about one and a*
half times longer than broad. They first appear as small, refractive
granules distributed at regular intervals, one in each ro<l. As the
.spore develops the mother-cell becomes less and less distinct, until it
disappears altogether, the complete oval spore being set free by its dis-
solution. (See Fig. 1.37.) Irregular spomlation sometimes takes
place, and occasionally there is no .spore formation, as in varieties of
non-spore-bearing anthrax.
THE ANTHRAX BACILLUS. 431
Staining. — The anthrax bacillus stains readily with all the aniline
colors, and also by Gram's method, when not left too long in the de-
colorizing solution. In sections good results may be obtained by
the employment of Gram's solution in combination with carmine,
but when only a few bacilli are present this method is not always reliable,
as some of the bacilli are generally decolorized.
Biology. — The anthrax bacillus grows easily in a variety of nu-
trient media at a temperature from 18° to 43° C, 37° C. being the
most favorable temperature. Under 12° C. no development takes
place, as a rule, though by gradually accustoming the bacillus to a
lower temperautre it may be induced to grow under these conditions.
Under 14° C. and above 43° C. spore formation ceases. The lower
limit of growth and of sporulation is of practical significance in de-
termining the question whether development can occur in the bodies
of animals dead from anthrax when buried at certain depths in the
earth. Kitasato has shown that at a depth 1.5 metres the earth in
July has a temperature of 15° C. at most, and that under these condi-
tions a scanty sporulation of anthrax bacilli is possible, but that at
a depth of 2 metres sporulation no longer occurs. The anthrax bacillus
is aerobic — that is, its growth is considerably enhanced by the presence
of oxygen — but it grows also under anaerobic conditions, as is shown
by its growth at the bottom of the line of puncture in stick cultures in
solid media; but under these conditions it no longer produces the pep-
tonizing ferment which it does with free access of air. Furthermore,
the presence of oxygen is absolutely necessary for the formation of
spores, while carbonic acid gas retards sporulation. This explains,
perhaps, why sporulation does not take place within the animal body
either before or after death.
It is also capable of leading a saprophytic existence. The bacillus
is non-motile.
Orowth in Oelatin. — In gelatin-plate cultures, at the end of twenty-
four to thirty-six hours at 24° C, small, white, opaque colonies are
developed, which, under a low-power lens, are seen to be dark gray
in the centre and surrounded by a greenish, irregular border, made
up of wavy filaments. As the colony develops on the surface of the
gelatin these wavy filaments spread out, until finally the entire colony
consists of a light grav, tangled mass, which has been likened to a
Medusa head (Fig. 138).
At the same time the gelatin begins to liquefy, and the colony is
soon surrounded by the liquefied medium, upon the surface of which
it floats as an irregular, white pellicle. In gelatin-stick cultures at
first development occurs along the line of puncture as a delicate
white thread, from which irregular, hair-like projections soon ex-
tend perpendicularly into the culture medium, the growth being
most luxuriant near the surface, but continuing also below. At the
end of two or three days liquefaction of the medium commences at
the surface and gradually progresses downward.
Orowth on Agar. — The growth on agar-plate cultures in the incu-
432 PA THOCEXIC MICRO-ORGAXISMS.
bator at 37° C, Is similar to thai on gelatin, and is still more char-
acteristic and beautiful in appearance. A grayish-white layer is
formed on the surface within twenty-four hours, which spreads rapidly
and is seen to be made up of interlaced threads.
Orovtb is BotUllDn.- — The growth is characterized by the formation
of flaky masses, which sink as a sediment to the bottom of the tube,
leaving the supernatant Hquid clear.
Spore formation, as already noted, only takes place in the presence
of oxygen, and at a temperature of 15° to 43° C, There ia no develop-
ment of spores at a greater depth than 1 .5 metres in the earth, or in
o onia o act lu an^ "'"fon^ei^^l'houn. X "so" (F. "uMe™ ^ ""
the bodies of living or dead animals; but spores may be found in
the fluids containing the bacilli when these come in contact with the
air, as in bloody <lischarges from the nostrils or from the bowels of the
(lead animal.
There are certain non-spore-bearing species of anthrax. Spore-
less varieties have also been produced artificially by cultivating the
typical anthrax bacillus under certain conditions, among which may
be mentione<l the addition of antiseptics, as carbolic acid, and of
continued high temperature (43" C). Varieties differing in their
pathogenic power may also be produced artificially, Pasteur pro-
<!ucefl an "attenuated virus" by keeping his cultures for a consider-
„ui„ .:™.. ijpfore replanting them upon fresh soil.
cultures containing spores retain their vitality for years;
■nee of spores the vitality is much more rapidly lost. When
liquids rich in albumin the bacilli attain a considerable
resistance; thus dried anthrax blood has been found to re-
rulence for sixty days, while dried bouillon cultures only
twenty-one days. Drie<] anthrax spores may be preserved
THE ANTHRAX BACILLUS. 433
for many years without losing their vitality or virulence. They also
resist a comparatively high temperature. Exposed in dry air they
recjuire a temperature of 140° C. maintained for three hours to destroy
them; but suspended in a liquid they are destroyed in four minutes
by a temperature of 100° C,
PathogeoesiB. — The anthrax bacillus is pathogenic for cattle,
sheep {except the Algerian race), horses, swine, mice, guinea-pigs,
and rabbits. Rats, cats, dogs, chickens, owls, pigeons, and frogs are
but little susceptible to infection. Small birds — the sparrow par-
ticularly— are somewhat susceptible. Man, though subject to local
infection and occasionally to in-
ternal forms of the disease, is
not as susceptible as some of the
lower animals.
In susceptible animals the
anthrax bacillus produces a true
septicemia. Among test animals
mice are the most susceptible,
succumbing to very minute in-
jections of a .slightly virulent
virus; next guinea-pigs, and
lastly rabbits, both of these ani-
mals dying after inoculation with
virulent bacilli. Infection is
most promptly produced by in-
troduction of the bacilli into the
circulation or the tissues, but J^^^
inoculation by contact with «ptir
wounds on the skin also causes
infection. It is difficult to produce infection by the ingestion even
of spores; but it may readily be caused by inhalation, particularly
of spores.
Subcutaneous injections of these susceptible animals results in
death in from one to three days. Comparatively little local reaction
occurs immediately at the point of inoculation, but beyond this there
is an extensive ceilema of the tissues. Very few bacilli are found in
the blood in the larger vessels, but in the internal organs, and especially
in the capillaries of the liver, the kidneys, and the lungs, they are present
in great numbers. In some places, as in the glomeruli of the kidneys,
the capillaries will be seen to be stuffed full of bacilli, and hemorrhages,
probably due to rupture of capillaries by the mechanical pressure of
the bacilli which are developing within them, may occur. The patho-
logical lesions in animals infected by anthrax are not marked except
in the spleen, which, as in other forms of septicaemia, is greatly enlarged.
Occarrence in Oattle and Sheep. — Cattle and sheep are affected
chiefly with the intestinal form of anthrax, infection in the.se ani-
mals commonly resulting from the ingestion of food containing
spores. The bacillus itself, in the absence of spores, is quickly de-
434 PATHOGENIC MICRO-ORGANISMS,
stroyed by the gastric juice. The disease usually takes a rapid
course, and the mortality is high — 70 to 80 per cent. The patho-
logical lesions consist of numerous ecchymoses, enlargement of the
lymphatic glands, serous, fatty, and hemorrhagic infiltration of the
mediastinum and mesentery, of the mucous membranes of the pharynx
and larynx, and particularly of the duodenum, great enlargement
of the spleen, and parenchymatous changes in the lymphatic organs.
The blood is very cfark and tar-like. Bacilli are present, especially in
the lymph spaces, in enormous masses.
Sheep are also subject to external anthrax, infection taking place
by way of the skin; cattle are seldom infected in this way. At the
point of inoculation there develops a hard, circumscribed boil — ^the
so-called anthrax carbuncle; or there may be diffuse oedema with
great swelling of the parts. When death occurs the appearances are
similar to those in intestinal anthrax, except that the duodenum is
usually less affected; but in all cases metastasis occurs in various
parts of the body, brought about, no doubt, by previous hemorrhages.
Occurrence in Man. — The disease does not occur spontaneously in
man, but always results from infection, either through the skin, the
intestines, or occasionally by inhalation through the lungs. It is
usually produced by cutaneous infection through inoculation of ex-
posed surfaces — the hands, arms, or face. Infection of the face or
neck would seem to be the most dangerous, the mortality in such
cases being 26 per cent., while infection of the extremities is rarely
fatal.
External anthrax in man is similar to this form of the disease in
animals. There are two forms: malignant pustule or carbuncle, and,
less commonly, malignant anthrax oedema.
In malignant pustule, at the site of inoculations, a small papule
develops, which becomes vesicular. Inflammatory induration extends
around this, and within thirty-six hours there is a dark brownish
eschar in the centre, at a little distance from which there mav be a
series of small vesicles. The brawny induration may be extreme.
There may also be considerable oedema of the parts. In most cases
there is no fever; or the temperature at first rises rapidly and the
febrile phenomena are marked. Death may take place in from
three to five days. In cases which recover the symptoms are slighter.
In the mildest form there may be only slight swelling.
Malignant anihrax cedeina occurs in the eyelids, and also in the
head and neck, sometimes the hand and arm. It is characterized bv
the absence of the papule and vesicle forms, and by the most exten-
sive oedema. The oedema may become so intense that gangrene re-
sults; such cases usually prove fatal.
The bacilli are found on microscopic examination of the fluid
from the pustule shortly after infection; later the typical anthrax
bacilli are often replaced by involution forms. In this case resort
may be had to cultures, animal inoculation, or examination of sec-
tions of the extirpated tumor. The bacilli are not present in the
THE ANTHRAX BACILLUS, 435
blood until just before death. Along with the anthrax bacilli pus
cocci are often found in the pustule penetrating into the dead tissue.
Internal anthrax is much less common in man; it does, however,
occur now and then. There are two forms of this: the intestinal
form, or mycosis intestinalis, and the pulmonic form, or wool-sorters'
disease.
Intestinal anthrax is caused by infection through the stomach and
intestines, and results probably from the eating of raw flesh or un-
boiled milk of diseased animals. That the eating of flesh from in-
fected animals is comparatively harmless is shown by Gerlief, who
states that of 400 persons who are known to have eaten such meat
not one was affected with anthrax. On the other hand, an epidemic
of anthrax was produced among wild animals, according to Jansen,
by feeding them on infected horse-flesh. It is evident, therefore,
that there is a possibility of infection being caused in this way. The
recorded cases of intestinal anthrax in man have occurred in persons
who were in the habit of handling hides, hair, etc., which were con-
taminated with spores; in those who were conducting laboratory ex-
periments, and rarely it has been produced by the ingestion of food,
such as raw ham and milk. The symptoms produced in this disease
are those of intense poisoning: chill, followed by vomiting, diar-
rhoea, moderate fever, and pains in the legs and back. The patho-
logical lesions are similar to those described in animals.
Wool-sorters' disease, or pulmonic anthrax, is found in large es-
tablishments in which wool and hair are sorted and cleansed, and
caused by the inhalation of dust contaminated with anthrax spores.
The attack comes on with chills, prostration, then fever. The breath-
ing is rapid, and the patient complains of pain in the chest. There
may be a cough and signs of bronchitis. The bronchial symptoms
in some instances are pronounced. Death may occur in from two
to seven days. The pathological changes produced are swelling of
the glands of the neck, the formation of foci of necrosis in the air
passages, oedema of the lungs, pleurisy, bronchitis, enlargement of
the spleen, and parenchymatous degenerations.
Prophylaxis against Anthrax Infection. — Numerous investiga-
tions have been undertaken with the object of preventing infection
from anthrax. The efforts of Pasteur to effect immunity in animals
by preventive inoculations of "attenuated virus" of the anthrax ba-
cillus opened a new field of productive original research. Follow-
ing in his wake many others have devised methods of immunization
against anthrax infection; but the one adopted by Pasteur, Chamber-
land, and Roux has alone been practically employed on a large scale.
According to these authors, two anthrax cultures of different degrees
of virulence attenuated by cultivation at 42° to 43° C, are used for
inoculation. Vaccine No. 1 kills mice, but not guinea-pigs; vaccine
No. 2 kills guinea-pigs, but not rabbits. The animals to be inoculated
— viz., sheep and cattle — are first given a subcutaneous injection of
one to several tenths of a cubic centimetre of a four-dav-old bouillon
436 PATHOGENIC MICRO-ORGANISMS.
culture of vaccine Np. 1 ; after ten to twelve days they receive a simi-
lar dose of vaccine No. 2. Prophylactic inoculations given in this way
have been widely employed with apparently good results.
Bacterial Cultures for Diac^osia. — The detection of the anthrax
bacillus is ordinarily not difficult, as this organism presents morpho-
logical, biological, and pathogenic characteristics which distinguish
it from all other bacteria. In the later stages of the disease, how-
ever, the bacilli may be absent or difficult to find, and cultivation on-
artificial media and experimental inoculation in animals are Dot al-
ways followed by positive results. Even in sections taken from the
extirpated pustule it is sometimes difficult to detect the bacilli. In
such cases only a probable diagnosis of anthrax can be made. It
should be remembered that the bacilli are not found in the blood
until shortly before death, and then only in varying quantity; thus
blood examinations often give negative results, though the bacilli
may be present in large numbers in the spleen, kidneys, and other
organs of the body. The suspected material should be streaked over
nutrient agar in Petri plates and inoculate<l in mice.
Differential Diagnosis. — Among other bacteria which may possibly
be mistaken for anthrax bacilli are BacUlvs sahlUis and the bacillus
of maglinant cedema. The former is distinguished by its motility,
by various cultural peculiarities, and by being non-pathogenic. The
latter differs from the anthrax bacillus in form and motility, in be-
ing decolorized by Gram's solution, in being a strict anaerobe, and in
various pathogenic properties.
The diagnosis of internal anthrax in man is by no means easy, un-
less the history points definitely to infection in the occupation of the
individual. In cases of doubt cultures should be made and inocula-
tions performed in animals.
BACILLUS AKTHBA0I8 8THPTOMATI0I (BACILLUS OT SYMP-
TOMATIC AHTHBAZ).
Like the bacilli of anthrax and of malignant cedema, both of which
it resembles in other respects also, the bacillus of symptomatic anthrax
is an inhabitant of the soil. It is found as the chief cause of the disease
in animals^princi pally cattle and sheep — known as "black leg,"
"quarter evil," or symptomatic anthrax (rauschbrand, German;
charbon symptomatique, French), a disease which is characterized by
a peculiar emphysematous swelling of the subcutaneous tissues and
muscles, especially over the quarters. Clinically it is sometimes con-
:h anthrax.
lOlogy. — Bacilli having rounded ends, from 0.5/i to 0.6«
d from 3,(1 to 5/i long; mostly isolated; also occurring in pair?,
id-to-end, but never growing out into long filaments, as the
liacilli in culture and the bacilli of malignant oedema in the
; animals are frequently seen to do. In the hanging drop
li are observed to be actively motile, and in stained prepara-
PATHOGENIC ANAEROBES. 437
tions flagella may be demonstrated surrounding the periphery. The
spores are elhptical in shape, usually thicker than the bacilli, lying
near the middle of the rods, but rather toward one extremity. This
gives to the bacilli containing spores a somewhat spindle shape.
Stains with the ordinary aniline dyes, but not with Gram's method
or only with difficulty and after long treatment or intense colors.
Biology. — Like the bacillus of malignant oedema, this is a strict
anaerobe, and cannot be cultivated in an atmosphere in which oxygen
is present. It grows best under hydrogen, and does not grow under
carbonic acid. This bacillus de-
velops at the room temperature in
the usual culture media, in the ab-
sence of oxygen, but it grows best
in those to which 1 . 5 to 2 per cent,
of glucose or 5 per cent, of glycerin
has been added.
Orowth on Agar. — The colonies on
agar are somewhat more compact
than tho.se of malignant cedema, but
they also send out projections very
often. In agarslick cultures, in the
incubator, growth occurs after a
day or two also some distance below
the surface, and is accompanied by . .
the production of gas and a peculiar" " ' "pS™.'"^A?ierZeti[iow!)* "'""'
disagreeable acid odor.
PathogetlflUS. — ^The bacillus of symptomatic anthrax is pathogenic
for cattle (which are immune against malignant a<<lema), sheep,
goats, guinea-pigs, and mice; horses, asses, and white rats, when in-
oculated with a culture of this bacillus, present only a limited reac-
tion; and rabbits, swine, dogs, cats, chickens, ducks, and pigeons are,
as a rule, naturally immune to the disease. The guinea-pig is the
most susceptible of test animals. When susceptible animals are in-
oculated subcutaneously with pure cultures of this organism, or with
spores attached to a silk thread, or with bits of tissue from the af-
fected parts of another animal dead of the disease, death ensues in
from twenty-four to thirty-six hours. At the autopsy a bloody serum
is found in the subcutaneous tissues, extending from the point of
inoculation over the entire surface of the abdomen, and the muscles
present a dark red or black appearance, even more intense in color
than in malignant osdema, and there is a considerable development
of gas. The lymphatic glands are markedly hyperfemic.
The disease occurs chiefly in cattle, more rarely in sheep and goats;
horses are not attacked spontaneously — i. e., by accidental infection.
In man infection has never been produced, though ample opportunity
by infection through wounds in slaughter-houses and by ingestion of
infected meat has been given. The usual mode of natural infection
by symptomatic anthrax is through woun'ds which penetrate not only
438 PATHOGENIC MICRO-ORGANISMS.
the skin, but the deep, intercellular tissues; some cases of infection
by ingestion have been observed.- The pathological findings present
the conditions above described as occurring in the experimental
infection.
Distribution Oatside ol the Body.— Symptomatic anthrax, like an-
thrax and mahgnant oedema, is a disease of the soil, but it shows a
more limited endemic distribution than the former, and is differently
distributed over the earth's surface than the second of these diseases,
being confined especially to places over which infected herds of cattle
have been pastured. It is doubtful whether the bacilli are capable
of development outside of the body like anthrax. In the form of
spores, however, reproduction may take place; by contamination wth
these, through deep wounds acquired by animals in infected pastures,
the disease is spread.
ToilnB. — Under favorable conditions extracellular toxins are formed
so that the filtrate of cultures is very poisonous. Injections of the
toxin into animals excite the production of antitoxins.
Diflwential Diagnosis. — The principal points of differentiating this
bacillus from the bacillus of malignant cedema, which it closely re-
sembles, are: it is smaller; it does not develop into long threads in
the tissues; it is more actively motile, and forms spores more readily
in the animal body than does the bacillus of malignant cedema. It
is pathogenic for cattle, while malignant oedema is not; and swine,
dogs, rabbits, chickens, and pigeonsj which are readily infected with
malignant oedema, are not, as a rule, susceptible to symptomatic
anthrax.
PreventiTe Inoculations. — It is well known to veterinarians that re-
covery from one attack of symptomatic anthrax protects an animal
against a second infection. Artificial immunity to infection can
also be produced in various ways: by inoculations with cultures which
have been kept for a few days at a temperature of 42° to 43° C. and
have thus lost their original virulence, or by inoculations of filtered
cultures, or of cultures sterilized by heat. For the production of
immunity in cattle it is advised to use a dried powder of the muscles
of animals which have succumbed to the disease, and which have
been subjected to a suitable temperature to ensure attenuation of the
virulence of the spores contained therein. Two vaccines are pre-
pared, as in anthrax — a stronger vaccine by exposing a portion of
the powder to a temperature of 85° to 90° C. for six hours, and a
weaker vaccine by exposing it for the same time to a temperature of
H)fl° to 104° C. Inoculations are made with this attenuated virus
f the tail^first the weaker and later the stronger. These
1 local reaction of moderate intensity, and the animal
ly immune from the effects of the most virulent material
disease. Fourteen days are allowed to elapse between
ulations. The results obtained from this method of
oculation seem to have been very satisfactory. Ae-
e statistics, including many thousand cattle treated, the
PATHOGENIC ANAEROBES, 439
mortality, which among 22,300 non-inoculated cattle was 2.20 per
cent., has been reduced to 0.16 per cent, in 14,700 animals inocu-
lated. When danger of immediate infection exists, it is advisable to
inject some antitoxin with the vaccine. This lessens the reaction and
gives immediate immunity.
If an antitoxic serum is at hand it should be given in cases seen early
in the disease.
THE OROUP OP MALIONANT CBDEBIA BAGILU.
This group is widely distributed, being found in the superficial
layers of the soil, in putrefying substances, in foul water, and by
invasion from the intestine, in the blood of animals which have been
suffocated. One such organism was discovered (1877) by Pasteur
in animals after infection with putrid flesh, and
named by him "vibrion septique." He recognized ^
its anaerobic nature, but did not obtain it in pure
culture. Koch and Gaffky (1881) carefully studied \y
\
In earlier times infection of man was quite often,
this microorganism, described it in detail, and gave
mis microorganism, aescnoea ii m aeiau, anu gave | ^
it the name '* Bacillus cedematis maligni'* (Fig. 141). l ^
In earlier times infection of man was quite often, • ^
now only occasionally, produced. This bacillus
belongs to a group which have lateral flagellae, **^* oMiema!*'**"*
produce oval spores, and grow only anaerobically.
Morphology. — The oedema bacillus is a rod of from 0.8// to 1// in
width, and of very varying length, from 2// to 10// or more, according
to the conditions of its cultivation and growth. It is usually found in
pairs, joined end to end, but may occur in chains or long filaments. It
forms spores, and these are situated in or near the middle of the body
of the rods. Exceptionally the spores are near the ends (see Fig. 141).
The spores vary in length and are oval in form, being often of greater
diameter than the bacilli, to which they give a more or less oval shape.
The bacilli siain readily by the usual aniline colors employed, but
are usually decolorized by Gram's method. Freytag found that very
young cultures were stained, while older ones were decolorized.
Biology. — A strictly anaerobic, liquefying, motile bacillus. Forms
spores which are very resistant. It grows in all the usual culture
media in the absence of oxygen. Development takes place at 20°
C, but more rapidly and abundantly at 37° C.
Orowth in Oelktin. — This bacillus may be cultivated in ordinary nu-
trient gelatin, but the growth is more abundant in glucose gelatin
containing 1 or 2 per cent, of glucose. After two or three days small,
almost transparent, circular colonies appear ^ to 1 mm. in diameter.
Later, as liquefaction increases, the colonies become grayish and then
confluent. Gas bubbles are formed and the gelatin liquefies.
Orowth on Agar. — On agar plates the colonies appear as dull, whitish
points, irregular in outline, and when examined under a low-power
lens are seen to be composed of a dense network of interlacing threads,
radiating irregularly from the centre toward the periphery.
440 PATHOGENIC MICRO-ORGANISMS.
Blood serum is rapidly li()uefied, with the production of gas. Cul-
tures of the malignant oedema bacillus give off gas with a peculiar,
disagreeable odor.
Resistance. — The spores are very resistant and because of this the
soil remains infected.
Pathogenesis. — The bacillus of malignant cedema is especially
pathogenic for mice, guinea-pigs, and rabbits, although man, horses,
dogs, goats, sheep, calves, pigs, chickens, and pigeons are also sus-
ceptible, A small quantity of a pure culture injected beneath the
skin of a susceptible animal gives rise to an extensive hemorrhagic
oedema of the subcutaneous connective tissue, which extends over the
entire surface of the abdomen and thorax, causing hypenemia and
redness of the superficial muscles. No odor is developed, ajid there
is little, if any, production of gas. In infection with garden earth,
owing to the presence of associated bacilli, the effused serum is frothy
from the development of gas and possesses a putrefactive odor.
The disease, in natural infection caused by the contamination of
wounds with earth or f^ces, runs the course above described. Simple
abrasion of the skin is not sufficient to produce infection; owing lo
the bacillus being capable only of an anaerobic existence, the poison
must penetrate deep into the tissues. Malignant cedema is coofined
mostly to the domestic animals, the horse, sheep, cattle, and swine,
but cases have also been reported in man.
Animals which recover from malignant oedema are subsequently
immune. Artificial immunity may be induced in guinea-pigs by
injecting filtered cultures of the malignant oedema bacillus in harm-
less ()uantities.
In man the chief .symptom is the sudden appearance of subcutane-
ous cedematous swelling accompanied by high fever. In light cases
this remains circumscribe<l ; in severe cases it spreads widely and the
case ends fatally. Appreciable quantities of gas usually fail. Au-
topsy shows a serous or hemorrhagic infiltration of the subcutaneous
ti.ssues and intramuscular connective tissue. lo the inflamed tissue
the bacilli with and without spores are found.
Prevention. — Most cases are produced by injecting subcutaneously
albuminous fluids infected by the bacilli. Care should be taken
that fluids to be injected do not Iwcome infected by dust or dirt.
BACILLUS AEBOGENES OAPSULATUS (BACILLUS WZLOHH).
Tk;., I — jiius ^f^^ found by Welch in the blood vessels of a patient
ith aortic aneurysm; on autopsy, made in cool weather.
: after death, the ves.sels were observed to be full of gas
iince then it has been found in a number bf cases in which
/eloped from within sixty hours of death until some hour*
. External cutting operations on the urethra and opera-
the uterus have been followed in a number of cases by
It has been found in ovarian abscesses and in infections of
PATHOGENIC ANAEROBES. 441
the genito-urinary tract. These cases are, as a rule, marked by de-
lirium, rapid pulse, high temperature, and the development of emphy-
sema and discoloration of the diseased area or of marked abdominal
distention when the peritoneal cavity is involved. This bacillus is
present, as a rule, in the intestinal canal of nian and animals and is apt
to be found in the dust of hospitals and elsewhere. Herter* has shown
that it is present in excessive numbers in certain diseases of the diges-
tive tract. These cases are apt to develop ansemia.
Morphology. — Straight or slightly curved rods, with rounded or
sometimes square-cut ends; somewhat thicker than the anthrax ba-
cilli and varying in length; occasionally long threads and chains are
seen. The bacilli in the animal body, and sometimes in cultures,!
are enclosed in a transparent capsule. Spores are usually absent in j'
the tissues and often in cultures. Dunham showed that the culture
isolated by Welch formed spores when grown on blood serum. Some
strains since isolated make spores readily. It is possible that these
differences may be due to the fact of there being several strains.
Biology. — An anaerobic, non-motile, non-liquefying bacillus. Dif-
ferent strains of this bacillus vary in their tendency to make spores.
It is stained by Gram, but is more easily decolorized than many
bacteria. Growth is rapid at 37° C, in the usual culture media
in the absence of oxygen, and is accompanied by the production of
gas. Nutrient gelatin is not liquefied by the growth of this bacillus,
but it is gradually peptonized. If agar colonies are developed which
are from 1 to 2 mm. or more in diameter, grayish-white in color,
and in the form of flattened spheres, ovals, or irregular masses, beset
with hair-like projections. Bouillon is diffusely clouded, and a
white sediment is formed. Milk becomes acidified and coagulated,
then partially digested, giving a worm-eaten appearance to the clot.
Pathogenesis. — Usually non-pathogenic in healthy animals, al-
though Dunham found that the bacillus taken freshly from human
infection is sometimes very virulent. When quantities up to 2.5 c.c.
of fresh bouillon cultures are injected into the circulation of rabbits
and the animals killed shortly after the injection, the bacilli develop
rapidly, with an abundant formation of gas in the blood vessels and
organs, especially the liver. This procedure is one of the best methods
of obtaining the bacilli : The material suspected to contain the bacillus
alone or associated with other bacteria is injected intravenously into
rabbits, which are killed five minutes later and kept at 37° C. for
sixteen hours, and cultures made from the liver and heart's blood.
It is suggested by Welch that in some of the cases in which death
has been attributed to the entrance of air into the veins the gas found
at the autopsy may not have been atmospheric air, but may have
been produced by this or some similar microorganism entering the
circulation and developing shortly before and after death. The same
may be true for gas in the uterine cavity.
* Journal of Biolog. Chem., 1906, ii., page 1.
442 PATHOGENIC MICRO-ORGANISMS,
BACILLUS ENTERTTIDIS SPOROOENES.
This very closely resembles B. Welchii. It produces spores readily.
Klein considers that when taken in milk it may produce diarrhoea.
This is disputed by others. It is also considered to be an evidence of
sewage pollution, but this is not at all certain since it occurs in culti-
vated soils (Jordan, Bacteriology, 1908, page 321).
CHAPTER XXXIV.
THE CHOLERA SPIRILLUM (SPIRILLUM CHOLERiE ASIATICS)
AND ALLIED VARIETIES.
In 1883 Koch separated a characteristically curved organism from
the dejecta and intestines of cholera patients — the so-called ** comma
bacillus/' This he declared to be absent from the stools and intes-
tinal contents of healthy persons and of persons suffering from other
affections. The organism was said to possess certain morphological
and biological features which readily distinguished it from all pre-
viously described organisms. It was absent from the blood and vis-
cera, and was found only in the intestines; and the greater the num-
ber, it was said, the more acute the attack. Koch also demonstrated
an invasion of the mucosa and its glands. The organisms were found
in the stools on staining the mucous flakes or the fluid with methylene
blue or fuchsin, and sometimes alone; by means of cultivation on
gelatin they were readily separated from the stools. Numerous con-
trol observations made upon other diarrhoeic dejecta and upon normal
stools were negative; the comma bacillus was found in choleraic
material only, or occasionally in small numbers in the stools of healthy
persons who came in contact with cholera. Soon, however, other
observers described comma-shaped organisms of non-choleraic
origin. Finkler and Prior, for instance, found them in the diarrhoeal
stools of cholera nostras, Deneke in cheese, Lewis and Miller in
saliva. All of these organisms, however, differed in many respects
from Koch's comma bacillus, and it has since been proved that none
of them is affected by the specific serum of animals immunized to
cholera. After a time, therefore, the exclusive association of Koch's
vibrio with cholera or those in contact with it became almost gener-
ally acknowledged, until now it is regarded by bacteriologists every-
where to be the specific cause of Asiatic cholera. Certain sporadic
cases of cholera-like disease, however, are undoubtedly due to other
organisms.
Morphology. — Curved rods with rounded ends which do not lie
in the same plane, of an average of 3 to 5// in length and about 0 . 4//
in breadth. The curvature of the rods may be very slight, like that
of a comma, or distinctly marked, particularly in fresh unstained
preparations of full-grown individuals, presenting the appearance
of a half-circle. By the inverse junction of two vibrios S-shaped
forms are produced. Longer forms are rarely seen in the intestinal
discharges or from the cultures grown on solid media, but in fluids, es-
pecially when grown under unfavorable conditions, long, spiral fila-
ments may develop. The spiral forms are best studied in the hang-
443
444 PATHOGENIC MICRO-ORGANISMS.
ing drop, for in the dried and stained preparations the spiral char-
acter of the long filaments is often obliterated. In film preparations
from the intestinal contents in typical cases it will be found that the
organisms are present in enormous numbers, and often in almost
pure culture (Figs. 142 and 143). In old cultures irregularly clubbed
and thickened involution forms are frequent, and the presence in the
organisms of small, rounded, highly refracttle bodies is often noted.
Staining. — The cholera spirillum stains with the aniline color.s
usually employed, but not as readily as many other bacteria; a dilute
aqueous solution of carbol fuchsin (1 .0 per cent.) is recommended as
rom agar. X TOO diuoelfln. (DuobKm.) pUU FuICun of cholen, X 800 dismelvn.
the most reliable staining agent with the application of a few minutes'
heat. It is decolorized by Gram's method. The organisms exhibit
one long, fine, spiral flagellura attached to one end of the rods, or, ex-
ceptionally, to both ends. {Cholera-like spirilla often have 1, 2, or
3 end flagella.) In sections they are stained best by alkaline methy-
lene-blue solution and washed in water slightly acidulated with acetic
acid.
Biology. — An aerobic (facultative anaerobic), liquefying, very
motile spirillum. Grows readily in the ordinary culture media,
best at 37° C, but also at room temperature (22° C); does not grow
at a temperature above 42° or below 8° C, and does not form spores.
In gelatifi-plale cuUures at 22° C. the colonies are quite character-
istic; at the end of twenty-four hours, small, round, yellowish-white
to yellow colonies may be seen in the depths of the gelatin, which
later grow toward the surface and cause liquefaction of the medium,
the colonies lying at the bottom of the holes or pocket thus formed.
The zone of liquefaction, which increases rapidly, at first remains
clear, then becomes cloudy, mostly gray, as the result of the growth
of the colonies. In many cases after a time concentric rings, in-
creasing from day to day, appear in the zone of liquefaction. (See
Fig.s. 144 and 145.) Examined under a low-power lens, at the end
of sixteen to twenty-four hours, the colonies appear as small, light
THE CHOLERA SPIRILLUM. 445
yellow, round, coarsely gratiular disks, with a more or less irregular
outline. In many cases at this stage an ill-defined halo is seen to
surround the granular colony. As the colonies become older the
granular structure increases, until a stage is reached when the surface
looks as if it were covered with little fragments of broken glass. Lique-
faction continues about the colonies, their structure appears fissured
and coarsely granular in texture, and occasionally a hair-like border
is formed at the periphery (Fig. 145), Sometimes the colonies may
be retained as compact masses in the zone of liquefaction, and then
they are dark yellow or brown in color, and forms occur which are
to Ihirty-sit hours' (mwth. X about 20 oing. x 'i*> Jismeten. {Duohato.)
absolutely unlike the typical cholera colonies. In gelatin-stick cultures
the growth is at first thread-like and uncharacteristic. At the end of
twenty-four to thirty-six hours a small, funnel-shaped depression
appears on the surface of the gelatin, which soon spreads out in the
form of an air bubble above, while below this is a whitish, viscid mass.
Later, the funnel increases in depth and diameter, and at the end of
from four to six days may reach the edge of the test-tube; in from eight
to fourteen days the upper two-thirds of the gelatin is completely
liquefied. (See Fig. 146.) Freshly isolated cholera vibrios liquefy
gelatin more rapidly than old laboratory cultures; a certain variation,
under some circumstances, in the characteristic fiquefacfion on the
gelatin, even in fresh cultures, should be borne in mind in malting a
diagnosis. Such variations in cultural peculiarities occur also with
other bacteria.
Upon the surface of agar the Comma bacillus develops a moist,
shining, grayish-yellow layer. In agar-plale cultures, for diagnostic
purposes, the growth of separated colonies is of some importance.
The nutrient agar after pouring in the plates and solidifying should
be slightly dried on the surface by putting the imcovered plate face
downward on the shelf of the incubator at 37° C. for thirty minutes,
or at 60° C. for five minutes. The cholera colonies develop fairly
characteristically, being more transparent than those of most other
bacteria except the cholera-like vibrios. Blood serum is rapidly
446 PATHOGENIC MICRO-ORGANISMS.
liquefied at the temperature of the incubator. On potato at incu-
bator temperature a moist growth of a dirty brown color occurs, it ilk
ia not coagulated. In bouillon the growth is rapid and abundant;
in the incubator at the end of ten to sixteen hours the liquid is diffusely
colored, and on the surface a wrinkled membranous layer is often
formed. In general the spirillum grows in any liquid containing a
small quantity of organic matter and having a slightly alkaline reaction.
An acid reaction of the culture medium prevents its development,
as a rule; but it has the power of gradually accommodating itself to
>wlli. (DuDhun.)
the presence of vegetable acids. Abundant development occurs in
bouillon which has been diluted with eight to ten parts of water and in
simple peptone solution.
The comma bacillus belongs to the class of aerobic organisms, inas-
much as it grows readily only in the presence of oxygen, and that it
develops active motility only when a certain amount of oxygen is
present. It does not grow in the total absence of oxygen, but a small
quantity of oxygen is all that is required for its development, as in
the intestines. This need of oxygen tends to send the spirilla to the
surface of fluid culture media.
Oholera-red Reaction. — When a small quantity of chemically pure
sulphuric acid is added to a twenty-four-hour bouillon culture of the
cholera bacillus containing peptone a red dish- violet color is produced.
Brieger separated the pigment formed in this reaction — the so-called
cholera-Ted — and showed that it was indol, and that the reaction was
nothing more fhnn the well-known indol reaction. Salkowski and
THE CHOLERA SPIRILLUM. 447
Petri then demonstrated that the cholera bacilli produced in thin
bouillon cultures, along with indol, nitrites by reducing the nitrates
contained in small quantities in the culture media. They showed
that it is the nitric acid, liberated by the addition of sulphuric acid
to the culture, which would give rise to the indol, the red body upon
which the cholera reaction depends. For a long time it was believed
that this nitroso-indol reaction was peculiar to the cholera bacillus
and great weight was placed on it as a diagnostic test. It has since
been shown, however, that there are a number of other vibrios which,
under similar conditions as the cholera vibrio, give the same red re-
action. The reaction is, nevertheless, a constant and characteristic
peculiarity of this spirillum and is of unquestionable value. . It is
even more valuable as a negative than as a positive test, as the ab-
sence of the reaction enables one to say of a suspected organism that
it is not the cholera spirillum. There are, however, certain pre-
cautions to be observed in its use. It has been shown that the re-
action may be absent, for instance, when the culture contains either
too much or too little nitrate. It is, therefore, advisable not to em-
ploy a bouillon culture the composition of which is uncertain, but
a distinctly alkaline solution of peptone, containing 1 per cent, pure
peptone and 0.5 per cent, of pure chloride of sodium (Dunham's
solution). With such a solution constant results can be obtained.
Development Outside of the Body. — It has been shown by experi-
ment that cholera spirilla multiply to some extent in sterilized river-
water or well-water, and preserve their vitality in such water for
several weeks or even months. Koch demonstrated the presence of
this spirillum in the foul water of a tank in India which was used by
the natives for drinking purposes. In his early investigations he
found that rapid multiplication may occur upon the surface of moist
linen.
Resistance and Vitality. — If a culture be spread on a cover-glass
and exposed to the action of the air at room temperature the bacilli
will be dead at the end of two or three hours, unless the layer of cul-
ture is very thick, in which case it may take twenty-four hours or
more to kill all the bacilli. This indicates that infection is not pro-
duced by means of dust or other dried objects contaminated with
cholera bacilli. The transmission of these organisms through the
air, therefore, can only take place for short distances, as by a spray
of infectious liquids by mechanical means — as, for instance, the
breaking of waves in a harbor, on water-wheels, etc., or in moist
wash of cholera patients.
The cholera bacillus is also injuriously affected by the abundant
growth of saprophytic bacteria. It is true that when associated with
other bacteria, if present in large numbers, and if the conditions for
their development are particularly favorable, the cholera bacillus
may at first gain the upper hand, as in the moist linen of cholera
patients, or in soil impregnated with cholera dejecta; but later, after
two or three davs, even in such cases, the bacilli die off and other
448 PATHOGENIC MICRO-ORGANISMS.
bacteria gradually take their place. Thus, Koch found that the fluid
contents of privies twenty-four hours after the introduction of coin-
man bacilli no longer contained the living organisms; in impure
river-water they were not demonstrable for more than six to seven
days, as a rule. In the dejecta of cholera patients they were found
usually only for a few days (one to three days), though rarely they
have been observed for twenty to thirty days, and on one occasion for
one hundred and twenty days. In unsterilized water they may also
retain their vitality for a relatively long time; thus, in stagnant well-
water they have been found for eighteen days, and in an aquarium
containing plants and fishes, the water of which was inoculated with
cholera germs, they were isolated several months later from the mud
at the bottom. In running river-water, however, they have not been
observed for over six to eight days. For the cholera organisms the
conditions favorable to growth are a warm temperature, moisture, a
good supply of oxygen, and a considerable proportion of organic ma-
terial. These conditions are fully met with outside the body in but
very few localities.
The comma bacillus has the average resistance of spore-free bac-
teria, and is killed by exposure to moist heat at 60° C. in ten minutes,
at 95° to 100° C. in one minute. The bacilli have been found alive
kept for a few days in ice, but ice which has been preserved for several
weeks does not contain living bacilli.
Chemical disinfectants readily destroy the vitality of cholera vibrios.
For disinfection on a small scale, as for washing the hands when
contaminated with cholera infection, a 0.1 per cent, solution of
bichloride of mercury, or a 2 to 3 per cent, solution of carbolic acid
may be used. For disinfection on a large scale, as for the disinfection
of cholera stools, strongly alkaline milk of lime is an excellent agent.
The wash of cholera patients, contaminated furniture, floors, etc.,
may be disinfected by a solution of 5 per cent, carbolic acid and soap
water.
Pathogenesis. — Not one of the lower animals is naturally subject to
cholera, nor has any contracted the disease as the result of the in-
gestion of food contaminated with choleraic excreta or from the in-
oculations of pure cultures of the spirillum, either subcutaneously
or by the mouth. It has been shown that the comma bacillus is
extremely sensitive to the action of acids, and is quickly destroyed
bv the acid secretions of the stomach of man or the lower animals,
when these secretions are normally produced. Koch sought to pro-
duce infection in guinea-pigs per vias naiurales by first neutralizing
the contents of the stomach with a solution of carbonate of soda —
5 c.c. of a 5 per cent, solution injected into the stomach through a
pharyngeal catheter — and then after a while administered through
a similar catheter 10 c.c. of a liquid into which had been put one or
two drops of a bouillon culture of the comma bacillus. The animal
then receives a dose of 1 c.c. of tincture of opium per 200 grams of
body-weight, introduced into the abdominal cavity, for the purpose
THE CHOLERA SPIRILLUM, 449
of controlling the peristaltic movements. As a result of this treat-
ment the animals are completely narcotized for about half an hour,
but recover from it without showing any ill eflfects. On the evening
of the same or the following day the animal shows an indisposition
to eat and other signs of weakness, its posterior extremities become
weak and apparently pjiralyzed, and, as a rule, death occurs within
forty-eight hours with the symptoms of collapse and fall of tempera-
ture. At the autopsy the small intestine is found to be congested
and filled with a watery fluid, containing the spirillum in great numbers.
These results, however, are somewhat weakened by the fact that
experiments made with some other bacteria morphologically similar
to the comma bacillus of Koch, but specifically different, occasionally
produced death when introduced in the same way into the small
intestines of guinea-pigs.
There are several cases on record which furnish the most satis-
factory evidence that the cholera spirillum is able to produce the
disease in man. In 1884 a student in Koch's laboratory in Berlin,
who was taking a course on cholera, became ill with a severe attack
of cholera. At that time there was no cholera in Germany, and the
infection could not have been produced in any other way than through
the cholera cultures which were being used for the instruction of
students. In 1892 Pettenkofer and Emmerich experimented on
themselves by swallowing small quantities of fresh cholera cultures
obtained from Hamburg. Pettenkofer was affected with a mild at-
tack of cholerine or severe diarrhoea, from which he recovered in a
few days i^ithout any serious effects, but Emmerich became very
ill. On the night following the infection he was attacked by fre-
quent evacuations of the characteristic rice-water type, cramps, tym-
panites, and great prostration. His voice became hoarse, and the
secretion of urine was somewhat diminished, this condition lasting for
several days. In both cases the cholera spirillum was obtained
in pure culture from the dejecta. Finally, there is the case of Dr.
Oergel, of Hamburg, who accidentally, while experimenting on a
guinea-pig, allowed some of the infected peritoneal fluid to squirt into
his mouth. He was taken ill and died a few days afterward of typical
cholera, though at the time of his death there was no cholera in the
city. These accidents and experiments would certainly seem to
prove conclusively the capability of pure virulent cholera cultures to
produce the disease.
Lesions in Man. — Cholera in man is an infective process of the
epithelium of the intestine, in which the spirilla clinging to and be-
tween the epithelial cells produce a partial or entire necrosis and final
destruction of the epithelial covering, which thus renders possible
the absorption of the cholera toxin formed by the growth of the
spirilla. The larger the surface of the mucous membrane infected
and the more luxuriant the development of bacilli and the produc-
tion of toxin, the more pronounced will be the poisoning, ending
fatally in a toxic paralysis of the circulatory and thermic centres.
29
450 PATHOGENIC MICRO-ORGANISMS,
On the other hand, however, there may be cases where, in spite of the
large number of cholera bacilli present in the dejecta, severe symp-
toms of intoxication may be absent. In such cases the destruction
of epithelium is not produced or is so slight that the toxic substance
absorbed is not in sufficient concentration to give rise to the algid
stage of the disease, or for some reason the spirilla do not produce
toxin to any extent. In no stage of the disease are living cholera
spirilla found in the organs of the body or in the secretions.
Distribution in the Body. — The cholera spirilla are found only in
the intestines and are believed never to be present in the blood or
internal organs. The lower half of the small intestine is most af-
fected, a large part of its surface epithelium becoming shed. The
flakes floating in the rice-water discharges consist mostly of masses
of epithelial cells and mucus, among which are numerous spirilla.
The spirilla also penetrate the follicles of Lieberkiihn, and may be
seen lying between the basement membrane and the epithelial lining,
which become loosened by their action. They are rarely found in
the connective tissue beneath, and never penetrate deeply. In more
chronic cases other microorganisms play a greater part and deeper
lesions of the intestines may occur.
Oommnnicability. Origin of Epidemics. — From this fact and
other known properties of the cholera spirillum, which have alreadv
been referred to, several important deductions may be made with re-
gard to the mode of transmission of cholera infection. In the first
place, the bacilli evidently leave the bodies of cholera patients, chiefly
in the dejections during the early part of the disease (they have usu-
ally disappeared after the fourth to the fourteenth day, but may re-
main for many months), and only these dejections, therefore, and
objects contaminated by them, such as bed and body linen, floors,
vaults, soil, well-water and river-water, green vegetables wet with
infected water, etc., can be regarded as possible sources of infection.
There is a special limitation even in these sources of infection, owing
to the fact that this spirillum is so easily destroyed by desiccation
and crowded out by saprophytic organisms. Thus, as a rule, onlj
fresh dejections and freshly contaminated objects are liable to con-
vey infection; a day after they have become completely dry there i>
little danger. Further, we must conclude from the distribution of
the cholera bacillus in the body and from experiments upon animal>
that the commonest mode of infection is by way of the mouth, and
chiefly by means of water used for drinking purposes, for the prep-
aration of food, etc. In recent times cholera spirilla have been found
not infrequently in water (wells, water-mains, rivers, harbors, and
canals) which has become contaminated by the dejections of cholera
patients.
As in other infectious diseases, not everyone who is exposed it>
infection is attacked by cholera. The bacilli have been found dur-
ing cholera epidemics in the dejections of healthy individuals with-
out any pathological symptoms. Abel and Claussen, for example,
THE CHOLERA SPIRILLUM, 451
in 14 out of 17 persons belonging to the families of 7 cholera pa-
tients, found cholera vibrios, in some of them for a period of four-
teen days. In Hamburg there were 28 such cases of healthy cho-
leraic individuals with absolutely normal stools. It is evident, there-
fore, that an individual susceptibility is requisite to produce the disease.
In the normal healthy stomach the hydrochloric acid of the gastric
secretions may destroy the spirilla; and, finally, *the normal vital resist-
ance of the tissue cells to the action of the cholera poison may be
taken into consideration. According to the greater or less power of
this vital resistance of the body the same infectious matter may give
rise to no disturbance whatever, a slight diarrhoea, or it may lead to seri-
ous results. Furthermore, it may be accepted as an established fact
that recovery from one attack of cholera produces personal immunity
to a second attack for a considerable length of time. This does not
appear to depend upon the severity of the attack, for cases are recorded
of persons who were apparently not sick at all and yet in whom an
acquired immunity was produced. How long this immunity lasts is
not positively known, but probably for a month or more, so that the
same person is not likely to be taken ill again with cholera during an
epidemic.
On the other hand, we may take it for granted that susceptibility
to cholera may be acquired or increased. For instance, there is no
doubt that gastric and intestinal disorders produced by overheating,
etc., may act as contributing causes to the disease. Other predis-
posing causes are general debility from poverty, hunger, disease, etc.
Flies which have fed or lighted on the discharges of cholera patients
or on things contaminated by them have been found to carry the organ-
isms not only on their feet, but also in their bodies for at least twenty-
four hours. Food contaminated by flies is therefore a possible souce
of infection. Even vegetables such as lettuce may be contaminated
by infected water. Cholera, as a rule, starts from Asia, travels to
Europe, and i^ carried by vessels to America.
Oholeiu Toxins. — Koch was the first to assume, as the result of
his investigations, that the severe symptoms of the algid stage of cholera
were due to the effects of a toxin produced by the growth of the comma
bacillus in the intestines.
In 1892 Pfeifler published an account of his elaborate researches
relating to the cholera poison. He found that recent aerobic cultures
of the cholera spirillum contain a specific toxic substance which is
fatal to guinea-pigs in extremely small doses. There is extreme
collapse, with subnormal temperature. This substance stands in
close relation with the bacterial cells, and is perhaps an integral
part of them. The filtrate of a recent cholera culture contains usually
only moderate amounts of toxic substances. The spirilla may be killed
by chloroform, thymol, or by desiccation, without apparent injury to
the toxic power of this substance, but subjected to 60° C. some of the
toxins are destroyed. Metchnikoff, Roux, and others have shown
that living, highly virulent cultures produce at times highly poisonous
452 PATHOGENIC MICRO-ORGANISMS.
toxins, the 0.2 c.c. of filtrate of a three- to four-day culture killing 100
grams of guinea-pig. The living culture in 2 to 4 c.c. of nutrient bouil-
lon contained in collodion sacs, when placed in the peritoneal cavity
of guinea-pigs, produced symptoms of poisoning and death in a few
days. Sacs containing the dead vibrios produced little eflfect. There
appears to be, therefore, considerable difference between the intracel-
lular and the soluble extracellular toxins.
Oholera Immanity and Bacteriolysins. — Koch found in his ani-
mal experiments that recovery from an intraperitoneal infection
with small doses of living cholera vibrios produced a certain immunity
against larger doses, though the animals inoculated were not very
much more resistant to the cholera poison than they were originally.
In 1892 Lazarus observed that the blood serum of persons who had
recently recovered from an attack of cholera possessed the power of
preventing the development in guinea-pigs of cholera bacilli, which
in these animals are rapidly fatal when injected intraperitoneally,
while the serum of healthy individuals had no such effect. This
specific change in the blood is observed to take place from eight to
ten days after the termination of an attack of cholera, and reaches its
maximum during the fourth week of convalescence, after which it de-
clines rapidly and disappears entirely in about two or three months.
Similar antitoxic or bactericidal substances develop in the serum of
guinea-pigs, rabbits, and goats, when these animals are immunized
artificially against cholera by subcutaneous or intraperitoneal injections
of living or dead cultures. These specific substances present in the
blood of cholera-immune men and animals act only upon organisms
similar to those with which they were infected; but, as Pfeiffer showed,
this specific relation, which is found to exist between the antibacterial
and protective substances produced during immunization and the bac-
teria employed to immunize the animals, is not confined to cholera.
The discovery, moreover, of this specific reaction of the blood serum of
immunized man and animals when brought in contact with the spirilla,
has given us an apparently reliable means of distinguishing the cholera
from all other vibrios, and the disease cholera from other similar affec*
tions, both of which have proved to be of great value, particularly in
obscure or doubtful cases, in which heretofore the only method of
differential diagnosis available — viz., by cultural tests — was often
unsatisfactory.
Anticholera Inoculations. — Within the last ten years Haffkine, in
India, has succeeded in producing an artificial immunity against
cholera infection by means of subcutaneous injections of cholera cul-
tures. Two or three injections are necessary to give the greatest
amount of protection. Animals treated by this method are refrac-
tory to intraperitoneal inoculations, but not to intestinal injections,
when fed bv Koch's method. In the intestines the bacteria seem to be
outside the influence of the bactericidal properties of the blood, and the
absorption of toxins is too great to be neutralized by the small amount
of antitoxin. In over 200,000 persons inoculated under his super-
THE CHOLERA SPIRILLUM, 453
vision the results obtained would seem to show a distinct protec-
tive influence in the preventive inoculations. Great care must be
taken that the vaccine is sterile. Through lack of care a number of
cases of tetanus were caused by a contaminated vaccine. The injec-
tions produce local swelling and a short rise of temperature with
possibly headache.
Serum Therapy. — Up to the present no successful results have
been reported. The outlook is also not very hopeful. A bactericidal
serum can be developed, but this has not saved animals showing toxic
symptoms.
Agglntinms. — ^Five to ten days after infection (natural or experi-
mental) agglutinins appear in the blood of man or animal. These
are at least in part specific. Their presence in the blood is of diag-
nostic importance, ^\^len present in great amount, such agglutinins
can be used for identifying doubtful spirilla. In their agglutina-
tion with a specific serum they are also alike. Some cultures ag-
glutinate with more difficulty than others, so that the same serum
may agglutinate different cultures in dilutions varying from 1 : 1000
up to 1 : 10,000. Such a serum would not agglutinate cholera-like
spirilla above a 1 : 50 dilution. Epecially among isolated cases of
cholera-like diseases spirilla are met with which do not agree in ag-
glutination characteristics. «
Variations of the Oholera Spirillam. — ^From the great majority of
all cases of epidemic cholera examined, cholera spirilla agreeing in
all essential characteristics have been obtained, usually in great
numbers and often in almost pure culture.
Biological Diagnosis of the Oholera Vibrio. Plan of Procedure.
— A, Dejecta (fluid) or intestinal contents of a cholera patient or
cholera suspect.
1. Use one drop (one platinum loop) for gelatin-plate cultures,
making two dilutions. Do this in duplicate or triplicate. Cultivate
at 22° C.
2. Inoculate a couple of bouillon tubes and a couple of Dunham's
1 per cent, peptone solution with one drop each, and place them in
the incubator (37° to 38° C.) for six to eight hours.
3. Examine a drop of the dejecta in a hanging drop.
4. Examine a drop of the dejecta in stained cover-glass prepara-
tion.'
5. Make gelatin plates from one drop taken from the surface of
each of the bouillon and peptone solution tubes and cultivate at
22° C.
* These direct microscopic examinations of the intestinal contents are, as a
rule, very unsatisfactory, at least in those in which the symptoms are not marked.
In a few the spirals will make up from 50 to 100 per cent, of the bacteria present.
In most of the cases during the last epidemic in New York Dunham found abun-
dance of columnar epithelium from the intestinal mucous membrane, numerous
straight, thick bacilli, and only a few curved bacilli or segments of spirals — too
few to identify. Plate cultures from these showed from 20 to 80 per cent, of all
the colonies developing to be cholera spirilla.
454 PATHOGENIC MICRO-ORGANISMS.
6. As soon as the plates (see 1 and 5} are sufficiently developeil
(thirty-six to forty-eight hours) fish the suspected cholera colonies
and use the material for the following procedures:
7. Inoculate six or eight peptone tubes (1 per cent, peptone and
0.5 per cent, NaCt in distilled water) and place them at once in the
incubator. Note the time.
8. Examine banging drop for form, size, and motility (and
arrangement).
9. Make stained cover-glass preparations and examine.
10. Then try indol reactibn with the same tubes.
11. While these tubes are incubating use material from the sus-
pected colonies on the plates (1 and 5) for hanging-drop cultures.
12. Meanwhile make stained cover-glass preparations from other
colonies of suspected cholera on the plates (1 and 5).
13. Make gelatin-tube cultures from colonies on plates (1 and h)
and study cultures daily for five or six days, to observe the shape of
growth along the line of puncture.
B. Suspected water.
Add to 500 c.c. or 1 litre of the water to be examined enough peptone-
salt solution (20 per cent, peptone and 10 per cent. NaCl) to make
a 1 per cent, solution of peptone. Then proceed as in A.
Bpedflc Ssnmi Reactions. — All authors now agree that the difTei^
entiation of the cholera vibrio from other similar vibrios cannot
always be made by the cultural method, nor is the usual inoculation
of animals sufficient. For this purpose serum is employed either by
making intraperitoneal injections of a surely fatal dose of the sus-
pected spirillum along with the serum of animals immunized tn
undoubted cholera cultures, so as to note whether specific protection
is afforded, or the agglutination test is carried out in .such a way a.s tn
determine if specific agglutination of the spirilla occurs.
SPIBILLA MORE OR LESS ALLIED TO THE CHOLERA 8PIBIU.nH.
The examinations of the stools of persons suffering from cholera
have revealed, in a small percentage of cases, spirilla resembling
either very closely or having a fair <iegree of .similarity to the true
cholera organisms. Further, in a small percentage of cases having
choleraic symptoms no true cholera vibrios have been found, but in-
stead other spirilla resembling them more or less closely.
vo or more end flagella, in size, in ,
■y may be identical in the tests com-
er in the specific agglutination and
. spirilla and among themselves.
:, Gottschlich obtained from sixteen
true spirilla, and found every one
rom all others. Some were patho-
ilation of a small quantity into the
cal in their development in nntrienl
THE CHOLERA SPIRILLUM. 455
gelatin. None of these microorganisms injected into animals in-
duced production of agglutinins for the true cholera spirilla.
Kolle and Gottschlich consider these various spirilla found by
them in Egypt as well as others found by different investigators in
India, Germany, and elsewhere to be saprophytes. It is more probable,
in the writer's opinion, that some of them must be considered as
bearing a part in exciting a cholera-like disease, but that they are not
very pathogenic and require very favorable conditions, probably
long-continued, before they can exert their action.
Some special varieties of spirilla resembling those of cholera have
received especial attention on account of having been obtained before
it was known that so many cholera- p,^ ,^7
like vibrios existed. The vibrio
Berolinensis, cultivated by Neisser
from Berlin sewage-water; the vibrio
Danuhicus, cultivated by Hauser
from canal-water, and the vibrio of
Massowah, cultivated by Pasquale i
from a case during an epidemic of
cholera, all are negative to the specific |
serum reactions, and differ in the
number of terminal flagella or in other
characteristics. Cunningham foimd
a number of such spirilla in cases of
apparently true cholera in India.
Some of these may have been true spirillum of Funkier and Prior,
ctioiera spirilla and others may nave
had some relationship to the disease in the person from which they
were derived.
Spirillmu of Tinkler and Prior.— Because of their prominence in
literature and their frequent use in, teaching, the spirillum of Finkler
and Prior, that of Metchnikoff, and that of Deneke are of consider-
able interest.
Finkler and Prior, in 1884, obtained from the fieces of patients
with cholera nostras, after allowing the dejecta to stand for some
days, a spirillum which is of interest mainly because it simulates
the comma bacillus of Koch, but differs from it in several cultural
peculiarities.
Morphology. — Somewhat longer and thicker than the spirillum of
Asiatic cholera and not so uniform in diameter, the central portion
being usually wider than the pointed ends.
Biology. — An aerobic and facultative anaerobic, liquefying spiril-
lum. Does not form spores. Upon gelatin plates small, white, punctt-
form colonies are developed at the end of twenty-four hours. These
are round, but less coarsely granular, <larker in color, and with a more
.sharply defined border than the comma bacillus. Liquefaction of
the gelatin around these colonies progresses rapidly, and at the end
of forty-eight hours is usually complete in plates where they are numer-
456 PATHOGENIC MICRO-ORGANISMS,
ous. In gelatinr-aiick cultures liquefaction progresses much more
rapidly than in similar cultures of the cholera spirillum, and a stocking-
shaped pouch of liquefied gelatin, already seen after forty-eight hours,
is filled with a cloudy Uquid. The liquefaction increases, and in
twenty-four hours more reaches the sides of the tube in the upper part
of the medium; by the end of the week the gelatin is usually completely
liquefied. Upon the surface of the liquefied medium a whitish film
is seen. Upon agar there is a somewhat more luxuriant growth
than is seen with the cholera vibrio. Upon 'potato this spirillum grows
at room temperature and produces a slimy, grayish-yellow, glistening
layer which soon extends over the entire surface. The cholera spiril-
lum does not grow at room temperature, and in the incubator produces
a thin, brownish layer. The absence of agglutination with a suitable
dilution of the serum of an animal immunized to the cholera spirillum
is a valuable diflFerential sign.
In 1884 Miller observed a curved bacillus in a hollow tooth, which
from its behavior in microscopic preparations, in cultures, and ani-
mal experiments, is probably identical with the Finkler and Prior
spirillum. Very similar spirilla have been found by others.
Spirillam of Metchnikoff.— Discovered in 1888, in Odessa, by
Gamaleia in the intestinal contents of fowls dying of an infectious
disease, which prevails in certain parts of Russia during the summer
months, and which presents symptoms resembling fowl cholera.
Gamaleia 's experiments show that this organism is the cause of the
disease mentioned. It has since been found by Pfuhl and Pfeiffer
in the water of the Spree at Berlin and in the Lahn by Kutchler.
Morphology. — Morphologically this spirillum is almost identical
with the cholera spirillum. In the blood of inoculated pigeons the
diameter is sometimes twice as great as that of the cholera spirillum,
and almost coccus-like forms are often found. A single, long, un-
dulating flagellum is attached to one end of the spiral filaments or
curved rods.
Stains with the usual aniline colors, but not by Gram's method.
Oultural Characters. — Upon gelatin plates the vibrio Metchnikoff
grows considerably faster than the cholera vibrio; small, white, punc-
tiform colonies are developed at the end of twelve hours; these rapidly
increase in size and cause liquefaction of the gelatin within twenty-
four to thirty hours. At the end of three days large, saucer-like areas
of liquefaction may be seen, the contents of which are turbid, as a
rule. In gelatin-stick cultures the growth is almost twice as rapid
as the cholera bacillus. In bouillon at 37° C. development is very rapid,
and the liquid becomes clouded and opaque, and a thin, wrinkled film
forms upon the surface. On the addition of the pure sulphuric acid
to twenty-four-hour peptone cultures a distinct nitrosoindol reaction is
produced. Milk is coagulated and acquires a strongly acid reaction.
The spirillum is not agglutinated by the specific cholera agglutinin.
PaUlOgenesis. — The vibria of Metchnikoff is pathogenic for fowls,
pigeons, and guinea-pigs. A small quantity of a virulent culture
THE CHOLERA SPIRILLUM. 457
fed to chickens and pigeons causes their death with the local and
general symptoms of fowl cholera. At the autopsy the most con-
stant appearance is hypercemia of the entire alimentary canal. A
grayish-yellow liquid, more or less mixed with blood, is found in
considerable quantity in the small intestine. In the watery fluid
large numbers of spirilla are found. A few drops of a pure culture
inoculated subcutaneously in pigeons produce septicaemia and cause
their death in twelve to twenty-four hours.
In contradistinction to the pathogenic virulence of these spirilla
for pigeons and guinea-pigs, the cholera spirillum is much less patho-
genic. Pigeons are not killed by the intramuscular inoculation of
pure fresh cultures of the vibrio cholerse. The pathogenic action of
the vibrio Metchnikoff upon pigeons and guinea-pigs, producing in
these animals general septicaemia and death, is, therefore, a char-
acteristic point of difference between this and the spirillum of Asiatic
cholera.
Within recent years numerous other somewhat similar spirilla, the
so-called "water vibrios," have been found while looking for the
cholera spirillum.
CHAPTER XXXV,
PATHOGENIC MICRO-ORGANISMS BELONGING TO THE HIGHER
BACTERIA (TRICHOMYCETES).
The members of the higher bacteria which are pathogenic for man
have as yet been incompletely studied and classified. The following
divisions serve as an attempt at differentiation:
1. Leptoihrix grows in stiff, almost straight threads, in which
division processes are seldom or never observed, and no branching has
been seen.
2. Cladothrix grows in threads which rapidly fragment and produce
false branching, that is, the terminal cell remains partly attached, but
is pushed to one side by further growth from the parent thread, thus
a Y-shaped growth is produced, and then bacillary characteristics in old
cultures.
3. Actinomyces grows in threads with true branching. No spores
have been observed. It is characterized by the radiating wreath-
like forms which it alone produces in the living body.
4. Noardia (Streptothrix) grows in threads which produce abund-
ant true branching, later there is fragmentation, and formation of
conidia, which serve as organs of propagation, and in this sense
may be considered as spores.
Foulerton considers all organisms in the group classed as higher bacteria
as belonging to a single genus, streptothrix, which he places with the hy-
phomycetes, or mold fungi, because of their growth in branching threads from
spore-like bodies. He says that streptothrix and actinomyces are absolutely
synonymous terms, and that the majority of pathologists consider them so.
It seems to us, however, that more minute work, both clinical and experi-
mental, should be done on this group of infections before this classification
can be accepted. The term streptothrix, too, is misapplied, since it was uscni
in 1839 for a mold (see p. 465).
These higher bacteria may rightly be considered, according to their
development, as a transition group between the simple bacteria and
the more highly developed fungi.
The nocardia group of microorganisms while having many affinities
with the bacteria seem to be more closely related to the true moulds
than any of the others. They develop from spore-like bodies into
cylindrical dichotomously branching threads, which grow into colonies
the appearance of which suggests a mass of radiating filaments, lender
favorable conditions certain of the threads become fruit hyphae, and
these break up into chains of round, spore-like bodies, which do not,
however, have the same staining reactions nor resisting powers as
bacterial spores. The tubercle, grass and diphtheria bacilli are by
458
MICRO-ORGANISMS BELONGING TO HIGHER BACTERIA, 459
some believed properly to belong in this group, on account of the
apparent branching forips developed by them under certain conditions,
but if not classed with the true bacteria, they should either be put ii)
a group by themselves or be classed with the cladothrix group since
their apparent branching takes place in a manner similar to that
described as occurring in the latter group.
Foulerton and his associates have made an extensive study of this
group of microorganisms both saprophytic and parasitic (see bibli-
ography), and they call attention to the acid-fast character of some
of the varieties and of the apparent relationship of the group to
B. tuberculosiSy B. mallei, and B, diphtherioB, To us, however, the re-
lationship does not seem to be close enough to place all of these or-
ganisms in one group. We have shown (see p. 17) that the apparent
branching in B. diphihericB is not a true branching.
Leptothriz Infections. — Leptothrix forms are frequently found in
the human mouth {Leptothrix huccalis), and one or two writers have
claimed that under certain conditions these may become pathogenic,
but since no corroborative work has been done, and very little is
known about the group, no opinion can be formed of the worth of these
observations.
Oladothriz Infections. — The organisms found in the comparatively
few cases which have been considered by their observers to be due to
cladothrix have not been minutely enough studied to decide definitely
as to their true or false branching, the characteristic chosen to separate
them from the nocardia; hence it is difficult to separate the two groups,
but an attempt should be made, since the difference said to exist
between them is a vital one, from a morphologic standpoint. Clinically,
however, according to the reports, the cases cited are very similar to
those said to be due to nocardia and to actinomyces.
Gasten found in a case of clinically typical actinomycosis, in which
abscess cavities were found along the spinal column, not the usual
actinomyces in the yellow, granular pus, but a fine mass of filament.
Cultures grew on all the ordinary media, best at incubator tempera-
ture, but also at lower temperature on gelatin. The gelatin stick
culture, which was especially characteristic, formed on the surface
a whitish button; delicate threads stretched out in all directions
from the point of inoculation. On agar and potato rumpled, folded
films with white deposit on the surface, which contained spores.
Animal inoculation gave positive results only in a few cases of intra-
peritoneal injection of rabbits and guinea-pigs. Purulent nodules
were found in the peritoneum. Gasten called the organism Cladothrix
liquefaciens,
Eppinger found in post-mortem examination of a case of chronic
cerebral abscess, which was the result of purulent meningitis, in the
pus and abscess walls, etc., a delicate fungoid growth which he suc-
ceeded in cultivating on various media. On sugar agar it formed
yellow, rumpled colonies which finally developed into a skin. On
potato it grew rapidly, but the colonies remained small, at first a
460 PATHOGENIC MICRO-ORGANISMS,
white, granular deposit, which afterward turned red, and on the
twentieth day resembled a crystallized almond. It did not grow well
on gelatin. In bouillon it formed on the surface a small white
granule, which became deeper in the centre as it grew and sank to
the bottom as a white deposit. The bouillon remained clear.
Microscopically, the fungus consisted of fine threads without branches
which exhibited distinct motility. No flagella were observed. It
was judged to be a cladothrix, to which the name **asteroides" was
given by the author. It proved to be quite pathogenic for rabbits
and guinea-pigs, and produced an infection of pseudo-tuberculosis.
Mice were not affected by inoculation.
THE MICRO-ORGANISM OF A0TINOMT0O8IS.
The little clumps produced by this parasite were first seen by Von
Langenbeck in 1845 and the organism was later discovered by Bol-
linger (1877) in the ox. It was given the name of actinomyces, or ray
fungus, by the botanist Harz.
The characteristics of the microorganisms, first described minutely
by Bostroem (1890) and by Wolf and Israel (1891), differed greatly
and have led to confusion. Bostroem's organism grew best aerobically
and developed well at room temperature. He noted the intimate
relation of the organism with fragments of grain, and this led to
the finding of similar microorganisms in the outer world on grains,
grasses, etc.
There is no doubt that some suppurative processes have been due
to organisms of these characteristics, but they do not seem to excite
true actinomycosis.
Wolf and Israel described a microorganism from two human
cases, which differs from that described by Bostroem, but agrees
with the microorganisms obtained by most of the more recent
investigators. It grew best under anaerobic conditions and did not
grow at room temperature. Its growth was much less luxuriant than
Bostroem's microorganism. On the surface of anaerobic agar slant
cultures on the third, fourth, and fifth day numerous minute iso-
lated dew-dro|>like colonies appeared, the largest pinhead in size.
These gradually became larger and formed ball-like, irregularly
rounded elevated nodules varying in size up to that of a millet-seed,
exceptionally attaining the size of a lentil or larger. As a rule, the
colonies did not become confluent, and an apparently homogeneous
layer of growth was seen to be made up of separate nodules if ex-
amined with a lens. In some instances the colonies presented a
prominent centre with a lobulated margin and appeared as rosettes.
A characteristic of the colonies was that they sent into the agar root-
like projections. In aerobic agar slant cultures no growth or a slow
and very feeble growth was obtained. In stab cultures the growth
was sometimes limited to the lower portion of the line of inoculation
or was more vigorous there. In bouillon, after three to five days,
MICRO-ORGAXISMS BELOSGISG TO HIGHER BACTERIA. 461
growth appeared as small white flakes, partly floating and partly
collected at the bottom of the tube. Growth occurred in bouillon
under aerobic conditions, but was better under anaerobic conditions.
The organisms here grow in branching and interlacing filaments, which
later tend to break into segments (see Fig. 148). The microorganism
in smear preparations from agar cultures appeared chiefly as short
homogeneous, usually straight, but also comma-hke or bowed rods,
whose length and breadth varied. In many cultures short plump
rods predominated, and in others longer, thicker, or thinner individ-
uals were more numerous. The ends of the rods often showed
oval or ball-like swellings. Swollen clubs were formed irregularly in
the presence of blood or serous fluids.
Some twenty guinea-pigs and rabbits were inoculated, most of them
in the peritoneal cavity, with pieces of agar culture. Eighteen animals
were killed after four to seventeen weeks, and four were still alive seven
to nine months after inoculation. Seventeen rabbits and one guinea-
pig showed at the autopsy tumor growths mostly in the peritoneal
cavity and in one instance in the spleen. In the four animals still
living tumors were to be felt in the abdominal wall. The tumors
in the peritoneal cavity were millet-seed to plum size, and were
situated partly on the abdominal wall and partly on the intestines,
the omentum, the mesentery, and in the liver or in adhesions. While
the surface of the smaller tumors was always smooth, the surface
of the larger tumors showed small hemispherical prominences, giving
them the appearance of conglomerates of smaller tumors. On
section the larger tumors presented a tough capsule from which
anastomosing septa extended inward inclosing cheesy masses. Micro-
scopic examination of the tumors showed in all cases but one the
presence of typical actinomyces colonies, in most cases with typical
462 PATHOGENIC MICRO-ORGANISMS.
"clubs." The general histological appearance of the tumors was
like that of actinomycotic tissue.
Wolf in a later paper reports that an animal inoculated in the
peritoneal cavity with a culture of the same organism had lived a
year and a half. At the autopsy several tumors were found in the
peritoneal cavity, and in the liver a large typical tumor in which were
many colonies which by microscopic examination were shown to l>e
typical club-bearing actinomyces colonie.s.
Wright in 1905 made an extensive study of actinomycosis ami
added greatly to our knowledge of it.
Naked-eye Appearance of Colonies of Parasite in Tissues.— In
both man and animals they can be readily seen in the pus from the
affected regions as smallj white yellowish or greenish granules of pin-
head size (from 0.5 to 2 mm. in diameter). When pus has not formed
they lie embedded in the granulation tissue.
Microscopic Appearance. — Microscopically, these bodies are seen
to be made up of threads, which radiate from a centre and present
bulbous, club-like terminations {Fig. 149), These club-like termi-
nations are characteristic of the actinomyces. They are generally
arranged in pairs, closely crowded together, and are very glistening
< 325 diuneton.
in appearance. They are more common in bovine than in human
lesions. They have been thought to be reproductive elements, but
they are probably simply a reaction of the filament end to the host
ti.ssue. The threads which compose the central mass of the granules
are from 0.3/i to 0.5/( in diameter: The threads show true branching
and in the older colonies show a segmentation which gives them the
appearance of chains of cocci. Sometimes the whole centre of the
colonies seems to be a mass of cocci, some of which may be true
cocci from a mixed infection; the clubs are from 6/( to 8/i in diameter.
The threads are stained with the ordinary aniline colors, also by
Gram's solution; when stained with gentian violet and by Gram's
MICRO-ORGANISMS BELONGING TO HIGHER BACTERIA. 463
method the threads appear more distinct than when stained with methy-
lene blue. The clubs lose their stain by Gram's method and take
the contrast strain.
Isolation of Actinomyces. — There are two cultural varieties, one
of which grows aerobically and the other anaerobically. The aerobic
variety is grown with difficulty. A large number of solidified blood
serum or serum agar tubes are inoculated with the hope that one or two
will develop a growth. The culture appears much like one of tubercle
bacilli. It grows, however, into the medium and takes on a yellowish
hue. Wright* recommends that granules, preferably obtained from
closed lesions, are first thoroughly washed in sterile water or bouillon
and then crushed between two sterile glass sides. In bovine cases
make sure the granule has filamentous masses, for if not no culture
will grow. The crushed granule is transferred to a tube of melted
1 per cent, glucose agar at 40° C. The material is thoroughly distrib-
uted by shaking and the tube placed in the incubator. A number
of granules after washing should be placed on the inside of a sterile
test-tube and allowed to dry. In this way, should the material be
contaminated, the drying of the granules for several weeks may kill
off the other bacteria. The tube should be examined daily. If a
number of living filaments were added to the agar a large number of
colonies will develop. These will be most numerous in the depth in a
zone five to twelve millimetres below the surface.
Experimental Inoculation in Animals. — True progressive infec-
tion is rarely or never obtained by the injection of pure cultures in
rabbits, guinea-pigs, or larger animals. It seems as if cultures on
artificial media must lose in virulence or that the disease is produced
by the entrance of the organism along with some irritating body.
\Vhen animals are inoculated, the cultures form the characteristic
** club ''-bearing colonies in the tissues of the experimental animals.
These colonies are either enclosed in small nodules of connective
tissue or are contained in suppurative foci within nodular tumors
made up of connective tissues in varying stages of development. The
most extensive lesions show little progressive tendency, and in only
a very few instances does multiplication of the microorganism in the
body of the inoculated animal take place.
Wright does not accept the prevalent belief, based on the work of
Bostroem, Gasperini, and others, that the specific infectious agent of
actinomycosis is to be found among certain branching microorgan-
isms, widely disseminated in the outer world, which differ profoundly
from actinomyces bovis in having spore-like reproductive elements.
He thinks that these forms belong to a separate genus, Nocardia, and
that those cases of undoubted infection by them should be called nocar-
diosis and not actinomycosis. The term actinomycosis should be
used only for those inflammatory processes the lesions of which contain
the characteristic granules or **drusen." That a Nocardia ever forms
* Journal of Medical Research, May, 1905.
J
464 PATHOGENIC MICRO-ORGANISMS.
these characteristic structures in lesions produced by it in man or
cattle has not been convincingly shown.
As the actinomycosis microorganism does not grow well on the
ordinary culture media and practically not at all at room tempiera-
ture, it seems very probable that it is a normal inhabitant of the buc-
cal cavity and gastrointestinal tract and does not grow outside the
body. Many good observers, however, believe the infection is pro
duced by infected grains upon which the organism has grown as a
saprophyte. The explanation of the diflFerent results is probably that
there are different varieties of this organism.
The cultures are (Juite resistant to outside influences; dried, they
may be kept for a year or more; they are killed by an exposure of
five minutes to a temperature of 76° C.
Occurrence. — Actinomycosis is quite prevalent among cattle, in
which it occurs endemically; it is more rare among swine and horses.
Over one hundred cases have been observed in man. The disease is
rarely communicated from one animal to another and no case is known
where a direct history of human contagion has been obtained. The
cereal grains, which from their nature are capable of penetrating
the tissues, have been repeatedly found in centres of actinomycotic
infection. This usually occurs in the vicinity of the mouth, where
injuries have been accidentally caused. The microorganism may
also be introduced by means of carious teeth. Cutaneous infection
has been produced by wood splinters, and infection of the lungs by
aspiration of fragments of teeth containing the fungus. The pres-
ence of the microorganism in cereal grains, which was formerly ac-
cepted, is denied by Wright and therefore certainly placed in doubt
The further distribution of the fungus after it is introduced into the
tissues is effected partly by its growth and partly by conveyance by
means of the lymphatics and leukocytes. Not infrequendy a mixed
infection with the pyogenic cocci occurs in actinomycosis.
Characteristics of Disease in Blan and Animals. — In the earliest
stages of its growth the parasite gives rise to a small granulation tumor,
not unlike that produced by the tubercle bacillus, which contains, in
addition to small round cells, epithelial elements and giant cells.
After it reaches a certain size there is great proliferation of the sur-
rounding connective tissue, and the growth may, particularly in the
jaw, look like, and was long mistaken for, osteosarcoma. Finally,
suppuration occurs, which, according to Israel, may be produced
directly by the fungus itself.
The course of the disease is very chronic. Usually the first sign is
a point of infiltration about the lower jaw or lower on the neck. This
almost painless swelling increases and finally softens in its centre. The
necrotic tissue finally forces a passage externally or, passing downward,
infects the pleura, lungs, mediastinum, or ribs. As a rule, the disease
is not accompanied by fever. In catde the disease is usually situ-
ated in some portion of the head, especially in the jaw, tongue, or
tonsils, hence called lumpy jaw, wooden tongue, etc. Primary lung,
MICRO-ORGANISMS BELONGING TO HIGHER BACTERIA. 465
intestinal, and skin lesions are not infrequent. These local lesions
sometimes scatter and produce a general infection and the udder
may be involved.
The experimental production of actinomycosis in animals with ma-
terial directly from cases has been followed by negative or very unsat-
isfactory results, as in the case of cultures. When artificially intro-
duced into the tissues the organism is either absorbed or encapsu-
lated. If introduced in large quantities multiple nodules are appa-
rently formed in some cases, which may suggest the production of
a general infective process; but on closer inspection of these nodules
the thread-like portion of the fungus is found to have disappeared,
leaving only the remains of the club-like ends, thus showing that
no growth has taken place.
Treatment. — In 1892 Nocard showed that cases in animals might
be cured by iodide of potassium, calling attention to the fact that
Thomassen had recommended this treatment in 1885. It is given in
doses of 1^ to 2i drams once a day. Salmon and Smith (U. S. Bureau
of Animal Industry, Circular No. 96) give directions as to its use.
Mycetoma (Madura Foot). — This is a purulent inflammation of
the foot occurring primarily in warm climates. The inflammation
is accompanied by much irregular enlargement of the foot. Three
varieties of this condition have been described based upon the color
of the granules found in the diseased area: (1) white, (2) black,
and (3) red. The white variety has been studied by Musgrave and
Clegg (1907), who have isolated an organism resembling somewhat
actinomyces and somewhat the organism isolated by Wright (1898)
from a black variety of the disease which is probably a true mould.
NOOARDIA (STREPTOTHRIX) INFECTIONS.
The most familiar name of this group of microorganisms is strepto-
thrix, but, this name had already been used for another genus; there-
fore, according to the rules of nomenclature, nocardia, which name
was proposed by Trevisan in 1889 for the organism discovered by
Nocard in farcin des hceufs of cattle should be employed. Wright
calls attention to the misuse of the term streptoihriXy and gives the
reasons for the employment of the term nocardia in its 'place.
From widely scattered Idealities and at long intervals of time re-
ports have been published describing unique cases of disease produced
by varieties of microorganisms belonging to the genus nocardia.
In some of these cases points of similarity can be recognized in the
clinical symptoms and the gross pathologic lesions, while others
differ widely in both respects. They have been found in brain ab-
scess, cerebrospinal meningitis, pneumonic areas, and in other patho-
logic conditions. Eppinger injected cultures into guinea-pigs
and rabbits, and observed that they caused typical pseudotubercu-
losis. Consolidation of portions of both lungs, thickening of the
peritoneum, and scattered nodules resembling tubercles were noted
30
466 PATHOGENIC MICRO-ORGANISMS.
by Flexner in a case of human infection as due to nocardia in which
the pathologic picture of the disease resembled so nearly that of tuber-
culosis in human beings that the two diseases could be separated only
by finding the causative microorganism in each case. But in no two
cases reported up to the present time have the descriptions of the
microorganisms found agreed in all particulars. In some cases no
attempt at cultivation was made. In other cases numerous and
careful plants on various culture-media failed to develop the specific
organism. In the remaining cases in which nocardia was obtained
in pure culture the descriptions of the growth characteristics es-
sentially diflFer. In a review of the literature Tutde was able
to find the reports of only twelve cases in which nocardia was
found in sufficient abundance to have been an important, if not the
principal, factor in producing disease. These cases were all
fatal, and only once was the character of the disease recognized
during life. As the clinical symptoms and the lesions in the human
subject as well as in the animals experimentally inoculated with
nocardia often resemble those of miliary tuberculosis, so that a num-
ber of these cases have been reported as pseudotuberculosis, the
question is naturally suggested whether such cases of nocardia
tuberculosis are not more numerous than the few reported cases
would indicate. The almost universal prevalence of genuine tuber-
culosis and the extreme gravity of the disease have so long occupied
the attention and study of the medical profession that much is taken
for granted, and in cases in which the symptoms and lesions resemble
with some closeness those characteristic of the well-known disease
they may easily be set down without question to the account of the
tubercle bacillus. The cases of nocardiosis reported which simulated
tuberculosis have been fatal, and the lesions for the most part have
been widely distributed, but in a number of cases old lesions have
been found which suggest that the disease may have been localized
for a longer or shorter time, and then, by some accident, may have
become rapidly general. In this respect also these cases may re-
semble tuberculosis. Whether all cases of nocardiosis in the human
subject are general and fatal or, as in tuberculosis and actinomycosis,
whether there may be cases of localized disease which recover, are ques-
tions which have not been decided at the present time. The methods
employed to demonstrate the presence of tubercle bacilli render nocar-
dia more or less invisible. Again, unless the observer keeps in mind
the possibility of nocardia infection, he may not appreciate the im-
portance of finding slender threads with or without branches, and may
consider them accidental bacilli, or varieties of leptothrix or non-
pathogenic fungi. As the lungs have appeared to be the seat of the
primary infection in most of the cases of human nocardiosis it is very
desirable that all cases presenting the physical signs of tuberculosis,
in which repeated examinations fail to discover the tubercle bacillus,
should be systematically examined for threads. In this way alone
can the frequency of the disease be determined. Gram's method of
MICRO-ORGANISMS BELONGING TO HIGHER BACTERIA. 467
staining or the Ziehl-Neelson solution decolorized with aniline oil
seem to be the most reliable agents for demonstrating these organisms.
Varieties of nocardia are widely distributed and are not very infre-
quently met with, but as yet, with the exceptions mentioned above,
very little is known about them.
Tuttle's report of the case of general nocardia infection at the
Presbyterian Hospital gives such a good clinical, bacteriologic, and
pathologic picture of a case of this infection that a considerable
portion of it is repeated here:
Six days before her admission to the hospital her illness began
with a severe chill and fever and pain in her left side and back.
The following day the pain in the side was worse and breathing was
diflBcult. She began to cough and had some expectoration, but no
blood was noticed in the sputa. At irregular intervals she had alter-
nating hot and chilly sensations.
On admision, the patient complained of pain in the left side of
the chest, cough, fever, weakness, and prostration. Her tempera-
ture was 103° and her pulse and respirations were rapid.
The history of the disease and the physical signs indicated an
attack of acute lobar pneumonia, the area of consolidation being
small and situated in the lower part of the left upper lobe in front.
Frequent and violent coughing, with almost no expectoration, pain
in the aflFected side and in the lumbar region, restlessness and
sleeplessness, and involuntary urination were the symptoms noted
during the first four days in the hospital. The pneumonic area in-
creased somewhat and extended backward to the posterior axillary
line, and the temperature was continuous at 103° to 103.5°. On the
fifth day the temperature fell two degrees, and signs of resolution
appeared in the consolidated area. The apparent improvement,
however, was of short duration. On the sixth day the temperature
rose to 104.5°, and continued to rise each day, reaching 107.5° shortly
before death, which occurred on the ninth day in the hospital and the
fifteenth day of the disease. There were repeated attacks of profuse
sweating. On the day before her death three indurated swellings be-
neath the skin were noticed. One, on the left forearm, about the
size of a walnut, apparently contained pus. Two, of smaller size,
were situated in the right groin.
Blood cultures from a vein in the arm, taken on the sixth day,
remained sterile. The leukocyte count on the seventh day was,
36,000.
Autopsy. — On the right arm, the left forearm, the abdominal wall,
and on both thighs there are eight or ten slightly projecting, rounded,
fluctuating, subcutaneous swellings from one-half inch to one inch in
diameter. The skin over most of these nodules is unaltered, but
over the larger ones there is a slight bluish discoloration. The nod-
ules are composed of bluish-gray, thick, mucilaginous matter, which
is very tenacious and can be drawn out into long threads. The
lower lobe is thickly studded with miliary tubercles, and scattered
468 PATHOGENIC MICRO-ORGAXISMS.
through the entire lung are suppurating foci. Liver and spleen
normal. Kidneys: The description of one applies to both. The
surface is everywhere and evenly dotted with minute white spots,
which suggest septic emboli rather than tubercles. A few prominent
StrepUillirix from bouilloD ci
(From TuUlr.)
white nodules, from one-quarter inch to one-half inch in diameter,
contain thick, tenacious matter (Fig. 152). Section shows that the
entire substance of the kidney is densely studded with these minute
white granules.
The gross pathological conditions were interpreted before nocardia
was found as follows: An old tuberculous nodule in the right lung;
acute miliary tuberculosis in the right lung and peritoneum; acute
lobar pneumonia, affecting the left lung; septic infarctions and pwinic
abscesses of both lungs, heart muscle, both kidneys, pancreas, mesenteric
lymph nodes, and subcutaneous connective tissue. The miliarj- tuber-
MICRO-ORGANISMS BELONGING TO HIGHER BACTERIA. 469
cles of the right lung and peritoneum presented the characteristic
appearance of genuine tuberculosis. They were minute, hard, gray,
almost translucent nodules, while the granules in the kidneys were of
an opaque white or yellowish-white color.
Microscopic Examination. — Smears from [the abscesses beneath
the skin and on the surface of the kindeys were stained with methyl-
blue, carbol-fuchsin, and bv Gram's method. The smears resemble
those made of tenacious sputum. There is a large amount of mucoid
material containing a considerable number of leukocytes. Occasion-
ally irregularly curved, thread-shaped microorganisms are found.
They vary considerably in length and thickness, and broken and ap-
parently degenerating fragments are seen. The more slender threads
are evenly stained, but some fragmentation or beading of the pro-
toplasm can generally be observed. The thicker threads and broken
fragments show deeply stained globules and irregular bodies in a
faintly visible rod or thread-shaped covering. Some branching
threads are observed, but more commonly they are not branching.
No other microorganisms are found in the smears. Sections from
the lower lobe of the right lung, stained with heematoxylin and eosin,
show in certain places the identical microscopic appearances which
are considered characteristic of tuberculosis. Stained by Gram's
method, with care not to decolorize too completely, threads like those
described in the abscesses are found in great abundance, but rather
faintly stained. No threads can be found within the typical tubercles
with giant cells, but in the zones of small cells around them they are
seen in great numbers, winding about among the cells and forming a
sort of network. In the minute foci of small cells one or two fragments
of threads are generally seen, and a moderate number in the small
abscesses. In the areas of more diffuse infiltration these threads are
abundant. No other microorganisms can be found except in the
pneumonic area of the left lung, where some groups of cocci are seen.
The most reliable staining method and the one requiring the least
time is a modified Gram's method. The sections stained with ani-
line-gentian violet are dipped for a short time in a diluted Gram's
iodine solution and then treated with aniline oil until sufficient color
has been removed. The aniline oil is then washed out with xylol
and the section is mounted in xylol balsam.
Culture I!xperiments. — Six tubes of Loeffler blood serum were in-
oculated from the kidneys and kept at 37° C. On the third day mi-
nute white colonies appeared in some of the tubes, and on the fifth
day all the tubes showed from three to ten or twelve similar colonies
in each. The colonies increased in size until some of them reached
a diameter of one-eighth of an inch. The color, at first white, changed
to yellowish-white and then to a decided pale yellow. The well-
developed colonies cling firmly to the surface of the medium and were
not easily detached or broken up. The growths in all of the tubes
were absolutely pure, and consisted of branching threads like those
found in the sections.
470 PATHOGENIC MICRO-ORGANISMS.
Loeffler's blood serum seems to be the most suitable medium for cul-
tures. The growth on this medium is more rapid and abundant than
on any of the other media tried.
On plain agar and glycerin agar the growth is the same as on blood
serum, but is less rapidly developed.
In bouillon the growth is slow. If the tube is not disturbed or
jarred, minute white tufts are seen clinging to the surface of the glass.
But if the tube is shaken even slightly thty sink slowly to the bottom,
forming a white, fluflFy layer. These growths when undisturbed re-
semble minute balls of thistle-down. The yellow color is not apparent
even in the mass at the bottom of the tube.
It is strictly aerobic.
Morphology. — When grown on blood serum the threads are com-
paratively thick and coarse, but those growing in bouillon are very
slender and delicate. The main trunk also is often thicker than the
branches. When unstained they are homogeneous gray threads,
without any appearance of a central canal or double-contoured wall.
There is never any segmentation of the threads. When properly
stained there is always a distinct beading or fragmentation of the
protoplasm, but overstaining with fuchsin produces rather coarse,
evenly red rods. The branching is irregular and without symmetry,
and the branches are placed at a wide angle, very nearly, and some-
times quite, at right angles. This is best seen in specimens taken
from liquid media. The irregularly stellate arrangement of the
branches, which was observed by Eppinger in his original specimen,
is often seen in young organisms floated out from a liquid medium.
Spore Formation. — On examining the deep-oralige or red-colored
growth upon potato, one is surprised to find that the threads have
entirely disappeared and that the specimen consists of moderately
large cocci. These cocci represent the spore form of the organism,
and when planted upon blood serum the branching threads again
appear. The spores stain readily with carbol-fuchsin and are not
easily decolorized. They are spherical, or nearly so, but often appear
somewhat elongated, apparently from beginning germination. They
are killed by exposure to moist heat of 65° to 70° C. for an hour,
but are more resistant to dry heat. Drying destroys the threads
after a comparatively short time, but the spores retain their vitality
for an indefinite period. A dried-up potato culture retains its vitality
at the end of almost four years.
The identity of this microorganism is not fully established. It is
undoubtedly a nocardia, but it does not agree in all particulars yaih
any of the varieties described.
Animal Inoculations. — A number of rabbits and guinea-pigs were
inoculated subcutaneously upon the abdomen and in the neighborhood
of the cervical, axillary, and inguinal lymph nodes with colonies
broken up in salt solution. Indurated swellings were producec^ at
the point of inoculation and a number of abscesses resulted. The
abscesses developed rapidly and some of them opened spontaneously,
MICRO-ORGANISMS BELONGING TO HIGHER BACTERIA. 471
while others were incised. The material evacuated did not resemble
ordinary pus, but was thick and mucilaginous and exceedingly tena-
cious, like that from the subcutaneous abscesses of the patient described
above. The microscopic appearance was the same, and the nocardia
threads were found in considerable numbers. Several rabbits and
guinea-pigs and two cats received peritoneal inoculations, but none
of them showed any sign of infection. When rabbits were inoculated
intravenously, a rapidly fatal general infection was produced, and the
lesions were similar in kind and distribution to those described in the
human subject.
Other Oases Reported.— Ferr^ and Faguet found in Bordeaux, in a
cerebral abscess in the centrum ovale, a branching fungus, colored by
Gram, which corresponded to nocardia. It grew on agar in round,
ochre-colored colonies; on potato there was little growth visible; slimy,
tough colonies, which became gray and remained free from white
dusting on the surface. Inoculations in rabbits and guinea-pigs were
negative.
Varieties of nocardia have been found in the human vagina. We
have found a variety of nocardia in several cases of still-birth with
infection of the placenta with the same organism. The organism is
being studied.
Nocardia in Oases Simulating Actinomycosis or Tuberculosis. —
Sabraces and Rivihe found, in a case of cerebral abscess and a
case of chronic lung disease ;vith occurrence of subacute abscesses,
fungi which differed from actinomyces. The organisms were con-
tained in the lungs and pus in the latter in pure culture. They grew
best at 37° C. in the presence of oxygen. On agar plates round,
wart-like colonies were found with yellowish under and whitish
upper surface. Grew particularly well on fat and glycerin media;
in milk a flesh-colored rim was developed; in gelatin agar a rough,
brownish deposit, becoming black with age. Gelatin was liquefied.
The culture had a strong odor of old mould. A yellowish pigment
was usually produced which dissolved in ether; in an atmosphere of
pure oxygen a brown pigment. Animal experiments gave positive
results only when to a fourteen-day-old bouillon culture lactic acid
was added; then pseudotuberculosis was produced.
Numerous cases have since been observed in which nocardia proved
to be the cause of chronic lung diseases, clinically suspected to be
tuberculosis.
Treatment. — Recently homologous vaccines have been tried in
certain cases, but it is yet too soon to determine with what result.
Bibliography.
Foulerton. The Strepotrichoses and Tuberculoses, The Lancet, 1910, clxxviii.,
551, 626 and 769.
Musgrave and Clegg. Phila. Jour. Sci., iii., 1907, 2, 477.
Wright. Jour. Exp. Med., 1898, iii., 421, and the Journ. of Med. Res, 1905, viii.
349, and Osier's Modern Medicine, 1907. i, 327.
CHAPTER XXXVI.
THE PATHOGENIC MOULDS (HYPHOMYCETES, EUM YCETESi ASI'
YEASTS (BLASTOMYCETES)— DISEASES DUE TO MICRO-
ORGANISMS NOT YET IDENTIFIED.
THE HTPHOMTOETES.
The majority of the moulds are not pathogenic and interest us merelj
as organisms which are apt to infect our bacteriologic media. Somf
are, however, true parasites, and produce a number of rather comiiK'-i
diseases; for example, ringworm, favus, thrush, and pitjTiasis versicolfir.
Certain of the commoner moulds have also been reported from time if
Chlttinydoniucor rscemqeuii
gium highly magnified ahowini
ydospore building: 5. dnvelopii
ing iporea. (AfW c BrefelJ.)
time as present in pathologic conditions in man as well as in the lower
animals. Many varieties have been found in plant diseases, sik)
others indirectly may be a source of danger to man. Indeed, vbf'
they form poisonous substances, as in the infection of grain by claricep^
purpurea {ergot poisoning), they are distinctly dangerous.
The relation of the moulds to the bacteria is shown on p. 458. I'il^*
the higher bacteria, these organisms grow in filaments, but the majoriiv
472
PATHOGENIC MOULDS AND YEASTS. 473
of them show more complicated structure in possessing a more dis-
tinct wall and a definite nucleus and in their reproductive organs.
Each filament is termed a hypha. The hjphre branch and grow into
a dense network called mycelium. In the lower forms each hypha is
a single cell, septa only occurring when fructification begins, while in
the higher forms the filaments are composed of rows of cells. Most
of the varieties form endospores in special spore sacks or sporangia,
produced by the end swelling of a hypha (Fig. 153). In certain va-
rieties a primitive sexual process has been obsened, a conjugation of
two cells with the formation of a zygo.spore, from which a sporangium
carrier may ari.se and immediately develop a sporangium. Spores
may also be produced in so-called gummte (chlamydospores), which
are swollen portions, segmented in the course of a hypha. (Fig. 153).
Finally spores may be formed as cunidia (Fig. 154).
The common moulds grow easily, especially in an acid medium,
hence they are often found on fruit. The more pathogenic varieties
grow with more difficulty. Among the common moulds, variou.s
species of mucor and of aspergillus have l>een reported pathogenic
for man. Paltauf reported the case of a man who died after enteritis
with secondary peritonitis. The autopsy showed multiple abscesses in
brain and lungs, besides the lesions in the intestines and peritoneum,
in all of which a species of mucor was found. Two other cases of
primary mucor infection in humans were reported by Furbringer.
A number of species of mucor have been found in ear and eye infec-
tions; for example, Mvcor corymbijer (Fig. 155) has been found in
ophthalmia. A number of varieties are pathogenic for lower animals.
Aspergillus is found still more frequently in lower animals, especially
in birds, where a kind of pseudotuberculosis is often produced.
Quite a number of similar cases have been reported in man, and it
474
PATHOGENIC MICRO-ORGANISMS.
is supposed that the infection may be carried from birds to man.
Aspergillus fumigatus (Fig. 156), is the most frequent variety found.
Penicillium minimum (similar to glaucum, Fig. 154) has been found
by Liebermann in inflammation of the ear. The more common patho-
genic fonns for man are those producing the various hair and skin
lesions mentioned above. These will now be described.
Trichophyton (Ringworm Fungus). — Ringworm of the body or
hairless parts of the skin, Tinea circinata, and ringworm of the hairy
parts, Tinea tonsurans and Tinea barbce or Tinea sycosis, are due
Fig. 155
Mucor coiymbifer, Cohn. Mycelium with underlying branched carriers. The sporangia at o haw
burst. ^\K (After Lichtheim.)
to the fungus trichophyton, discovered by Gruby in the human hair,
and between the epidermal cells by Hebra, and obtained in free cul-
tures by gravity.
According to Sabouraud, whose conclusions are based on an exten-
sive series of microscopic examinations of cases of tinea in man and
animals, of cultivation in artificial media, and of inoculation on man
and animals, there are two distinct types of the fungus trichoph\tc>n
causing ringworm in man — one with small spores (2 to 3 mm.) which
he calls T. microsporon, and one with large spores (7 to 8 mm.) which
he calls T. megalosporon. They differ in their mode of growth on
artificial media and in their pathological effects on the human skin
and its appendages. T. microsporon is the common fungus of Tinea
tonsurans of children^ especially of those cases which are rebellious
to treatment, and its special seat of growth is in the substance of the
hair. T. megalosporon (Fig. 157) is essentially the fungus of ring-
PATHOGENIC MOULDS AXD YEASTS. 475
■worm of the beard and of the smooth part of the skin; the prognosis
as regards treatment is good. One-third of the cases of T. tonsurans
of children are due to trichophyton megalosporon. The spores of T.
microaporon are contained in a mycelium; but this is not visible, the
spores appearing irregularly piled up like zoogloea masses; and, growing
outside, tiiey form a dense sheath around the hair. The spores of T.
megalosporon are always contained in distinct mycelium filaments,
which may either be resistant when the hair is broken up or fragile and
a lungiis. Mfsulosporoi
easily breaking up into spores. The two types when grown in artificial
cultures show distinct and constant characters. The cultures of T.
microsfOTon show a downy surface and white color; those of T. megalo-
sporon a powdery surface, with arborescent peripheral rays, and often a
476 PATHOGENIC M[CR(M)RGAyrSMS.
yellowish color. Although the morphological appearances, mode of
growth, and clinical effects of each type of trichophyton show certain
characters in general, yet there are certain constant minor differences
which point to the fact that there are several different kinds of species
of fungus included under each type. The species included under T.
microsforon are few in number, and, with the exception of one which
causes the common contagious "herpes" of the horse, almost entirely
human. The species of T. megatosporon are numerous and fall
under several natural groups, the members of which resemble one
another both from clinical and mycological aspects (Fig. lo8). Many
animals are subject to the growth upon their skins of particular varie-
ties of T. megalosporon.
Achorion Schoenlemii (Fams).— ^Favus is due to a fungus di^
covered by Schoenlein in 1839, and called by Remak Ackorion ackom-
leinii: The disease is communicated by contagion, the fungus being
often derived from animals, especially cats, mice, rabbits, and fowls:
dogs also are subject to it. It grows much more slowly than the
ringworm fungus, and is, therefore, not so easily transmitted. Wanl
of cleanliness is a predisposing factor. The fungus seems to find a
PATHOGES/C MOULDS A\D YEASTS. 477
more favorable soil (or its development on the skin of persons in weak
health, especially from phthisis, than in others.
Pathologically, the disease represents the reaction of the tissues to
the irritation caused by the growth of the fungus. The spores gen-
erally find their way into the hair follicles, where they grow in and
about the hair (Fig. 159). The favus fungus grows in the epider-
mis, the density of the growth causing pressure on the parts below,
thus crushing out the vitality of the hair and giving rise to atrophic
scarring. The disease shows a marked preference for the scalp,
but no part of the skin is exempt, and even the mucous membranes
are liable to be attacked. Kaposi has reported a case in which a
patient suffering from universal favus died, with symptoms of severe
gastrointestinal irritation, which was found after death to be due to
the presence of the favus fungus in the stomach and intestines. On
the scalp it first appears as a tiny sulphur-yellow disk or acutulum,
depressed in the centre like a cup and pierced by a hair. This is the
characteristic lesion. The cup shape is attributed by Unna to growth
at the sides proceeding more vigorously than at the centre.
The favus fungus is readily cultivated at the body temperature,
and also at rootn temperature, in the ordinary culture media, as agar,
blood serum, gelatin, bouillon, milk, infusion of malt, eggs, potato, etc.
(Fig. 160). The growth develops slowly and shows a preference to
growth beneath the surface of the medium— except on potato, upon
which it develops on the surface in layers. The characteristic form of
growth is that of moss-like projections from a central body. The
color is at first grayish-white, then yellowish. As seen under the micro-
scope, ray-like mycelium filaments are developed, which divide into
branches. The ends are often swollen or clul>-shaped, and there are
various enlargements along the body of the filament.
Pityriasis Versicolor. — This organism belongs to a group of fungi
which, in contrast to the more parasitic fungi, favus and trichophyton,
invades only the most superficial layers of the skin. It does not
penetrate the deeper layers nor does it give rise to any considerable
478 PATHOGENIC MWRO-ORGASISMS.
pathological changes in the skin or hair. Although the Tegetative
elements of these fungi are much more numerous in the affected
portions of the skin than is the case with the more parasitic spedes,
they are not nearly as contagious as the latter.
By preference Pityriasis versicolor attacks the chest, abdomen,
back and axillse; less frequently neck and arms, while exceptionally
it attacks also the face. The growth shows itself as scattered spols
varying in color from that of cream-coffee to reddish-brown. The
spots are readily scraped off and show fine lamellation or scaling.
Occasionally the spots are confluent, and sometimes arranged in
ring form like Herpes tonsurans.
In spite of their slight contagiousness this is one of the most fr^
quent derma to mycoses. Although it is distributed widely over the
Fia. leo earth, it is more frequently observed in
southern than in northern countries.
Persons with a tender skin and a dis-
position to perspire freely are particularly
affected by Pityriasis versicolor, and this
is undoubtedly the only, reason why the
affection is so frequently observed in con-
sumptives. Women are more frequently
attacked than men, while children and
old people are rarely affected.
The source of infection is unknown,
since the absence of contagion has fre-
quently been demonstrated. It seems
likely that the spores of this fungus are
so widely distributed that susceptible individuals are easily infected.
The arrangement of the fungus in the scales of epidermis is char-
acteristic. The short and thick-curved hyphse (7^ to 13;< long and
3/1 to 4/i wide) surround large clumps of spores. The spores are
coarse, doubly contorted (4/( to 7ft) or round. On staining with
Ziehl's solution the spores are seen to contain deeply stained globules
lying, in all probability, on the inner surface of the cell membrane.
The rest of the protoplasm is but little stained, or not at all. One
frequently finds that these globules have disintegrated into numerous
fine granules. The globules are also found free; what their nature
is does not appear; they are not found in cultures, the freshly de-
veloped spores showing only a single globular mass of protoplasm
pos.sessing a fine blue lustre.
Soor Fungua (Thrush) (Fig. 161).— Soor, as is well known, occurs
most frequently in the oral mucous membrane of infants during tie
early weeks of life. It is also found as a slight mycosis in the vagina,
especially of pregnant women. In rare cases the disease attacks
adults, and then especially those whose system has been undermined
by other disease.-^, such as diabetes, typhoid patients, etc. A few
cases are recorded in the literature in which this fungus has given
rise to constitutional di.seasc. In these cases autopsy has shown
PATHOGENIC MOULDS AND YEASTS. 479
abscesses in various parts of the body, such as in the lungs, spleen,
kidney, and brain.
In the lesions of the disease as well as in cultures, this fungus appears
both as a yeast and a mycelium. It thus seems to stand between the
true moulds and the yeasts. The yeast cells are oval in form, about 5/e
to Gp long and 4/i wide, and can in no way be distinguished from other
yeast cells either by their appearance or their method of propagation.
Influnmstianof romenby thnuh. ff. Cnnidia: «. pua will. (From Plautia Kolleand WuHmmnn.)
The threads of the mycelium vary markedly in length and thickness,
and show all intermediate forms between a typical and a budding
mycelium.
Soor is not much influenced by acids*or alkalies, growing well both
in acid and in alkaline media. On the other hand, it is very sus-
ceptible to the common disinfectants, especially salicylic acid, cor-
rosive sublimate, phenol, etc. This fact is made use of in local
treatment.
BLA8TOHTOKTE8 (TEASTS).
These microorganisms have been for many centuries of the great-
est importance in brewing and baking (p. 102). They are not uncom-
monly present in the air and in cultures made from the throat. Cer-
tain recent experiments have shown that some varieties when injected
are capable of producing tumor-like growths. Certain varieties are
pathogenic for mice, and in recent years (since 1894) there have been
reported a number of cases (twenty-three) of human infection from
yeasts.
480 PATHOGENIC MICRO-ORGAXISMS,
The position which the yeasts occupy in systematic biology has not,
thus far, been accurately determined. In fact, it is even undetermined
by some whether they constitute distinct fungi or whether they should
be classed under the moulds.
The chief characteristic of the yeasts is their peculiar method of
reproduction which in most cases is by means of budding. For this
reason these organisms go by the name of hlaMomycetes in contrast
to the fission fungi, or schizomycetes, and the thread fungi, or Ay-
phamycetes. The fact was mentioned above that a transition between
the blastomycetes and the hyphomycetes is formed by the soar fungus,
which at one time grows to long threads, at another time (under cer-
tain conditions almost exclusively) multiplys by building. But no
hard-and-fast line exists between these classes, for the veasts can at
times develop short hyphse, at other times, in rare cases, form new in-
dividuals by segmentation.
The most important property of yeasts, though one not possessed
by all to the same degree, is that of producing alcoholic fermenta-
tion. In practice we distinguish between the yeasts that can be
employed practically, ''culture yeasts," and those which often act
as disturbing factors, so-called **wild" yeasts.
The shape of most of the culture yeasts is oval or elliptical (Fig.
162). Round or globular forms are more often met with among the
wild species which usually excite only a slight degree of fermenta-
tion. They are known as "torula" forms. But sausage-shaped and
thread forms are also met with.
The individual yeast cells are strongly refractive, so that under
the microscope at times they have almost the lustre of fat droplets.
This is important because in examining fresh tissues the yeast cells
may be hard to distinguish from fat droplets, often requiring the aid
of certain reagents for their identification.
The size of the individual yeast cells varies enormously, even in
those of the same species or the same culture. In old colonies indi-
viduals may be found hardly larger than cocci, I to 2fi in diameter,
while in other colonies, especially on the surface of a liquefied me-
dium, giant yeast cells are found often attaining a diameter of 40jtt
or more. In spite of these wide fluctuations, however, the various
species are characterized by a fairly definite average in size and form.
Each cell contains a definite nucleus, which is demonstrated by the
usual chromogenic stains.
During the process of budding the nucleus of the cell moves toward
the margin, where it divides. At this point the limiting membrane of
the cell ruptures or usually a hernia-like protrusion develops which has
the appearance of a button attached to the cell. The daughter-cell so
formed rapidly increases in size and gradually assumes the shape and
size of the mother-cell.
A fact of the utmost importance for the propagation of the blasto-
mycetes and continuation of the species is the formation of spores (Fig.
162). In this also the cell nucleus takes part, dividing into several frag-
PATHOGENIC MOULDS AXD YEASTS. 481
nienls, each of which becomes the centre of a new cell lying within
the original cell. These new cells possess a firm membrane, a cell
nucleus, and a little dense protoplasm. The number of spores de-
veloped in the yeast cells varies, but is constant for a given species.
As a rule, one cell does not produce more than four endogenous spores,
.so-called astrospores; but species have been observed — e. g., Schizo-
.tacckaromycea odoaporus (Beljerlnck) P^^ jg2
^in which eight spores are found.
Guilliermond has described conju-
gation in yeasts before the formation
of spores.
The vitality of yeasts Is truly |
enormous. Hansen as well as Lind-
ner were able to obtain a growth from Sac-clmromyciB cereviBi*. I., Han*™.
cultures twelve years old. Busse sue- ro'?o.'"jF"m''Hl^^ )"'"'* "' ''"™
ceeded in getting a luxuriant growth
from a dry potato culture seven and a half years old, which was almost
as hard as l>one.
The pathogenic blaslomycetes may be briefly summarized as follows:
Saccharomyces Busse, isolated in 1894 by O. Busse from the tibia
of a thirty-one-year-old woman, who died thirteen months after the
first .symptoms appeared. The autopsy showed numerous broken-
down nodules on several of the bones, in the lungs, spleen, and kid-
nevs. The yeast was cultivated from all these foci (Fig. 163).
haccharomyces tumejaciens, isolated in 1895 by Curtis. The patient
was a young man showing multiple
tumors on the hips and neck having
the gross appearance of softened
myxosarcomata.
This yeast is pathogenic for rats,
/ mice, and dogs, only slightly so for
rabbits, and not at all for guinea-
f P'«^- ...
' Various similar cases have since
■ » been described, a number of them
^B l)ecoming generalized, and ending
a fatally. In generalized blastomy-
cosis the lung seems frequently to
be the seat of primary infection.
— The ca.ses described first by Rix-
sa.:,h.™^d«^B^.^.x^sodi«new™. f^j^ ^^^ Gllchrist as coccidlosis
due to "Coccidioides imitis" —
thought to be a protozoon — must be classed here, since Ophiils and
others have shown that the "coccidtum" formed hyphie and elliptical
forms on culture. Cultures from fresh tissue do not grow readily,
but after they are once started they grow with ease on the usual
laboratory media. The growth is more mould-like than yeast-like,
except on potato when budding yeast-like cells are produced. Dogs,
482 PATHOGENIC MICRO-ORGAXIS.VS.
rabbits, and guinea-pigs are susceptible to tbe fungus and show lesion."
similar to those in human beings.
A typical case of systemic blastomycosis has just been rcporl«l
by Fontaine, Hasse, and Mitchell from Memphis, Tenn., accompanied
by very good illustrations of tissue sections. Fig. 164 shows the rfiar-
acteristic microscopic appearance of the lung lesion.
Luodsgaard reported a case of ophthalmia due to a yeast. Hi:
patient, a man thirty-four years old, had a severe hypopyon kera-
titis, in the pus of which many yeast cells were present. Pure cul-
Flo. IM
SeclloD of lung, x ISO; blutomyMUa in Urse BvocyiJiil nil imw- (From FDoUioe, HuK.uil
Mitchell.)
tures of these inoculated into guinea-pigs produced abscess both atthr
site of inoculation and in the lymph glands.
Buschke isolated a yeast from a cervical discharge in which no
gonococci were present. The yeast was pathogenic for guinea-pigs.
In 1895 Dr. G. Tokishige reported an epidemic quite common
among horses in Japan, known as "Japane.se worm," "benign worm,
or "pseudoworm," which is caused by one of the saccharoroyces-
This disease begins in the skin in the form of hard, painless nodules
from the size of a pea to that of a walnut. These break down and pve
rise to gradually extending ulcers. Pure cultures of the saccharo-
rayces are pathogenic only for horses, not for rabbits, guinea-pigs, of
hogs. In the districts where the disease prevails among horses it is
also frequently seen in cattle.
Shortly after Tokishige's publication a similar disease occurring
in horse.s in Italy and southern France was identified as being caused
by saccharomyce.s. Cultures of this yeast, however, differ somewhat
from that obtained in Japan, so that Busse is inelii^ed to regard the two
as two different species of blastomycetes.
PATHOGENIC MOULDS AXD YEASTS. 483
Recently Kartulis, in Alexandria, has described about a hundred
cases of a skin affectfon occurring in the gluteal regions of men and
characterized by an elongated finger-like swelling, which breaks and
emits a purulent discharge forming an unhealed sinus. In the dis-
charge and surrounding tissues are numerous blastomycetes which
Xartulis after cultivation and study considered a variety of the ordinary
fermenting yeast (Saccharomyces cerevisire — Hansen). The cases
were cured by excising the growth,
Kessler reported a skin lesion in an infant (see Fig. 165) probably
due to a similar blastomycete, since the lesions healed after treatment
with potassium iodide. The description of the yeast isolated is too
incomplete to identify it.
Some years ago the attempt was made to connect the develop-
ment of cancerous growth with blastomycetes. This was due in a
measure to a certain similarity between the yeasts and the cell in-
clusions or so-called "parasites" of cancer, and, further, to the fact
that when yeasts are injected into the animal body tumor-like nod-
ules are often developed at the site of inoculation and in the internal
organs. But these nodules are not tumors in the pathological sense
of the term, but merely masses of blastomycetes mixed with inflam-
matory tissue proliferations to a very variable degree. At the present
time Sanfelice and. his pupils are perhaps the only ones who regard
the thickenings produced in the tissues by SacchaTomyces neoformana
as true tumors. His work, however, is not at all convincing.
BiBLIOQRAPHT.
Rieketla. Journ. Med. Rea., 1901, vi.. 377.
Kloeker. Trans, by Allan and Millar, 1903, New York and London.
Bu»«€. In KoUe u. WaBsermann'a " Handbuch d. path. Mikrofirg.," 1903, Jena.
FoiUaine, Has»e and Mitchelt. The Arch, of Int. Med., 1909, iv., 101.
Keaaler. The Journ. o( the Am. Med. Assoc., 1907, xlix., 550.
484 PATHOGEXIC MICRO-ORGANISMS,
DISEASES IN WEIGH THE laORO-OROANISMS EXCITIHa THSM
ARE AS TET XTHDETECTED.
Measles. — Many bacteria as well as bodies supposed to be protozoa
have been described by various investigators as occurring on the
mucous membranes or in the blood of those sick of measles. None
of these have been established as the exciting factor. For recent work,
see p. 620.
Scarlet Fever. — Both streptococci and protozoa have been described
as the exciting factors in this disease. The streptococci are certainly
present, but are looked upon by most as secondary invaders. They
undoubtedly add greatly to the gravity of the disease. The bodies
described by Mallory as protozoa are still under investigation, and
will be described in the section on Protozoa. Serum treatment has
been used to overcome the streptococcus infection. The best results
have been obtained in Vienna, and by Moser. He uses a serum
obtained from horses receiving multiple cultures from cases of scar-
let fever. Only about one horse in three gives a sufficiently curative
serum. The doses used are very large (100 to 200 c.c). The results
claimed are very striking.
Typhus Fever. — Nothing has as yet been determined concerning
the microorganisms exciting this disease. For work on the typhus
fever of Mexico see p. 427.
Smallpox. — Streptococci as secondary invaders add here, as in
scarlet fever, a dangerous infection. The status of protozoa is de-
scribed fully under the section on Protozoa.
Babies (Hydrophobia). — No bacteria have been discovered that
are considered as factors. The probability of the Negri bodies being
protozoa and the exciting factor is considered under Protozoa. The
virus of rabies has been shown to be partially filterable (Remlinger,
Bertarelli, and others) through the coarser Berkefeld filters. The
retained portion is always more virulent than the filtrate. This
indicates that there are some forms just within the limits of visibility
and others larger, which corresponds with what we know of the
variations in size of the Negri bodies (see p. 626).
Whooping-cough. — Jochmann and Krause (1901), in Germany,
and Wollstein, in this country, have shown that bacilli differing slightly
in cultural reactions and in agglutination from typical influenza ba-
cilli can be detected in practically all cases of whooping-cough dur-
ing the acute stages. Wollstein proved that the blood of cases of
whooping-cough agglutinated these bacilli frequently in dilutions of
1 : 200 and over. Bordet and Gengou (1906) described a bacillus differ-
ing slightly from this, which they consider the specific organism because
they obtain with it the complement fixation reaction. Wollstein
(1909) was not able fully to corroborate their work.
Pemphigus Neonatorum. — Several micrococci have been described
as the cause of infection.
Impetigo Contagiosa. — The findings have been similar to those in
pemphigus.
PATHOGENIC MOULDS AND YEASTS. 485
Scurvy. — This disease is probably not due to microorganisms.
Mumps. — Diplococei have been considered by several investiga-
tors as possibly being the exciting organisms.
Noma. — It is as yet undecided whether this disease is due to one
or to several microorganisms. A special predisposition of the tissues
is necessary. A streptothrix, pseudodiphtheria bacilli, and diphtheria
bacilli have been the organisms most frequently present.
Articular Rheumatism. — ^The specific organisms of this disease
have been sought in the synovial fluid, blood, vegetations on heart
valves, and in the exudates on tonsils, etc. Streptococci have been,
of all bacteria, most frequently found. They grow in short chains
or as diploccocci. Most bacteriologists believe the exciting factor
has not yet been identified and that the streptococci and other cocci
are important secondary infections.
Beriberi. — Microorganisms, both of bacterial and protozoan na-
ture, have been considered as the exciting factor, but nothing definite
has been proven.
Pellagra. — ^This disease has been much studied recently. The
theory that it is due to the ingestion of damaged corn has received
added evidence. Perhaps because of this fact or perhaps because it
is true the disease seems to be on the increase. Whenever there is
defective development of corn there is an increase in the prevalence
and severity of the disease, while with the use of well-dried and
healthy corn the disease decreases. By some observers the specific
cause is supposed to be an intoxication similar to that of ergot poi-
soning, but others think it is a microorganism.
Invisible or Ultra-microscopical Organisms.— There exists a class
of infectious diseases from which it has been quite impossible up to
the present time to demonstrate visibly any microorganism, although
infective material from such diseases may, with certain precautions,
be passed through stone filters of varying degrees of porosity; the
filtrates will contain the virus and be capable of reproducing the
disease with all its characteristic symptoms when inoculated into a
susceptible animal.
Examined microscopically, even with the highest powers, the fil-
trate is limpid, and, except in one or two diseases, which will be de-
scribed in detail later on, not the faintest sign of particulate matter
can be seen.
Certain precautions must be observed in such filtrations. In the
first place, the filter must be shown by actual test to be free from
imperfections — any and all of the known bacteria must be abso-
lutely retained and none pass into the filtrate. (The bacillus of guinea-
pig pneumonia, which is 0 . o/i X 0 . 7/1, passes Berkefeld No. 5 (Wherry) ;
a spirillum isolated by von Esmarch passes, according to him, Berkefeld,
-Chamberland F, and other filters; finally, a minute water flagellate was
found by Borrel to pass through the coarser filters.) The filtration
must be completed within a moderate time, because even bacteria as
large as the typhoid bacillus may, in media containing a certain amount
486 PATHOGENIC MICRO-ORGANISMS,
of albuminous material, grow through the filter. The material to be
filtered should be greatly diluted and first filtered through filter-paper
in order to avoid the clogging action of extraneous material.
If, after all the proper precautions have been taken, the filtrate
is pathogenic, one must be certain that the symptoms are due to a
microorganism and not to a toxin — this may be decided with almost
absolute certainty by inoculating a series of animals successively with
the filtrate obtained from a previously so inoculated animal — it is im-
possible with our present knowledge to conceive that toxin or enzyme
can be potent enough to cause symptoms after the enormous dilution
it receives in passage through the animals, using a filtered virus for
the first animal and a filtered virus for the second obtained from a
portion of the infected material of the first.
Among the best known ultra-microscopical diseases are:
Anterior Poliomyelitis. — Recent Experimental Study. — Landsteiner
and Popper * reported the transmission of acute poliomyelitis to apes.
They inoculated spinal cord intraperitoneally and produced typical
symptoms and lesions, but did not succeed in transmitting from ape
to ape, probably because they used a mild case. They thought the
virus belonged to the class of invisible protozoa.
Knoepfelmacher also failed to retransmit the disease, probably be-
cause he used a chronic case.
Flexner ' transmitted the disease from monkey to monkey by means
of intracerebral inoculations.
Landsteiner and Levaditi' also transmitted it from monkev to
monkey. They found that virus lives four days outside of body; that
the degenerative nerve cells are taken up by the phagocytes, and that
there is an analogy between the lesions of poliomyelitis and those
produced by rabies from street virus, as well as that it is filterable.
Leiner and Weisner transmitted the disease from monkey to monkey,
found young animals more susceptible than older ones, and the spinal
fluid, blood, and spleen negative.
Flexner* transmitted the disease by means of inoculation into the
blood or peritoneal cavity, and transmitted the disease by means of
the subcutaneous method, and found the virus to be filterable. Cul-
tures so far have been negative.
Landsteiner and Levaditi * found the virus in the salivary glands
and suggested the saliva, moist or dry, as a source of contagion.
Foot and Mouth Disease. — A highly infectious disease of cows.
Other domestic animals, as well as man, may also be attacked, the
latter becoming infected by drinking the milk of animals suffering
* Landsteiner and Popper. May 25, 1909, Zeitschrift fiir Immunltatsforschung,
Band ii, H. 4.
^Flexner. Journal Amer. Med. Assn., Nov. 13, 1909.
^Landsteiner and Levaditi. Nov. 27. Compt. Rendus See. de Biologie, Dec. 3,
1909.
* Flexner. Journ. Amer. Med. Assn., Dec. 4 and Dec. 18.
^Landsteiner and Levaditi. Compt. Rendus Soc. de Biologie, Dec. 24, 1909,
(read Dec. 18, 1909).
PATHOGENIC MOULDS AND YEASTS, 487
from this disease. It is characterized by the appearance of vesicles
in the mouth, around the coronet of the foot as well as between the
toes. The organism was discovered by Loffler and Frosch in 1898,
who obtained it as follows: after diluting the contents of an unbroken
vesicle with 20 to 40 times its volume of water, the resulting fluid
was passed through a Berkefeld filter. The filtrate contains the
virus and remains infectious for some time.
Yellow Fever. — ^The undiluted serum from cases of this disease
has been shown by the American commission and others (see p. 638)
to pass Berkefeld and Chamberland F filters and in this form to be
infectious; therefore some forms at least of the specific organism are
probably ultramicroscopic.
Mosaic Diseases of Tobacco. — The young leaves become devoid of
chlorophyll in spots, which enlarge, turn brown, and the underlying
tissue becomes necrotic. Beijerinck in 1899 showed that the fil-
trate from a porcelain filter promptly reproduced the disease on to-
bacco leaves, and he was inclined to believe the virus was in true
solution.
South African Horse Sickness. — It occurs in warm weather, as a
rule, and is said to be more common in animals which do not pass
the night under cover. The horses are uneasy, have diflSculty in
breathing, and a reddish froth exudes from their mouth. The tem-
perature rises in the daytime, but has a tendency to drop at night.
In severe cases an oedematous swelling of the head and neck may
appear. MacFadycan succeeded in passing blood serum of a dis-
eased horse (diluted) through a Berkefeld and Chamberland F, but
not through a B filter.
Rinderpest. — Rinderpest, the fatal European and African disease
of cattle, is characterized by inflammation of the intestinal mucous
membi'ane. The blood is infectious, and filtrates of it through Berke-
feld and Chamberland F (Nicolle and Adel Bey) produce the disease.
No organism can be seen.
Dengue. — Recently Ashburn and Craig claim to have reproduced
dengue in susceptible individuals by a similar procedure to that em-
ployed in yellow fever. The virus passes a Berkefeld filter. The
intermediary host in natural infection is claimed by them to be Cidex
fatigans.
In a certain number of diseases, at the highest limit of our present
magnification, the cause of the disease is seen as minute granules. We
know, surely in one disease at least, that these are the cause, because
they have been made to grow and to produce the disease in new
animals. This disease is known as
CSontagious Pleuro-pneumonia of Cattle. — This malady affects
bovines, but not other species. Typically there is an inflammation
of the lungs and the pleura which is invasive and causes necrosis of
the diseased parts. Nocard and Roux succeeded in cultivating the
organism in collodion sacs placed in the peritoneal cavity of rabbits,
using a mixture of serum and bouillon. After two weeks a very
J
488 PATHOGENIC MICRO-ORGANISMS.
faint turbidity appeared in the sacs: coincidently the fluid became
infective. The organisms will pass a Berkefeld and Chamberiand
F filter, but not a Chamberiand B.
Ghlamydozoa (Strongyloplasmse). — Lipschutz in a recent article
calls attention to the fact, that though it can be seen, this organism
will pass certain filters, and he claims to have discovered with Borrel
similar organisms in moluscum contagio sum of man and birds. He
calls them microscopically visible filterable organisms, and says that
probably the organisms of vaccinia (Volpino's motile granules discov-
ered in vaccinia by the ultramicroscope) and of rabies (Prowazek's
chlamydozoa) belong to this class. He thinks they should be given the
name " strongyloplasmen " because of their prevailing round form.
CHAPTER XXXVII.
THE BACTERIOLOGICAL EXAMINATION OF WATER, AIR, AND
SOIL— THE CONTAMINATION AND PURIFICATION OF
WATER— THE DISPOSAL OF SEWAGE.
The bacteriological examination o^ water is undertaken for the
purpose of discovering whether any pathogenic bacteria are liable
to be present. The determination of the number of bacteria in water
was for a time considered of great importance, then it fell into disrepute,
and the attempt was made to isolate the specific germs of diseases
which were thought to be water-borne. At first these attempts seemed
very successful in that supposed typhoid bacilli and cholera spirilla
were found. Further study revealed the fact that there were common
water and intestinal bacteria which were so closely allied to the above
forms that the tests applied did not separate them. Even the use of a
serum from an animal immunized to injections of the typhoid bacillus
was found to agglutinate some other bacteria in high dilutions; so that
the test as usuallv carried out was insuflScient. With the latest tech-
nique it is probable but not certain that absorption tests with the serum
from an immunized animal will be suflBcient to decide whether a sus-
pected bacillus is the typhoid bacillus or not. The improbability of
getting typhoid bacilli from suspected water except under unusually
favorable conditions caused a return to the estimation of the number
of bacteria in water and above all to the estimation of the number of
intestinal bacteria. It is known that the group of colon bacilli have
a somewhat longer existence than the typhoid bacilli, and as the colon
bacilli come chiefly or wholly from the intestinal passages of men and
animals, it was fair to assume that typhoid bacilli could not occur
without the presence of the colon bacillus except in rare cases, as, for
example, pollution with urine alone. The latter could, of course,
occur abundantly without the typhoid bacillus.
During the past few years the attention of sanitarians has been
seriously devoted to the interpretation of the presence of smaller or
larger numbers of colon bacilli in water, uutil at present upon the
quantitative analysis (measuring, within certain limits, decomposing
organic matter) and the colon test (indicating more specifically that
pollution derived from intestinal discharges of man or animals) the
bacteriological analysis of water is based. The determination of the
number of bacteria is also of value.
Technique for Quantitative Analysis.— The utmost care is neces-
sary to get reliable results. A speck of dust, a contaminated dish, a
delay of a few hours, an improperly sterilized agar or gelatin, a too
high or too low temperature, may introduce an error or variation in
results which would make a reliable test impossible.
489
490 PATHOGENIC MICRO-ORGANISMS.
OoUection of Samples. — The small sample taken must represent the
whole from which it was drawn. If a brook-water, it must be taken
some distance from the bank; if from a tap, the water in the pipes
must first be run oflF, for otherwise the effect of metallic substances
will invalidate the results; if from lake or pond, the surface scum
or bottom mud must be avoided, but may be examined separately.
The utensils by which the water is taken should be of a good quality
of glass, clean and sterile. From a brook the water can be taken
directly into a bottle, the stopper being removed while it fills, avoid-
ing the surface film and its attending excessive numbers of bacteria;
from a river or pond it can be taken from the bow of a small boat,
or from a bottle properly fastened on the end of a pole so as to avoid
contamination; from a well a special apparatus has been devised
by Abbott, where a bottle with a leaded bottom is so held that when
lowered to the proper depth a jerk will remove the cork and allow the
bottle to fill. The same device or another accomplishing the same pur-
pose can be rigged up readily by anyone. The sample of water should
be tested as soon as possible, for the bacteria immediately begin to
increase or decrease. In small bottles removed from the light preda-
tory microorganisms and many bacteria begin to increase, and among
these are the members of the colon group. Thus, the Franklands
record a case in which in a sample of well-water kept during three days
at a moderate temperature the bacteria increased from 7 to 495,000;
while Jordan found that in a sample the bacteria in forty-eight hours
fell from 535,000 to 54,500. In a sample we took from the Croton
River the colon bacilli during twenty-four hours increased from 10 to
100 per c.c. The only safe way to prevent this increase is to plate and
plant the water in fermentation tubes within a space of one or two
hours. It is far better to make the cultures in the open field or in a
house rather than to wait six to twelve hours for the conveniences and
advantages of th^ laboratory. If sent to the laboratory, water should
be kept in transit at about 5° C. (41° F.).
The third matter of great importance is the adding of proper amounts
of water to the broth in the fermentation tubes and the media for plant-
ing. Usually 1 c.c, 0.1 c.c, and 0.01 c.c. are added to the fermenta-
tion tubes and to 10 c.c. of the melted nutrient agar or gelatin. If
possible duplicate tests should always be made. When it is desired to
know whether colon bacilli are present in larger amounts than 1 c.c,
quantities as great at 10 or 100 c.c. can be added to bouillon, and then
after a few hours 1 c.c added to fermentation tubes. Less than twenty
colonies and more than two hundred on a plate give inaccurate counts,
the smaller number being too few to judge an average and the larger
number interfering with each other. When as many as 10,000 colonies
develop in the agar contained in one plate, it will be found that there
will develop in a second plate containing but one-tenth the amount of
water from 20 to 50 per cent, as many colonies. This shows that the
crowding of the colonies had prevented the growth of all but one-fifth
to one-half of them.
BACTERIOLOGICAL EXAMINATION. . 491
The chemical composition of the medium on which the bacteria are
grown affects the result of the analysis. Nutrient 1.5 per cent, agar
gives slightly lower counts than gelatin, but on account of its con-
venience in summer and its greater uniformity it is being more and
more generally used for routine quantitative work. There is an
optimum reaction for every variety of bacteria, and to ensure uniformity
the committee of the American Public Health Association adopted a
standard reaction of +1.0 per cent., which was as near as possible
to the average optimum for water bacteria. Such a uniform standard
is a necessity to secure comparability of results. At best only a
certain proportion of bacteria develop, and it is only important that
our counts represent a section through the true bacterial flora which
fairly represents the quick-growing sewage forms. Comparability is
the vitally essential factor.
The temperature at which the bacteria develop is of great importance,
and they should be protected from light. The access of oxygen
which prevents the growth of anaerobes must also not be forgotten.
As a rule, the plate cultures are developed for four days at 20° to 21°
C, and for twenty-four or forty-eight hours at incubator temperature.
Some bacteria do not develop colonies in four days, but these are
neglected. The number of bacteria growing at room temperature is
usually much greater than those growing at 37°. As all the intestinal
groups of bacteria grow at body temperature, while many of the water
types do not, some investigators believe it important to develop the
bacteria at both temperatures so as to compare the results. We
have not found this to be of any advantage when tests are also made
for the colon group of bacilli.
The lactose broth is placed at 37° C. for the development of the
colon bacilli. The fermentation tubes not showing gas are recorded
as negative and usually discarded. Those showing gas are suspected
to contain colon bacilli. To a number of tubes containing melted
litmus-lactose agar at about 44° C. are added 1, 0.1, and 0.01 loop
of the culture fluid. Plates are poured and the whole placed in the
incubator. The Bacillus coli ferments lactose and thus produces
acid, so that if colon bacilli are present we have a number of red
colonies on a blue field. Later, if many colon bacilli were present,
the whole medium becomes acid. At forty-eight hours, on account
of alkali being produced by the formation of NH,, the blue may
return. If after inspection red colonies are seen, four or five are
picked and planted into lactose bouillon and other media. Litmus-
lactose agar is frequently used for the original plating of water samples,
the absence or presence of acid-producing colonies being thus im-
mediately noted. The colon-like cultures should be subjected to the
Vosges reaction (page 499), and should be kept for 1 month at 20° C.
in gelatin before a decision is made. Colon bacilli do not liquefy
gelatin nor give the Vosges reaction. There are a few colon-like
bacilli in the intestinal tract that give the Vosges reaction. For a
more complete understanding of the technique and the irUerpretation
492 PATHOGENIC MICRO-ORGANISMS.
of results of the bacteriological examination of water see Elements of
Water Bacteriology — ^Prescott and Winslow.
For the characteristics of the colon bacilli the Massachusetts State
Board of Health uses six media — ^gelatin, lactose agar, dextrose broth,
milk, nitrate solution, and peptone solution, determining, respectively,
absence of liquefaction, production of gas, turbidity, coagulation
without liquefaction of the coagulaum, products of nitrite, and indol.
Lactose-bile-peptone solution has been much used. In badly con-
taminated waters this has a distinct advantage in that the bile inhibits
many varieties of bacteria more than those of the colon-typhoid group.
In good waters the results are very similar from the lactose-peptone
and lactose-bile-peptone solutions.
Significance of the Colon Bacillus. — ^The colon test has been re-
ceived by the majority of engineers and practical sanitarians with
great satisfaction, and has been applied with confidence to the exami-
nation not only of water, but of shell-fish and other articles of food as
well. On the other hand, some have denied its value. Bacteriologists
have found bacilli like certain members of the colon group in ap-
parently unpolluted well-water. The discovery that animals have
colon bacilli identical in the usual characteristics studied with those
of man has complicated matters. Thus a fresh hillside stream may be
loaded with colon bacilli from the washings of horse or cow manure
put on the fields through which it runs or polluted by a stray cow or
horse. Swine, hens, birds, etc., may contaminate in unsuspected
ways. The number of colon bacilli rather than their presence in anv
body of surface water is therefore of importance. In well- and spring-
water the presence of colon bacilli indicates contamination. The
absence of colon bacilli in water proves its harmlessness so far as
bacteriology can prove it. When the colon bacillus is present so as to
be isolated from 1 c.c. of water in a series of tests, it is reasonable proof
of animal or human pollution and the conditions should be investigated.
Ten colon bacilli in 1 c.c. indicates serious pollution. Surface waters
from inhabited regions will always contain numerous colon bacilli
after a heavy rain storm or shower. The washings from roads and
cultivated fields contain necessarily large numbers. Winslow reports
that in only two out of fifty-eight samples of presumably non-polluted
waters did he get colon bacilli in the 1 c.c. samples. Even in twenty-
one stagnant pools he only found colon bacilli in five of the 1 c.c.
samples.
The experience of all who have studied the subject practically is
that in delicacy the colon test surpasses chemical analysis; in con-
stancy and definiteness it also excels the quantitative bacterial count.
All these tests must, however, be supplemented by inspection.
Interpretation of the Quantitative Analysis. — ^The older experi-
menters attempted to establish arbitrary standards by which the
sanitary quality of water could be fixed automatically by the number
of germs alone. This has been largely given up. Dr. Sternbei;p
considers that a water containing less than 100 bacteria is presumably
BACTERIOLOGICAL EXAMINATI02\. 493
from a deep source and uncontaminated by surface drainage; that
one with 500 bacteria is open to suspicion; and that one with over
1000 bacteria is presumably contaminated by sewage or surface
drainage. Even this conservative opinion must be applied with
caution. The source of the sample is of vital importance in the
interpretation; thus, a bacterial count which would condemn a spring
or well might be normal for a river. In woodland springs and lakes
several hundred bacteria per c.c. are frequently found. In lakes the
point at which the sample is taken is of great importance, as the
bacterial count varies with the distance from the shore and with the
depth. The weather also is an influence, since the wind causes cur-
rents and waves which stirs up the bottom mud, bringing up organisms
which have been sedimented. Rains greatly influence streams by
flooding them with surface water bringing a huge number of bacteria
at times. The season of the year is an important factor. The counts
are highest in the winter and spring months, and lower from April to
September.
The following figures illustrate this point:
Water Observer Year Jan. Feb. March April May June
New York aty tap-water Houghton 1904 890 1100 660 240 360 370
Boston tap-water Whipple 1892 135 211 102 62 63 86
Merrimac River tap-water Clark 1899 4900 6900 6300 2900 1900 3600
The winter and spring increases are not exceptions to the rule
that high numbers indicate danger, but an indication of its truth;
for it means a melting of the snow and a flow of surface water into
the streams without the usual filtering soil filtration. A number of
severe epidemics of typhoid fever have been produced in this way.
It is only the fact that typhoid fever is at a minimum in, winter that
prevents more frequent pollution. Although, as a rule, a series of
tests are necessary to pass judgment on a water, a single test may be
very important. A large increase in the number in tap-water a day
after a storm points unerringly to surface pollution, and if towns exist
in the water-shed, to street and sewer pollution. The Croton water
frequently jumps from hundreds to thousands after such a storm.
In a typhoid epidemic at Newport, Winslow reports that a test
of the water supply showed but 334 bacteria per cubic centimetre,
but one from a well thowed 6100. The svspicion aroused was justi-
fied by finding all the typhoid cases had gotten water from this well.
The study of the bacterial eflBuent from municipal water filters
is the only way in which the efficiency of the filter and the accidents
which occur can be determined. In Germany these regular tests
are obligatory. The filter should remove about 99 per cent, of the
bacteria. Elaborate studies have recently been made of the exact
distribution of streams of sewage in bodies of water into which they
flow, their disappearance by dilution and sedimentation, and their
removal by death. Under peculiar conditions bacteria in water may
increase for a time, but here the prevailing bacteria belong almost
exclusively to one type.
494 fA THOGEXIC MICRO-ORGA\lSMS.
Streptococci in Sewage. — The varieties of streptococci found most
often in polluted water correspond to the streptococci described hj
Houston. In some water in which these are found no B. coli have
been found and there is considerable doubt in such cases as to whether
the streptococci imply serious pollution. The streptococci remain
alive longer than the colon bacilli. In England the examination for
streptococci in water is much more regularly done than in America.
Other Bacteria. — Most of the bacteria which develop in the intes-
tines of man and animals necessarily occur in polluted water, and
an examination for some of these has been advocated by many, su(4
as the B. enteritidis sporogenes, other anaerobic spore formers, the
various members of the typhoid-colon group, and the proteus group.
Isolation of the Typhoid Bacilltia from Water.— If it were possi-
ble to readily obtain the typhoid bacilli from water, when they were
present in small numbers, its examinalion for that purpose would
he of much greater value than it is now; but we have to remember
that we can only examine at one time a few cubic centimetres d
water by bacteriological methods, and that although the typhoid
bacilli may be sufBciently abundant in the water to give, in the quan-
tity that we ordinarily drink, a few bacilli, yet it must be a very lucky
chance if they happen to be in the small amount which we examine.
Still, further, although it is very easy to isolate typhoid bacilli from
water when they are in considerable numbers, yet when they arc a
very minute proportion of all the bacteria present it is almost im-
possible not to overlook them. Many attempts have been made to
devise some method by which the relative number of the typhoid
and other parasitic bacteria present in water could be increased at
the expense of the saprophytic bacteria. Thus, to 100 c.c. of water
25 c.c. of a 4 per cent, peptone nutrient bouillon is added, and the
whole put in the incubator at 37° C. for twenty-four hours. From
this, plate cultures are made. As a matter of fact, the typhoid bacillus
is rarely found, even in specimens of water where we actually know
that it is or has been present because of cases of typhoid fever which
have developed from drinking the water. From these facts we must
consider our lack of finding the bacillus in any given cases as abso-
lutely no reason for considering the water to be free from danger. An-
other serious drawback to the value of the examinations for the typhoid
'""""" '" that they are frequently made at a time when the water is
Tom contamination, though both earlier and later the bacij-
?senl. It is hardly worth while, therefore, except in care-
iiental researches, to examine the water for the typhoid
It rather study the location of the surrounding privies and
contamination. A number of observers, resting on th*
jn test, have thought they have isolated typhoid bacilli
oil and water, but these investigators had not considered
the matter of group agglutinins, and their results are not
BACTERIOLOGICAL EXAMINATION. 495
CONTAMINATION AND PURIFICATION OF DRINKING WATERS.
Brook-water and river-water are contaminated in two ways: through
chemicals, the waste products of manufacturing establishments,
and through harmful bacteria by the contents of drains, sewers, etc.,
the latter method being by far the more dangerous.
When water, which has been soiled by waste products of manu-
factories only, becomes so diluted or purified that the contamina-
tion is not noticeable to the senses and shows no dangerous products
on chemical analysis, it is probably safe to drink. When sewage is
the contamination, this rule no longer holds, and there may be no
chemical impurities and no pathogenic bacteria found, and yet dis-
ease be produced. That river-water which has been fouled by sew-
age will, by oxidization, dilution, sedimentation, action of sunlight,
and predatory microorganisms, become greatly purified is an indis-
putable fact. The increase in bacteria which occurs from contami-
nation is also largely or entirely lost after ten to twenty miles of river
flow. Nevertheless, the history of many epidemics seems to show
that a badly contaminated river is never an absolutely safe water
to drink, although with the lapse of each day it becomes less and
less dangerous, nor will sand filter-beds absolutely remove all danger.
These statements are founded upon the results of numerous inves-
tigations; thus the marked disappearance of bacteria is illustrated
by the following: Kummel found below the town of Rosbock 48,000
bacteria to the cubic centimetre; twenty-five kilometres farther down
the stream only 200 were present — about the same number as before
the sewage of Rosbock entered. On the other hand, the doubtful
security of depending on a river purification is proved by such ex-
periences as the following: In the city of Lowell, Massachusetts, an
alarming epidemic followed the pollution of the Merrimac River three
miles above by typhoid faeces, and six weeks later an alarming epidemic
attacked Lawrence, nine miles below Lowell. It is estimated that
the water took ten days to pass from Lowell to Lawrence and through
the reservoirs. Typhoid bacilli usually die in river-water in from
three to ten days, but they may live for twenty-five days in other water;
the Lawrence epidemic is easily explained. Newark-on-Trent,
England, averaged seventy-five cases a year from moderately well
filtered water and only ten when it was changed to deep-well supply.
Purification of Water on a Large Scale.— For detailed informa-
tion on this subject the reader is referred to works on hygiene. Sur-
face waters, if collected and held in sufficiently large lakes or reser-
voirs usually become so clarified by sedimentation, except shortly
after heavy rains, as to require no further treatment so far as its
appearance goes. The collection of water in large reservoirs allows
not only the living and dead matter to subside, but allows time also
for the pathogenic germs to perish through light and antagonistic
bacteria and other deleterious influences, sand or mechanical coagu-
lant. Filtration of water exerts a very marked purification, taking
496 PATHOGENIC MICRO-ORGANISMS.
out 99.8 per cent, of the organisms in those best constructed and at
least 95 per cent, in those commonly used in cities. The construc-
tion of filters is too large a subject to enter on minutely here; sand
■filters consist, as a rule, of several layers, beginning with fine sand,
and then smaller and larger gravel, and finally rough stones. A
certain time elapses before the best results are obtained; this seems
to wait for the formation of a film of organic material on the sand,
which is full of nitrifying bacteria. Even the best filters only greatly
diminish the dangers of polluted water. Spring-water and well-water
are, in fact, filtered waters.
Water which is subject to serious pollution must be submitted to
a preliminary purification before it can be considered a suitable
source for a drinking-water supply. The means employed for its
purification depend to a large extent upon the character of the water
and the nature of the pollution. Filtration through slow sand filters,
three to five feet in depth, removes 98 to 99.5 per cent, of the bacteria
and organic matter; so that eflluents from the best constructed sand
filtration beds constitute safe and reliable drinking waters. Five
hundred thousand to one or two million gallons, depending some-
what upon the extent of pollution and the fineness of the sand, can
be filtered daily per acre. Only the surface of the sand filter becomes
in any way clogged and as thin a layer as can be scraped off is removed
one or more times a month. This surface sand is washed with clean
water and several scrapings replaced at one time. Sand filtration
beds are very widely used abroad and are coming into extensive use
in this country. The filter-beds at Lawrence, Mass., have been used
over ten years with marked success; when properly managed, they
render the highly polluted Merrimac River a fairly safe drinking water;
the filter-beds there are scraped about thirteen times a year.
Mechanical filtration plants find considerable favor where clarifi-
cation as well as bacterial purification is desired. A coagulant such
as sulphate of aluminum is employed and forms in the water a floc-
culent precipitate which carries down with it all suspended matter;
125,000,000 or more gallons of water can be filtered on an acre
daily, but the filters must be washed daily by reversing the flow and
cleansing the clogged filter with a stream of the purified water.
Chlorinated lime when added to drinking water to the extent of one-
eighth to one-twelfth of a grain per gallon will destroy all intestinal
bacteria of the typhoid-colon group within a few hours. This is a
very useful means of purification for emergencies. It does not injure
the water.
Under special conditions other methods, such as the passage of
ozone, have proved successful.
Domestic Purification. — Water which requires private filtering
should not be supplied for drinking purposes. Unhappily, however,
it often is. Domestic filters may be divided, roughly, into those for
high and low pressure. The former are directly connected with the
water main, while the others simply have the slight pressure of the
BACTERIOLOGICAL EXAMINATION. 497
column of water standing in the filter. Many household filters
contain animal charcoal, silicated carbon, etc., either in a pressed
condition or in one porous mass. These filters remove much of the
deleterious matter from the suspected waters, but the majority cannot
be depended upon to remove all bacteria. Even those which are
equipped for self-cleansing become in a little while foul, and, if not
cleaned, unfit for use. The best of the filters are of porous stone,
such as the Berkefeld and Pasteur filters. These yield a water, if
too great a pressure is not used, almost absolutely free from bacteria,
and if they are frequently cleansed they are reliable. A large Berke-
feld filter will allow sixty gallons of water to pass per hour. The
Pasteur filter is more compact and slower. From the best Pasteur
filters sterile water may be passed for two to three weeks; from the
Berkefeld usually only a few days. A single typical low-pressure
filter is that of Bailey Denton. The upper compartment contains the
filtering material, which may be sand or charcoal, and is fed from a
cistern or hydrant. After a certain quantity of water has passed in,
the supply is automatically cut off until the whole amount is filtered.
A fairly eflScient filter is the following: Take a large-sized earthenware
pot and plug the hole in the bottom with a cork, through which pass
a short glass tube. Upon the bottom place an inch of small pieces
of broken flower-pot; upon this a couple of inches of well-washed small
gravel, and upon this six to twelve inches of well-washed, fine, sharp
sand. Cover the sand with a piece of filter-paper and hold this down
with a few small stones. Mount the pot on a tripod, and it is ready
for use. The paper prevents the sand being disturbed when water is
added, and as it also holds most of the sediment, this can be readily
removed. Every few months the sand can be washed and replaced.
Animal charcoal is not a good substance for permanent filters, as
bacteria grow well in it. Whenever water is suspected, and there
is any doubt as to the filters, it should be boiled for ten minutes; this
will destroy all bacteria. This precaution should always be taken
in the presence of typhoid fever and cholera epidemics.
THE DISPOSAL OF SEWAGE.
The disposal of sewage is becoming a vital question with all towns
and cities which are not situated near salt-water outlets, since the
present tendency in legislation is to compel such towns to dispose of
their waste so that it shall not be a menace to drinking-water streams,
destructive to fisheries, or a nuisance to harbors.
Methods of sewage purification depend upon the character of the
sewage and the kind of effluent desired.
Two hundred thousand gallons of crude sewage may be filtered
upon an acre of land daily and an eflBuent obtained which will com-
pare favorably in every way known to the chemist and bacteriologist
with the best mountain springs. This is, however, a slow process,
and it is rare that such a pure effluent is required. Similar results
32
498 PATHOGENIC MICRO-ORGANISMS,
may be obtained by utilizing the septic-tank method, running the
sewage from the septic tank to contact beds and thence to sand filter-
beds; where because of the partial "self-purification of the sewage"
in the septic tank and contact beds 2,500,000 gallons of sewage can
be filtered daily on an acre' of surface. In this process less land is
required and both these eflBuents can be safely turned into drinking-
water streams.
If, however, a merely non-putrescible eflBuent is required, one
which, though high bacterially, will not be offensive in any way, or
subject to further decomposition, it may be obtained by passing crude
sewage to septic tanks, thence to double contact beds, the resulting
eflBuent having merely an earthy, humus-like odor and being non-
putrescible.
Where acid wastes, tannery wastes, dyestuffs, etc., from various
factories enter into sewage, its disposal becomes a more complicated
problem and chemical precipitation by the use of lime or other chemicals
is generally employed for such sewage purification, which at best is
only partial and is sometimes supplemented by sand filtration.
Sea-water. — This is only feebly bactericidal. The salty tidal
waters of rivers allow typhoid bacilli and other members of the
typhoid-colon group to live for a number of days.
BAOTERIOLOOIOAL EXAMINATION OF AIR.
Saprophytic bacteria are always present in considerable numbers
in the air except far out at sea or on high mountains. They are more
abundant where organic matter abounds and in dry and windv
weather. Pathogenic bacteria, on the other hand, are only occasionallr
present in the air. The practical results obtained from the examina-
tion of air for pathogenic bacteria have been slight. We know that
at times they must be in the air, but unless we purposely increase
their numbers they are so few in the comparatively small amount of
air which it is practicable to examine that we rarely find them. Ex-
amination of dust, however, in hospital wards and sick-rooms, in
places where only air infection was possible, have revealed tubercle
bacilli and other pathogenic bacteria.
The simplest method of searching for the varieties of bacteria in
the air and their number in any place is to expose to the air for longer
or shorter periods nutrient agar spread upon the surface of the Petri
dish. After exposure the plates are either put in the incubator at 37^
C. or kept at room temperature. The more careful quantitative
examination is made by drawing a given quantity of air through tubes
containing sterile sand, which is kept in by pieces of metal gauze.
When the operation is completed the sand is poured into a tube con-
taining melted nutrient gelatin or nutrient agar, and after thoroughly
shaking, the mixture is poured into a Petri dish and the bacteria allowed
to develop, either at 37° or 23° C, according as the growth of the
parasitic or saprophytic varieties is desired. Instead of agar or gelatin.
BACTERIOLOGICAL EXAMINATION. 499
ascitic broth or animals may be inoculated. Such examinations are
occasionally made of the air of theatres, crowded streets in cities, etc.
They give interesting, but hardly valuable results.
BAOTERIOLOOIOAL EXAMINATION OF THE SOIL.
The subject from its agricultural side is considered on p. 95. Speci-
mens of deep soil can be gathered in sterile, sharp-pointed, sheet-iron
tubes. Through the examination, we wish to learn either the number
of bacteria or the important varieties of bacteria present. To esti-
mate the number, small fractions of a gram are. taken and planted in
nutrient agar or in special media contained in Petri dishes. Anae-
robic as well as aerobic cultures should be made.
According to Houston, uncultivated sand soil averages 100,000
bacteria per gram, garden soil 1,500,000, and sewage-polluted 115,-
000,000. The most important bacteria to be sought for are bacilli
of the colon group and streptococci. Both of these suggest fairly
recent excremental pollution.
The period during which typhoid bacilli remain alive in soil is
variable, since it depends on so many unknown factors and differs
so in different places. The typhoid bacilli probably rarely increase
in the soil and probably rarely survive a month in it. The main dan-
ger of soil bacteria is their being washed into water supplies by rains
or carried to them by the wind.
Reaction of Vosges and Proskauer. — Grow the culture in 1 per
cent, glucose-peptone water in a fermentation tube for four days at
37° C. Add 1 c.c. of 50 per cent. KOH solution to the open end
and allow the mixture to stand for two days at room temperature.
With certain varieties of bacteria a red color like that of eosin de-
velops after twenty-four to forty-eight hours. With true B, coli this
color does not develop.
CHAPTER XXXVni.
THE BACTERIOLOGY OF MILK IN ITS RELATION TO DISEASE.
From the standpoint of the dairy many of the diCFerent varieties
of bacteria found in milk are of importance, which have little or no
medical interest. We have space here only to consider the bacteri-
ology of milk so far as it is related to health and disease. The sapro-
phytic bacteria taken collectively have importance because one can
determine from their number something as to the care taken in han-
dling the milk and also because, when numerous, they produce chem-
ical changes in the milk which are harmful for infants.
Numerical Estimation of Bacteria. — The number of bacteria in
a c.c. of milk is usually estimated from the colonies developing in
nutrient agar plate cultures during a period of three to four days,
when kept at 20*^ to 27° C. Some authorities prefer a temperature of
37° for 48 hours, but this allows in market milk in which bacteria have
developed at low temperature only a certain proportion of the varieties
of bacteria to develop colonies. Sometimes fully twice as many
colonies develop at 20° to 27° C. as at 37°. For the technique see pages
43-46. This culture method necessarily underestimates their number,
as many of the bacteria remain after vigorous shaking in pairs or
small groups. In order to overcome this and also to note the mor-
phological types, the direct microscopical examination of smears of
the sediment has been urged. A great practical objection to this is
that, if a heated milk is examined, the dead as well as the living bac*
teria are counted. This method has, however, great advantages in that
one can immediately tell whether a sample has few or many bacteria
and also note the presence of streptococci and leukocytes.
Smear Method for Direct Exiumnation of Milk. — 1. The sample
of milk to be examined is shaken thoroughly, not less than twenty-
five times.
2. One cubic centimeter is withdrawn and put into a tube of small
calibre having two rubber corks and is centrifugalized for 10 minutes.
3. After centrifugalization the upper cork is removed and the
supernatant cream and milk are gently poured off; the lower cork
which holds the sediment is then removed and the sediment is spread
as evenly as possible on slides in areas of two square centimetres upon
which a drop of sterile water has been previously placed.
4. After drying in the air, the smears are fixed with methyl alcohol
and stained with a watery solution of methylene blue. By turning the
slides the excess stain drains off and washing with water is avoided
with its danger of removing bacteria. The sediment contains about
500
BACTERIOLOGY OF MILK. 501
33 per cent, of the bacteria in the whole milk. If there is any fat in
the sediment this can be removed by flooding the slide after fixing
with i per cent. NaOH solution.
5. Ten fields are counted in each smear, four at the top and bot-
tom and one at either end. A net micrometer, fitted into the eye-
piece and marking a field equal to i^linF ^^ ^ square centimetre
is used with the oil immersion, the average counts of the ten fields
are multiplied by 20,000 and the results are therefore the bacterial
count of the sediment from one cubic centimetre or milk.
6. If a leukocyte count only is desired, the same technique is followed
except that before centrifugalization the milk is heated to 65° to 70°
C. for 10 minutes, after which it is thoroughly shaken and put into
the centrifugal tubes.
Identification of Bacteria. — ^The milk is plated in a 2 per cent,
lactose-litmus nutrient gelatin or agar, and the bacteria, after devel-
opment of colonies, isolated and grown upon the usual identification
media. The pathogenic properties of the different bacteria can be
tested by intraperitoneal and subcutaneous inoculation in guinea-
pigs with 2 c.c. of a forty-eight-hour broth culture, and by feeding
young kittens for several days with 3 to 6 c.c. daily of a twenty-four-
hour broth culture by means of a medicine dropper.
Varieties. — Bacteria in milk can be divided into two great groups —
those which get into the milk after it leaves the udder and those which
come from the cow. The first group comprises bacteria from dust,
hands, milking pails, strainers, etc.
The extraneous bacteria are of importance because they produce
changes in the chemical composition of the milk when they have de-
veloped in great numbers. The number of bacteria in any sample
of milk depends on three factors: the number deposited in the milk
from the cow's udder, from the air, and utensils; the time during
which they have developed, and the temperature at which the milk
has stood. The last is perhaps the most important factor. The at-
tempt was made during a period of one year to connect illness in in-
fants and children with special varieties of saprophytic bacteria in
milk. As a matter of fact, no such connection was made out.
From the milks altogether 239 varieties of bacteria were isolated
and studied. These 239 varieties, having some cultural or other
differences, were divided into the 31 classes, each class containing
from 1 to 39 more or less closely related organisms.
As to the sources of bacteria found in milk, we made suflBcient
experiments to satisfy us that they came chiefly from outside the
udder and milk-ducts.
Bacteria were isolated from various materials which under cer-
tain conditions might be sources of contamination for the milk, and
the cultures compared with those taken from milk. Thus there were
obtained from 20 specimens of hay and grass, 31 varieties of bac-
teria; from 15 specimens of faeces, manure, and intestinal contents,
28 varieties; from 10 specimens of feed, 17 varieties. Of these 76
502 PATHOGENIC MICRO-ORGANISMS,
varieties there were 26 which resembled closely those from milk —
viz., 11 from grass or hay; 26 from manure; 5 from feed.
During the investigation a number of the varieties isolated from
milk were shown to be identical with types commonly found in water.
From the few facts quoted above and from many other observa-
tions made during the course of the work, it would seem that the
term **milk bacteria" assumes a condition which does not exist in
fact. The expression would seem to indicate that a few varieties,
especially those derived in some way from the cow, are commonly
found in milk, which forms having entered the milk while still in the
udder or after its withdrawal, are so well fitted to develop in milk
that they outgrow all other varieties.
As a matter of fact, it was found that milk taken from a number
of cows, in which almost no outside contamination had occurred,
and plated immediately, contained, as a rule, very few bacteria, and
these were streptococci, staphylococci, and other varieties of bacteria
not often found in milk sold in New York City; the temperature at
which milk is kept being less suitable for them than for the bacteria
which fall into the milk from dust, manure, etc. A number of speci-
mens of fairly fresh market milk averaging 200,000 bacteria per
cubic centimetre were examined immediately, and again after twelve
to twenty-four hours. In almost every test the three or four pre-
dominant varieties of the fresher milk remained as the predominant
varieties after the period mentioned.
The above experiments seem to show that organisms which have
gained a good percentage in the .ordinary commercial milk at time of
sale will be likely to hold the same relative place for as long a j>eriod
as milk is usually kept. After the bacteria pass the ten or twenty
million a change occurs, since the increasing acidity inhibits the
growth of some forms before it does that of others. Thus some
varieties of the lactic acid bacteria can increase until the aciditv is
twice as great as that which inhibits the growth of many bacteria.
Before milk reaches the curdling point, the bacteria may have reached
over a billion to each cubic centimetre. For the most part speci-
mens of milk from different localities showed a difference in the
character of the bacteria present, in the same way that the bacteria
from hay, feed, etc., varied. Even the intestinal contents of cows,
the bacteriology of which might be expected to show common char-
acteristics, contained, besides the predominating colon types, other
organisms which differed widely in different species and in different
localities. Cleanliness in handling the milk and the temperature
at which it had been kept were also found to have a marked influence
on the predominant varieties of bacteria present.
Pathogenic Properties of the Bacteria Isolated.— Intraperitoneal
injection of 2 c.c. of broth or milk cultures of about 40 per cent, of
the varieties tested caused death. Cultures of most of the remainder
produced no apparent deleterious effects even when injected in larger
amounts. The filtrates of both cultures of a number of varieties
BACTERIOLOGY OF MILK, 503
were tested, but only one was obtained in which poisonous products
were abundantly present. Death in guinea-pigs weighing 300 grams
followed within fifteen minutes after an injection of 2 c.c; 1 c.c. had
little effect.
As bacteria in milk are swallowed and not injected under the skin,
It seemed wise to test the effect of feeding them to very young ani-
mals. We therefore fed forty-eight cultures of 139 varieties of bac-
teria to kittens of two to ten days of age by means of a glass tube.
The kittens received 5 to 10 c.c. daily for from three to seven days.
Only one culture produced illness or death. A full report on the
identification of the varieties of bacteria met with in this investiga-
tion can be found in an article by Dr. Letchworth Smith in the 1902
Annual Report of the Department of Health of New York City,
After five years of effort to discover some relation between special
varieties of bacteria found in milk and the health of children the
conclusion has been reached that neither through animal tests nor the
isolation from the milk of sick infants have we been able to establish
such a relation. Pasteurized or ** sterilized'* milk is rarely kept in
New York lomrer than thirty-six hours, so that varieties of bacteria
which after long standing develop in such milk did not enter into
our problem. The harmlessness of cultures given to healthy young
kittens does not of course prove that they would be equally harmless
in infants. Even if harmless in robust infants, they might be in-
jurious when summer heat and previous disease had lowered the
resistance and the digestive power of the subjects.
Streptococci in Relation to Disease.— In an investigation by Dr.
D. H. Bergey connection between diarrhoea and pus and streptococci
was sometimes found.
The results of this investigation appear to warrant the following
conclusions:
1. The occurrence of an excessive number of leukocytes in cows'
milk is probably always associated with the presence in the udder of
some inflammatory reaction brought about by the presence of some
of the ordinary pyogenic bacteria, especially of streptococci.
2. When a cow's udder has once become infected with the pyo-
genic bacteria, the disease tends to persist for a long time, probably
extending over several periods of lactation.
3. Lactation has no causative influence per se upon the cellular
and bacterial content of cows' milk, though it probably tends
toward the aggravation of the disease when the udder is once infected.
It is impossible to differentiate in routine milk examinations the
pathogenic streptococci of diseased cows from saprophytic varieties.
Thus it happens that a milk which contains great numbers of strep-
tococci may or may not be more dangerous than one w^hich contains
an equal number of other apparently less harmful bacteria.
The Deleterious Effect of Bacteria in Milk on Infants. — We have
tested this ourselves in the following way: During each of the sum-
mers of 1902, 1903, and 1904 a special lot of milk was modified for
504 PATHOGENIC MICRO-ORGANISMS.
a group of fifty infants, all of whom were under nine months of age,
and distributed daily. To one half the milk was given raw; to the
other half a similar milk heated at 60° for 20 minutes.
The modified milk was made from a fairly pure milk mixed with
ordinary cream. The bacteria contained in the milk numbered on
the average 45,000 per cubic centimetre, in the cream 30,000,000.
The modified raw milk taken from the bottles in the morning aver-
aged 1,200,000 bacteria per cubic centimetre, or considerably less
than the ordinary grocery milk; the Pasteurized, about 1000; taken
in the late afternoon of the same day they had, respectivelv, about
20,000,000 and 50,000.
Twenty-one predominant varieties of bacteria were isolated from
six specimens of this milk collected on diflFerent days. The varieties
represented the types of bacteria frequently found in milk. The in-
fants were selected during the first week in June, and at first all were
placed on Pasteurized milk. The fifty infants which had been selected
were now separated into two groups as nearly alike as possible. On
the 15th of June the milk was distributed without heating to one half
the infants, the other half receiving as before the heated milk. In
this way the infants in the two groups received milk of identically the
same quality, except for the changes produced by heating to 165° F.
for thirty minutes. The infants were observed carefully for three
months and medical advice was given when necessary. \\Tien severe
diarrhoea occurred barley-water was substituted for milk.
The first season*s trial gave the following results: Within one
week 20 out of the 27 infants put on raw milk suffered from moderate
or severe diarrhoea; while during the same time only 5 cases of moderate
and none of severe diarrhoea occurred in those taking Pasteurized milk.
Within a month 8 of the 27 had to be changed from raw back to
heated milk, because of their continued illness; 7, or 25 per cent., did
well all summer on raw milk. On the other hand, of those receiving
the Pasteurized milk, 75 per cent, remained well, or nearly so, all
summer, while 25 per cent, had one or more attacks of severe diarrhoea.
There were no deaths in either group of cases.
During the second summer a similar test was made with 45 infants.
Twenty-four were put on raw modified milk; 13 of these had serious
diarrhoea, in 5 of whom it was so severe that they were put back
upon heated milk; 10 took raw milk all summer without bad effects;
2 died, 1 from gross neglect on the part of the mother, the other from
diarrhoea. Of the 21 on Pasteurized milk, 5 had severe attacks of
diarrhoea, but all were kept on this milk except for short periods,
when all food was omitted; 16 did well throughout the summer. One
infant, markedly rachitic, died. The third summer's results have
not been tabulated, but were similar to those of the first two tests.
The outcome of these observations during the first two summers
are summarized in the following table:
BACTERIOLOGY OF MILK.
505
Kinds of milk
I Re-
I k1?S weU for
beroi «_*;_«
linfanU ^,^"*
sum-
mer
Pastetmsed milk. 1.000 to 50.000 \
bacteria per c.c. ;
Raw milk. 1.200.000 to 20.000- t,
000 bacteria per c.c. j
41 ,
5P
31
17
N am.ber ^^e^je ^^
seve^Sr <i-r."^ -?«^ . bTSf
mode?ate Z.nu^'' ^^Jht ^Vs
diarrhopa "^J!^' '^**«*** diar-
™®'^ rhoea
Deaths
\
10
33
3
5.6
4.0 OB.
3.9
3.50X. I 11.5
1
2
Although the number of cases was not large, the results, almost
identical during the three summers, indicate that even a fairly pure
milk, when given raw in hot weather, causes illness in a much larger
percentage of cases than the same milk given after Pasteurization.
A considerable percentage of infants, however, do apparently quite
as well on raw as on Pasteurized milk.
Bacteria in Milk. Effect on Older Children— The children over
three years of age who received unheated milk, containing at different
times from 145,000 to 350,000,000 bacteria per cubic centimetre,
showed almost no gastrointestinal disturbance. The conditions at
three institutions will serve as examples.
In the first of these an average grade of raw milk was used which,
during the summer, contained from 2,000,000 to 30,000,000 bacteria
per cubic centimetre. This milk was stored in an ice-box until re-
quired. It was taken by children unheated and yet no case of diar-
rhoea of sufficient gravity to send for a physitian occurred during the
entire summer. This institution was an orphan asylum containing
650 children from three to fourteen years of age — viz., three to five
years, 98; five to eight years, 162; eight to fourteen years, 390.
A second institution used an unheated but very pure milk which
was obtained from its own farm. This milk averaged 50,000 bac-
teria per cubic centimetre. The inmates were 70 children of ages
ranging from three to fourteen years. In this institution not a single
case of diarrhoeal disease of any importance occurred during the
summer.
In a third institution an average grade of milk was used which
was heated. This milk before heating contained 2,000,000 to 20,-
000,000 bacteria per cubic centimetre. The institution was an in-
fant asylum in which there were 126 children between the ages of
two and five years. There were no cases of diarrhoea during the
summer.
These clinical observations taken in connection with the bacterio-
logical examination at the laboratory show that although the milk
may come from healthy cattle and clean farms and be kept at a tem-
perature not exceeding 60° F., a very great increase in the number
* Thirteen of the fifty-one infants on raw milk were transferred before the end
of the trial to Pasteurized milk because of serious illness. If these infants had
been left on raw milk it is believed by the writers that the comparative results
would have been even more unfavorable to raw milk.
506 PATHOGENIC MICROORGANISMS.
of bacteria may occur. Furthermore, this may occur without the
accumulation in the milk of sufficient poisonous products or living
bacteria to cause appreciable injury in children over three years (rf
age, even when such milk is consumed in considerable amount and
for a period extending over several months. Milk kept at tempera-
tures somewhat above 60° F, was not met with in our investigations,
but the histories of epidemics of ptomain poisoning teach thai such
milk may be very poisonous. It is also to be remembered that milk
abounding in bacteria on account of its being carelessly handled is
also always liable to contain pathogenic organisms derived from
human or animal sources.
Resolts witli Very Impure Milk Heated vs. Those with Pnn
or Average Milk Heated. — ^During the summer of 1901 we were able to
observe a number of babies fed on milk grossly contaminated by hac-
teria. In 1902 systematic supervision of all stores selling milk was
instituted by the Health Department, so that the very worst milk was
not offered for sale that summer.
The obser\'ations upon the impure milk of 1901 are of sufficieni
importance to be given in detail, although already mentioned in the
report of the observations upon infants of both summers which n-ere
fed on "store milk." A group of over 150 infants was so divided
that 20 per cent, were allowed to remain on the cheapest store milk
which they were taking at the time. To about the same number was
given a pure bottled milk. A third group was fed on the same qiialiiv
of milk as the second, but sterilized and modified at the Good SalDa^
itan Dispensary. A fourth group received milk from an ordinatr
dairy farm. This milk was sent to a store in cans and called for bv
the people. A few infants fed on breast and condensed milk were
observed for control.
In estimating the significance of the observations recorded in the
tables, one should bear in mind that not only do different infants
possess different degrees of resistance to disease, but that, try as hard
as the physicians could, it was impossible to divide the infants into
groups which secured equal care and were subjected to e.vactly the
.same conditions. It was necesskry to have the different groups in
somewhat different parts of the city. It thus happened that the in-
fants on the cheap store milk received less home care than the aver-
age, and that tho^se on the pure bottled milk lived in the coolest por-
tion of the city. Certain results were, however, so striking that Uieir
interpretation is fairly clear. It is to be noted that the number of
infants included in each group is small.
'T'i,„.„ :„ "othing in the observations to show that fairly fresh milk
cows, living under good hygienic conditions and con-
jme days, when delivered, as many as 200,000 bacteria
itimetre, had any bacteria or any products due to har-
mained deleterious after the milk was heated to near
lint.
ler hand, it is possible that certain varieties of bacteria
BACTERIOLOGY OF MILK,
507
Table Showing the Results of Feeding during July and August, 1901, in
Tenement Houses, of 112 Bottle-fed Infants under 1 Year of Age, and
OF 47 Bottle-fed Infants between 1 and 2 Years op Age with Milk
FROM Different Sources, and the Number of Bacteria Present in the
Milk.
Character of the milk
Pure milk. 24 hours old,
sent in quart bottles to ten-
ements, heated and modi-
fied at home. 20,000 to
200.000 bacteria per c.c.
when delivered.
3.
4.
5.
Ordinary milk, 36 hours
old, from a selected group
of farms, kept cool in cans
during transport; 1,000,-
000 to 25,000,000 bacteria
per c.c, heated and modi-
fied at home before using. .
Cheap milk, 3d to 60 hours
old, from various small
stores, derived from var-
ious farms, some fairly
clean, some very dirty;
400,000 to 175,000.000 bac-
teria per c.c.
Condensed milk of differ-
ent brands. Made up
with hot water. As given,
contained bacteria from
5,000 to 200,000 per c.c.
Infants under one year
Infants over one year
-
-
-
- —
—
1
-
-
—
•o
Diarrhoea
•s
Diarrhoea
. «
Average
weekly
gain
i
1
umber
infants
III
umbel
infant
(ild
£
>
[ild
iS
>
1
•«s
^
2:
^
^
1. Pure milk boiled and modi-
fied at dbpensary or sta-
tions; given out in small
bottles. Milk before boil-
ing averaged 20,000 bac-
teria per c.c; after boiling
2 per c.c.
41
21
9
6. Breast milk 16
3oi.
23 4i oi.
^ 18 4 OS.
Q
10 i 8
8
6 6
i oz. 4
i oz. 5
2i oz. i 5
13
V
0 24 I 4i oz. 8
0
1» 12
4 oz.
0
ioz. 1
0
3 4 , 3i oz.
0
0
may, under conditions that are unsanitary, find entrance to milk and
survive moderate heat or may develop poisonous products resistant
to heat in sufficient amount to be harmful, even when they have ac-
cumulated to less than 200,000 per cubic centimetre.
Turning now to the results of feeding with milk which has been
heated and which before sterilization contained from 1,000,000 to
25,000,000 bacteria per cubic centimetre, averaging about 15,000,-
000, though obtained from healthy cows living under fairly decent
conditions and although the milk was kept moderately cool in transit,
we find a distinct increase in the amount of diarrhoeal diseases.
* This infant died from enteritis and toxsemia.
' This infant died of pneumonia. There had been no severe intestinal disorder
noted.
* One of the four had pertussis, the remaining three died from uncomplicated
enteritis.
508 PATHOGENIC MICRO-ORGANISMS.
Though it is probable that the excessive amount of diarrhoea in lW\s
group of children was due to bacterial changes which were not neu-
tralized by heat or to living bacteria which were not killed, yet it is
only fair to consider that the difference was not very great and thai
the infants of this group were under surrounding.s not quite as good
at those on the purer milk.
Finally, we come in this comparison to the infants who received
the cheap store milk after heating. This milk had frequently to be
returned because it curdled when boiled, and contained, according
to the weather, from 4,000,000 to 200,000,000 bacteria per cubic
centimetre. In these infants the worst results were seen. This is
shown not only by the death rate, but by the amount and by the
severity of the diarrhoeal diseases, and the general appearance of tbe
children as noted by the physicians. Although the average number
of bacteria in the milk received by this group is higher than that re-
ceived by the previous group, the difference in results between this
group and the previous one can hardly be explained by the difference
in the number of bacteria. The varieties of bacteria met with in
this milk were more numerous than in the better milk, but we were
unable to prove that they were more dangerous. Probably the higher
temperature at which the milk was kept in transit, and the longer
interval between milking and its use, allowed more toxic bacterial
products to accumulate.
Bacterial Oontaminatlon of Milk — General Goncliuiona' as to
Relative Importance. — 1. During cool weather neither the mortality
nor the health of the infants observed in the investigation was appre-
ciably affected by quality of the market milk or by the number of bac-
teria which it contained. The different grades of milk varied much
less in the amount of bacterial contamination in winter than in summer,
the store milk averaging only about 750,000 bacteria per cubic
centimetre.
2. During hot weather, when the resistance of the children was
lowered, the kind of milk taken influenced both the amount of illness
and the mortality; those who took condensed milk and cheap store
milk did the worst, and those who received breast milk, pure bottled
milk, and modified milk did the best. The effect of bacterial con-
tamination was very marked when the milk was taken without pre-
vious heating; but, unless the contamination was verj' excessive, only
"' ' ■ ■ ' ■■ IS employed shortly before feeding.
bacteria which may accumulate before milk
armful to the average infant in summer differs
? bacteria present, the age of the milk, and the
1 it has been kept. When the milk is taken
I'teria present the better are the results. Of the
1,000,000 bacteria per cubic centimetre are
o the average infant. However, many infants
BACTERIOLOGY OF MILK. 509
take such milk without apparently harmful results. Heat above
170® F. (77® C.) not only destroys most of the bacteria present, but,
apparently, some of their poisonous products. No harm from the
bacteria previously existing in recently heated milk was noticed
in these observations unless they had amounted to many millions, but
in such numbers they were decidedly deleterious.
4. When milk of average quality was fed, sterilized and raw, those
infants who received milk previously heated did, on the average,
much better in warm weather than tfiose who received it raw. The
difference was so quickly manifest and so marked that there could
be no mistaking the meaning of the results.
5. No special varieties of bacteria were found in unheated milk
which seemed to have any special importance in relation to the sum-
mer diarrhoeas of children. A few cases of acute indigestion were seen
immediately following the use of Pasteurized milk more than thirty-
six hours old. Samples of such milk were found to contain more
than 100,000,000 bacteria per cubic centimetre, mostly spore-bear-
ing varieties. The deleterious effects, though striking, were neither
serious nor lasting.
6. After the first twelve months of life, infants are less and less
affected by the bacteria in milk derived from healthy cattle. Ac-
cording to these observations, when the milk had been kept cool, the
bacteria did not appear to injure the children over three years of age
at any season of the year, unless in very great excess.
7. Since a large part of the tenement population must purchase
its milk from small dealers, at a low price, everything possible should
be done by health boards to improve the character of the general
milk supply of cities by enforcing proper legal restrictions regarding
its transportation, delivery, and sale. Sufficient improvements in
this respect are entirely feasible in every large city to secure to all
a milk which will be wholesome after heating. The general prac-
tice of heating milk, which has now become a custom among the
tenement population of New York, is undoubtedly a large factor in
the lessened infant mortality during the hot months.
8. Of the methods of feeding now in vogue, that by milk from
central distributing stations unquestionably possesses the most ad-
vantages, in that it secures some constant oversight of the child, and,
since it furnishes the food in such a form that it leaves the mother
least to do, it gives her the smallest opportunity of going wrong.
This method of feeding is one which deserves to be much more ex-
tensively employed, and might, in the absence of private philan-
thropy, wisely be undertaken by municipalities and continued for
the four months from May 15th to September 15th.
9. The use, for infants, of milk delivered in sealed bottles, should
be encouraged whenever this is possible, and its advantage duly ex-
plained. Only the purest milk should be taken raw, especially in
summer.
10. Since what is needed most is intelligent care, all possible means
510 PATHOGENIC MICRO-ORGANISMS,
should be employed to educate mothers and those caring for in-
fants in proper methods. This, it is believed, can most effectively
be done by the visits of properly qualified trained nurses or women
physicians to the homes, supplemented by the use of printed
directions.
11. Bad surroundings, though contributing to bad results in feed-
ing, are not the chief factors. It is not, therefore, merely by better
housing of the poor in large cities that we will see a great reduction
in infant mortality.
12. While it is true that even in tenements the results with the
best bottle feeding are nearly as good as average breast feeding, it is
also true that most of the bottle feeding is at present very badly done;
so that, as a rule, the immense superiority of breast feeding obtains.
This should, therefore, be encouraged by every means, and not dis-
continued without good and sufficient reasons. The time and money
. required for artificial feeding, if expended by the tenement mother
to secure better food and more rest for herself, would often enable her
to continue nursing with advantage to her child.
13. The injurious effects of table food to infants under a year old,
and of fruits to all infants and young children in cities, in hot weather,
should be much more generally appreciated.
Influence of Temperature upon the Multiplication of Bacteria in
Milk. — ^Few, even of the well informed, appreciate how great a dif-
ference a few degrees of temperature will make in the rate of bac-
terial multiplication. Milk rapidly and suflSciently cooled keeps al-
most unaltered for thirty-six hours, while milk insufficiently cooled
deteriorates rapidly.
The majority of the bacteria met with in milk grow best at tem-
peratures above 70° F., but they also multiply slowly even at 40°
F.; thus of 60 species isolated by us, 42 developed good growths at
the end of seven days at 39*^ F. Our observations have shown that
the bacteria slowly increase in numbers after the germicidal prop-
erties of the milk have disappeared, and the germs have become ac-
customed to the low temperature. In fact, milk cannot be perma-
nently preserved unaltered unless kept at 32° F. or less. The degree
of cooling to which ordinary supplies of milk are subjected differs
greatly in various localities. Some farmers chill their milk rapidly,
by means of pipe coils over which the milk flows; others use deep
wooden tanks filled with water into which the cans of milk are placed
soon after milking. In winter these methods are very satisfactory
for the water runs into the pipes or tanks at about 38° F. In warmer
weather they are unsatisfactory, unless ice is used, as the natural
temperature of the water may be as high as 55° F. A considerable
quantity of milk is not cooled at all at the farms. It is sent to the
creamery or railroad after two to six hours, and is then more or less
cooled. These few hours in summer, when the milk is left almost at
blood heat, allow an enormous development of bacteria to take place,
as is shown in the table below.
BACTERIOLOGY OF MILK. 511
Table I. — Showing the development of bacteria in two samples of milk main-
tained at different temperatures for twenty-four, forty-eight, and ninety-six hours,
respectively. The first sample of milk was obtained under the best conditions
possible, the second in the usual way. When received, specimen No. 1 contained
3000 bacteria per c.c, specimen No. 2, 30,000 per c.c.
Time which elapsed before making test.
Temperature.
N
Fahrenheit.
24 hrs.
48 hrs.
96 hrs.
168 hrs.
32°
2400
2100
1850
1400
30,000'
27,000
3600
24,000
19,000
39°
2500
218,000
4,209,000
38,000
2600
66,000
4,300,000
38,000,000
42°
3600
500,000
11,200,000
43,000
3100
210,000
6,760,000
120,000,000
46°
12,000
1,480,000
80,000,000
42,000
360,000
12,200,000
300,000,000
50°
11,600
540,000
300,000,000
1,000,000,000*
89,000
1,940,000
1,000,000,000'
55°
18,800
187,000
3,400,000
38,000,000
60°
180,000
900,000
28,000,000
168,000,000
68°
450,000
4,000,000
500,000,000
1,000,000,000'
86°
1,400,000,000*
14,000,000,000'
Observations on Bacterial Multiplication in Milk at 90° F., a Temperature Com-
mon in New York in Hot Summer Weather.
Table II. — Number of Bacteria per I c.c.
Milk I.
Milk II.
MUk III.
Fresh and of good
Fair quality from
Bad quality from
quality
store.
store.
Original number
5200
92,000
2,600,000
After two hours
8400
184,000
4,220,000
After four hours
12,400
470,000
19,000,000
After six hours
68,500
1,260,000
39,000,000
After eight hours
654,000
6,800,000
124,000,000
A sample of milk No. I. removed after six hours and cooled to 50° F. contained
145,000,000 at the end of twenty-four hours. Some of this milk, kept cool from
the beginning, contained but 12,800 bacteria per c.c. at the end of twenty-four
hours.
Pasteurization of Milk. — The two dominant factors which control
the temperature and time at which the milk should be heated are
(1) the thermal death points of pathogenic bacteria, and (2) the
thermolabile food constituents of the milk. The first factor is al-
most equally important for milk used by persons of all ages, while
the second factor is only important for milk used in very young
children.
The exposure of bacteria for a short time at a high temperature
is equivalent to a longer time at a lower temperature. The ferments
and other labile food constituents, on the other hand, are altered much
more by the higher temperature. It is well, therefore, to choose the
lowest possible temperature which will kill the non-spore-bearing patho-
* The figures referring to tests of the second sample are printed in heavy-face
type.
^ These figures are fairly accurate estimates.
Degree of heat.
Time exposed.
60° C.
15 min.
60° C.
20 min.
60° C.
30 min.
70° C.
0.5* min.
70° C.
1 min.
70° C.
2 min.
512 PATHOGENIC MICRO-ORGANISMS.
genie bacteria in a practicable length of time. Such an exposure is
60*^ C. (140° F.) for 20 minutes or 70° C. 158° F.) for 5 minutes. Yen
much shorter exposures, as one minute at 70° C, will kill the great ma-
jority of pathogenic and other bacteria in the milk and add much of
safety as seen in the tables below, but it is better to be on the safe side.
Table showing e£fect of heat upon tubercle bacilli in milk heated instantly.
Amount milk. Result in guinea-pigs.
1 c.c. Infection
1 c.c. No infection
1 c.c. No infection
1 c.c. Infection*
1 c.c. No infection
1 c.c. No infection
Control not heated . 001 Infection
^ This milk was infected by adding one-fifth of its quantity of sputum rich in
tubercle bacilli.
Development of Bacteria in Heated Milk, — ^There is a common idea that bacteria
develop much more rapidly in milk that has been heated than in raw milk. This
is only true for freshly drawn milk which has slight bactericidal power.
The table below shows the effect on bacteria in milk of heating to 70^ C. for
one-half and one minute. Not only the immediate reduction in number is seen
to be great, but the difference continues when the milk is kept cold for two days.
Two samples mixed from 100 samples of inspectors. Pasteurized at 16(f F.
Plates made same day.
Sample I. Sample II.
Control 600,000 Control 5,400,000
im 2000 im 7400
Im 1000 Im 600
Same Samples Kept in Ice-box 24 hrs. at 45^ F. (7® C).
Control 6,300,000 Control 21,600,000
im 18,000 im 12,000
Im 900 Im 3600
In Ice-box 48 hrs, at 45'" F. (7° C).
Control 16,200,000 Control 63,000,000
i m 120,000 i m 276,000
Im 10,000 Im 90,000
In Room at 71° F. (22° C).
Control 36,600,000 Control 150,000,000
i m 5,460,000 ' i m 4,500,000
1 m 5,400,000 1 m 3,600,000
Number of Bacteria in Milk Produced under Different Conditions.
1. The number of bacteria present at the time of milking and twenty-four,
forty-eight, and seventy-two hours afterward in milk obtained and kept under
correct conditions.
No preservatives were present in any of the following specimens:
Pure milk obtained where every reasonable means was taken to ensure cleanli-
ness. The long hairs on the udder were clipped; the cows roughly cleaned and
placed in clean barns before milking; the udders were wiped off just previous
to milking; the hands of the men were washed and dried; the pails used had small
(six-inch) openings, and were thoroughly cleaned and sterilized by steam before
use. Milk cooled within one hour after milking to 45® F., and subsequently kept
at that temperature. The first six specimens were obtained from individuial
cows ; the last six from mixed milk as it flowed at different times from the cooler.
Temperature of barns 55® F.
* Most of the guinea-pigs were not infected by the milk heated for one-half
minute.
BACTERIOLOGY OF MILK. 513
X umber of Bacteria in 1 c.c. of Milk.^
From sij: individual cows.
5 hrs.
after milking. After 24 hrs. After 48 hrs. After 72 hrs.
500 700 12,500 Not counted.
700 700 29,400 Not counted.
19,900 5200 24,200 Not counted.
400 200 8600 Not counted.
900 1600 12,700 Not counted.
13,600 3200 19,500 Not counted.
Average 6000 1933 17,816
From mixed milk of entire herd.
6900
12,000
19,800
494,000
6100
2200
20,200
550,000
4100
700
7900
361,000
1200
400
7100
355,000
6000
900
9800
445,000
1700
400
8700
389,000
Average 4333 2766 10,583 329,000
Twenty-five samples taken separately from individual cows on another day and
tested immediately averaged 4550 bacteria per c.c. and 4500 after twenty-four
hours. These twenty-five specimens were kept at between 45® and 50° F.
2. Milk taken during winter in well- ventilated, fairly clean, but dusty barns.
Visible dirt was cleaned off the hair about the udder before milking. Milkers'
hands were ^nped off, but not washed. Milk pails and cans were clean, but the
straining cloths dusty. Milk cooled within two hours after milking to 45° F.
Number of Bacteria in 1 c.c. of Milk.
At time of milking. After 24 hrs. After 48 hrs.
12,000 14,000 57,000
13,000 20,000 65,000
21,500 31,000 106,000
Average 15,500 21,666 76,000
Number in City Milk.
3. The condition of the average city milk is very different, and is shown in the
following tables.
The twenty samples were taken late in March by Inspectors of the Department
of Health of New York City from cans of milk immediately upon their arrival in
the city.
The temperature of the atmosphere averaged 50° F. during the previous twenty-
four hours. The temperature of the milk when taken from the cans averaged
45° F. Much of this milk had been carried over two hundred miles. From the
time of its removal from the cans, which was about 2 a. m., until its dilution in
nutrient agar, at 10 a. .m., the milk was kept at about 45° F.
* Number of bacteria obtained from development of colonies in nutrient agar in
Petri plates. The nutrient medium contained 2 per cent, peptone and 1.2 per
cent, agar, and was faintly alkaline to litmus. One set of plates were usually
left four days at about 20° C, and one set twenty-four hours at 37° C, and then
twenty-four hours at 20° C. From 5 to 300 per cent, more colonies developed, as
a rule, in the plates kept at room temperature than in those kept for twenty-four
hours at 37° C. The milk was diluted as desired with 100 or 10,000 parts of
sterile water, and 1 c.c. of the diluted milk was added to 8 c.c. of melted nutrient
agar. Plates containing over 1000 colonies were found to be inaccurate, in that
they gave too low totals. Apparently a considerable number of bacteria failed
to develop colonies when too many were added to the nutrient agar. Nutrient
gelatin was found to be more troublesome and not to yield more accurate results
than nutrient agar.
33
514 PATHOGENIC MICRO-ORGANISMS.
From New York and Hxidson River Railroad. From Harlem Railroad.
No. of bacteria No. of bacteria
No. of sample. in 1 c.c. No. of sample. in 1 c.c.
50 35,200,000 48 6,200,000
51 13,000,000 49 2,200,000
52 2,500,000 50 15,000,000
53 1,400,000 51 70.000
54 200,000 52 80,000
55 600,000 53 320.000
While the above figures indicate that much of the milk sold is fair,
even in summer, they show an appalling condition for most of that
sold to the poorer classes — those who not only comprise the larger
part of the population, but who are also compelled to keep their chil-
dren in town during the hot weather.
It must be kept in mind that milk averaging 3,000,000 bacteria
per cubic centimetre will, when kept at the temperature common in
the homes of the poor, soon contain very largely increased numbers
and show its dangerous condition by turning sour and curdling.
Cleanliness Used in Obtaining Milk, and Its Influence.— The pres-
ent conditions under which much of the milk is obtained are not
pleasant to consider. In winter, and to a less extent at other seasons
of the year, the cows in many stables stand or lie down in stalls in
the rear portion of which there is from one to four inches of manure
and urine. When milked the hands of the milkers are not cleansed,
nor are the under portions of the cows, only visible masses of ma-
nure adhering to the hair about the udder being removed. Some
milkers even moisten their hands with milk, to lessen friction, and
thus wash off the dirt of their hands and the cow's teats into the milk
in the pails. Some may regard it as an unnecessary refinement to
ask that farmers should roughly clean the floors of their stalls once
each day, that no sweeping should be done just before milking, and
that the udders should be wiped with a clean damp cloth and the
milkers should thoroughly wash and wipe their hands before com-
mencing milking. The pails and cans should not only be carefully
cleansed, but afterward scalded out with boiling water. The wash-
ing of the hands would lessen the number of ordinary filth bacteria
in the milk, and diminish risk of transmitting to milk human infec-
tious diseases, like scarlet fever, diphtheria, and enteric fever, by the
direct washing off of the disease germs from infected hands. It
would also inculcate general ideas of the necessity of cleanline5>
and of the danger of transmitting disease through milk. The value
of cleanliness in limiting the number of bacteria is demonstrated bv
the figures contained in the tables.
General Oonclusions. — Because of its location and its hairy covering,
the cow's udder is always more or less soiled with dirt and manure
unless cleaned. On account of the position of the pail and the acce^^
of dust-laden air it is impossible to obtain milk by the usual methods
without mingling with it a considerable number of bacteria. With
suitable cleanliness, however, the number is far less than when filthy
BACTERIOLOGY OF MILK. 515
methods are used, there being no reason why fresh milk should contain
in each cubic centimetre, on the average, more than 12,000 bacteria
per c.c. in warm weather and 5000 in cold weather. Such milk,
if quickly cooled, to 46° F., and kept at that temperature, will at the
end of thirty-six hours contain on the average less than 50,000 bacteria
per cubic centimetre, and if cooled to 40° F. will average less than its
original number.
With only moderate cleanliness such as can be employed by any
farmer without adding appreciably to his expense, namely, clean
pails, straining cloths, cans or bottles, and hands, a fairly clean place
for milking, and a decent condition of the cow's udder and the adjacent
belly, milk when first drawn will not average in hot weather over
30,000, and in cold weather not over 25,000 bacteria per cubic centi-
metre. Such milk, if cooled and kept at 50° F., will not contain at
the end of twenty-four hours over 100,000 bacteria per cubic centi-
metre. If kept at 40° F. the number of bacteria will not be over
100,000 per cubic centimetre after forty-eight hours.
If, however, the hands, cattle, and barns are filthy and the pails
are not clean, the milk obtained under these conditions will, when
taken from the pail, contain very large numbers of bacteria, even up
to a million or more per cubic centimetre.
Freshly drawn milk contains a slight and variable amount of bac-
tericidal substances which are capable of inhibiting bacterial growth.
At temperatures under 50° F. these substances act efficiently (unless
the milk is filthy) for from twelve to twenty-four hours, but at higher
temperatures their effect is very soon completely exhausted, and the
bacteria in such milk will then rapidly increase. Thus the bacteria
in fresh milk which originally numbered 5000 per cubic centimetre
decreased to 2400 in the portion kept at 42° F. for twenty-four hours,
but rose to 7000 in that kept at 50° F., to 280,000 in that kept at 65°
F., and to 12,500,000,000 in the portion kept at 95° F.
As we have seen, the milk in New York City is found on bacterio-
logical examination to contain, as a rule, excessive numbers of
bacteria. During the coldest weather the milk in the shops averages
over 300,000 bacteria per cubic centimetre, during cool weather
about 1,000,000, and during hot weather about 2,000,000. The
milk in other large cities is, from all accounts, in about the same
condition.
The above statement holds for milk sold at the ordinary shops,
and not that of the best of the special dairies, where, as previously
stated, the milk contains only from 1000 to 30,000 bacteria, accord-
ing to the season of the year.
The question might be raised, Are even these enormous numbers
of bacteria often found in milk during hot weather harmful ?
Our knowledge is probably as yet insufficient to state just how
many bacteria must accumulate to make them noticeably dangerous
in milk. Some varieties are undoubtedly more harmful than others,
and we have no way of restricting the kinds that will fall into milk.
516 PATHOGENIC MlCRO-ORGAMSAfS.
except by enforcing cleanliness. We have also to consider that milk
is not entirely used for some twelve hours after being purchased, and
that during all this time bacteria are rapidly multiplying, especially
where, as among the poor, no provision for cooling it is made. Slight
changes in the milk which to one child would be harmless, would in
another produce disturbances which might lead to serious disease.
A safe conclusion is that no more bacterial contamination should
be allowed than it is practicable to avoid. Any intelligent farmer can
use sufficient cleanliness and apply suflScient cold, with almost no
increase in expense, i cent per quart, to supply milk twenty-four to
thirty-six hours old which will not contain in each cubic centimetre
over 50,000 to 100,000 bacteria, and no milk containing more bacteria
should be sold.
The most deleterious changes which occur in milk during its trans-
portation are now known not to be due to skimming off the cream or to
the addition of water, but to the changes produced in the milk by mul-
tiplication of bacteria. During this multiplication, acids and distinctly
poisonous bacterial products are added to the milk, to such an extent
that much of it has become distinctly deleterious to infants and invalids
It is the duty of health authorities to prevent the sale of milk rendered
unfit for use through excessive numbers of bacteria and their products.
The culture tests to determine the number of bacteria present in
any sample of milk require at least forty-eight hours; so that the sale
of milk found impure cannot be prevented. It will, however, be the
purpose of the authorities gradually to force the farmers and the middle-
men to use cleanliness, cold, and dispatch in the handling of their milk,
rather than to prevent the use of the small amount tested on any one
day.
If the milk on the train or at the dealer's were found to contain ex-
cessive numbers of bacteria, the farmers would he cautioned and
instructed to carry out the simple necessary rules, which would be
furnished.
Transmisson of Contagious Diseases through Blilk. — Pathogenic
Bacteria in Milk. — Tuberculosis, typhoid fever, scarlet fever, diphtheria
and tonsillitis are the chief diseases transmitted by means of milk
in this locality. In other countries cholera, malta fever and possibly
other diseases mav be due at times to milk infection. The obscure
disease trembles is also believed to be due to milk.
The tubercle bacilli are in the majority of cases derived from the cow,
but may come from human sources, the typhoid bacilli are entirely from
man, the contagion of true scarlet fever conveyed in milk is probably
always from man, but the contagion of a disease closely allied to it i>
certainly given off by cows suffering from certain septic diseases as yet
not fully identified. Diphtheria bacilli are probably always of human
origin, as animals, except cats, practically never suffer from the dLsease
and these only under exceptional conditions. The streptococci exciting
tonsillitis are probably usually from cases of septic inflammation of the
udder, but possibly may at times come from man. As milk is usuaUy
BACTERIOLOGY OF MILK. 517
kept below 60° F. the typhoid bacilli and the streptococci are the only
germs that we believe increase in any appreciable extent.
The following epidemics and cases have been recorded in the bulle-
tin of the Marine Hospital service, as produced by cow's milk:
Epidemics. Cases.
Typhoid fever 179 6900
Scarlet fever 51 2400
Sore throat 7 1100
Diohtheria 23 960
Tuberculosis
The cases of trembles (milk sickness), believed to be due to milk,
have not been collected with suflBcient care to be reported. No case
of measles, smallpox, whooping cough, or mumps has been clearly
traced to milk.
The Relation of the Typhoid Carrier to Blilk Infection. — Many epi-
demics of typhoid fever have until recently puzzled investigators be-
cause, though evidently milk-borne, yet no case of typhoid fever could
be found. The discovery that about 2 per cent, of those who have
recovered from typhoid fever remain infected and continue during the
rest of their lives to pass typhoid bacilli has cleared up the mystery.
Epidemics due to these carriers have already been traced both in New-
York City and elsewhere. Many observers have already discussed the
relation of typhoid cases to milk infection. Hands, water, flies, etc.,
may all aid in the transfer of the bacilli from the dejecta to the milk.
Last year we traced over four hundred cases to infection of a milk supply
by a typhoid carrier who had the disease forty-seven years ago. Just
recently we traced fifty cases to a man who had the disease seven years
ago.
The Oonveyance of Scarlet Fever by Means of Blilk. — As we do not
know the organism which excites scarlet fever, we are not as clear as to
the means by which it is spread as we are in the case of tuberculosis,
typhoid fever and diphtheria. We know, however, that the throat
secretions and the peeling scales of skin are dangerous. Where the
infection has been traced it has usually been found that the milker has
^flFered from an unrecognized case or is convalescent. It seems as if
the contagion must either increase in milk or be capable of infecting
when greatly diluted, for cases have developed from milk after great
dilution. A small number of epidemics have appeared to come from
the milk of diseased cows. Many are skeptical about this, but after
personal experience we cannot doubt it. The history of this case was
as follows: The milk from a septic cow was delivered to two schools.
About thirty of the boys who drank the milk developed the disease
while none of the dav scholars who went home to lunch did. Some of
the cases developed at first only sore throats, others only the rash.
On the second dav the cases resembled verv closelv scarlet fever. There
was no scarlet fever in the town. The milk contained immense numbers
of long-chained streptococci.
518 PATHOGENIC MICRO-ORGANISMS.
Diphtheria and septic sore throats are occasionally produced bv milk.
The diphtheria bacilli usually originate from a mild case, the nature of
which is not detected. Septic sore throats produced by milk are usually
caused by infection from cows suffering from some form of uddet
disease.
PART III.
PROTOZOA
CHAPTER XXXIX.
GENERAL CHARACTERISTICS AND CLASSIFICATION.
Introduction. — Recent discoveries relating to the origin of human
diseases are adding to the number caused, or probably caused by
Protozoa. Indeed, the fact that the specific etiological factor in malaria
is a protozoon, has not been known long, though this organism is the
first protozoon shown to be pathogenic for man. The evidence which
has been accumulating in favor of the idea that a certain form of
dysentery is due to an ameba is gaining ground; quite recently sleeping
sickness and kala-azar have been added to the list of protozoan dis-
eases, and it is now thought that some of the members of the group
of contagious diseases, known as the exanthemata, may be due to
infection with organisms belonging to this sub-kingdom; therefore
it becomes more and more necessary for those interested in the etiology,
course, and prevention of disease to obtain a more definite under-
standing of this great group of microorganisms.
In any treatise on pathogenic organisms which is intended to aid
the medical student and practitioner special attention should be given
to the effect of the organism upon the host and to methods of diag-
nosis and treatment. Therefore symptoms of the disease, tissue
changes in the host, and special staining and other methods for diag-
nosing the organisms are described. Only those characteristics of the
organisms are given which will help in recognizing them in disease.
For minutia of morphology, theories in regard to relationship, and
other special points relating to the organisms themselves the student
should consult such books as Calkins' "Protozoology."
Definition. — A protozoon (the lowest form of animal life) is a mor-
phologically single-celled organism, composed of protoplasm which
is differentiated into cytoplasm and nucleus (or nuclear substance),
both of which show many variations throughout the more or less
complicated life cycle that each individual undergoes.
Relationship to Other Microdrganisms. — They are classed as the low-
est animals, but they are so closely related to the protophyta or lowest
plant forms on one side and the metazoa or many-celled animals on the other,
that it is difficult to mark out a sharp line of distinction on either side. Fol-
lowing Haeckel, some authors group them with bacteria and other closely re-
lated forms as protista, but in such a group they should be regarded as of a
519
520 PATHOGENIC MICRO-ORGANISMS.
higher grade than bacteria because of their greater complexity in gtnielure
and life cycle.'
Whether one of the simplest microorganisms is a plant or an animal is
often difRcult to decide, hence there are a number of forms which are claimfd
by both botanists and zoologista. For example the Mycttozoa are described
by both, and some members of the group are contested for by each. Again it
is not yet decided whether the spirochetes belong to the bacteria or to ihe
protozoa.
The dilliculty in deciding as to the plant or animal nature of these low
organisms is due to the fact that the obvious differences which exist between
a higher plant and a higher animal are not seen here. There is no one di^
tinctive characteristic which separates the lowest plants from the lowest
animals. In the broad sense, vegetal-nutrition is the using of more simple
nitrogenous substances than the proteids or peptones needed by animals,
as well as the mineral substances and the organic carbon compounds re-
quired to build up their protoplasm. But if we classify organisms according
to their morphology, we find that many forms placed with the bacUn'a re-
quire complex specially prepared food similar to that needed by animals:
likewise, if we depend upon a physiologic distinction, we find that chlo-
rophyl which is supposed to be a characteristic plant substance is pos-
sessed both by some bacteria and by some protozoa; and so on, through the
whole list of supposed differential characteristics. Even when we do the
better thing and make a third kingdom, the protista, some doubtful forms will
always be found on the border line.
Historical Notei.'— The history of protoioa begins with that of bacteria
in the discoveries of A. Van Leeuwenhoeck and his followers during the
latter part of the seventeenth century.' At that time all of the micro««ipJ(
organisms seen were classed together as little animals. Indeed, all of tbe
microorganisms first described at any length were probably protozoa and
only after further improvement of lenses and a more minute study of the
organisms were bacterial forms gradually recognized as a separate claa?.
The same scepticism tliat is seen in the acceptance of most new disrovMies
was displayed by doubters of the truth of these early reports of microscopif
findings. Chief among the sceptics must be placed Linnjeus who in the fir^i
edition of his Syslema Naturte (1735) absolutely denies the existence of
Leeuwenhoeck's animalcula, though in the later editions he grudgingly adm lis
them under the significant generic name of Chaos (Chaos protevs {Amabai.
etc.).
The first ideas of the structure of the protozoa were drawn from analogj-.
The early, ol)servers thought that each tiny organism possessed an internal
structure made up of organs and tissues similar to those in metazoa. Ther
could not conceive of motion without articulation, tendons, and muscles;
nor of food absorption without an alimentary tract, and they were so im-
pressed with the ideas of what they thought they ought to see that they were
convinced that they really saw many of the complicated structures possessed
by metazoa. For example, the contractile vacuole, a characteristic pubatinj
vesicle of the protozoa, discovered by Joblot in 1754 was thought by many
said to be stomachs, the mouths were often
ry tract was supplied from the imagination:
forms were interpreted as true e\'e*i. etr
these views, however, and the idea of the
which was advanced by Schleiden in 183S.
protozoa were single cells with no definite
abstracted from Calkins' excellent re\-ie« io
CLASSIFICATION AND GENERAL CHARACTERISTICS. 521
With the publications of Dujardin (1835-41) a correct idea of the struc-
tural simplicity of the microorganisms gained ground. But for some time
after, the controversy regarding the simple nature of protozoa was strenu-
ously carried on. It is a most instructive bit of history in research work,
showing how the lack of minute observation, the exercise of a too vivid
imagination, and the close reasoning from analogy may lead one astray,
while the proper use of these functions may bring out the truth.
Kolliker, Butschli, Engelmann, and Hertwig, with many others (1870-80)
finally demonstrated fully the unicellular nature of the protozoa.
The most important characteristic of a protozoon, its life history, was
first partially made out by Trembley in 1744-47. Btitschli helped determine
the sexual activities of the members of this group, while Maupas (1889)
was the first to demonstrate the conditions leading to their conjugation.
Origin of Protozoa. — Though Leeuwenhoeck and one or two others be-
lieved that the animalcules developed from minute eggs or germs, the great
majority of investigators thought that these low forms of life arose by spon-
taneous generation,* and it was not until late in the nineteenth century that
it was finally proved that under known conditions each living organism
arises from a specific spore or its prototype. When and how life began no
one is yet able to say. That spontaneous generation did take place in the
remote past is possible, that it may even be taking place now under unknown
conditions is conceivable, but all such ideas are purely hypothetical.
Though it was known comparatively early in the study of protozoa that
many forms grow on and in the higher animals and plants as parasites and
that probably they have an etiological relationship to certain diseases, it was not
until recently that definite forms were shown to be the cause of definite dis-
eases in human beings. Up to the present time, however, the pathogenic
forms worked out are so few, that in their further study and in the study of
new forms we must still find many of our analogies m the more distantly
related but better known non-pathogenic types. For this reason we include in
this section some types of the common protozoa which are easily obtained
for class study.
Materials Required for the Study of Protozoa. — Most of the appa-
ratus and chemicals described in Part I as necessary for the study
of bacteria are also used in examining protozoa. Attention may be
called to the following essential things:
Small glass pipettes with rubber caps. Some of these should be very
finely drawn out for the purpose of isolating individual protozoa in fluid
media.
Platinum needles.
Shallow glass dishes with ground-glass covers for studying large numbers
of protozoa in fluid media. *
Cover-glasses and plain hollow glass slides.
Petri dishes for the "pure mixed" cultures of amebae.
Glass jars, with ground-glass covers, for holding fixing and staining fluids.
A microscope similar to the one described under bacteria.
Drawing materials.
The general fixing fluids are: (1) Sublimate alcohol j two parts of con-
centrated watery corrosive sublimate solution and one part absolute alcohol ;
5 per cent, glacial acetic acid may be added to this mixture just before using.
For the use of this in smear and section preparations see page 537. Saturated
sublimate -f- 5 per cent, glacial acetic acid is also good as a fixative.
(2) Two per cent, osmic acid (to be kept in a red glass with a ground-glass
stopper). Moist smears are exposed to its fumes for a few seconds, small
* See Part I, Introduction.
522 PATHOGENIC MICRO-ORGANISMS,
pieces for sections, four to eight hours, then carried through the various
alcohols and xylol and mounted or embedded in the usual way.
(3) Hermann^ 8 Fluid. — Platinum chloride 15 cc, a 1 per cent, solution
osmic acid 4 cc, a 2 p^r cent, solution, glacial acetic acid 1 cc. Moist
spreads may be fixed for several minutes; very small pieces of tissue for
twenty-four hours.
(4) Zenker*8 Fluid. — Add to a solution of Miiller (bichromate of potash,
2-2 J parts; sulphate of soda, 1 part; water, 100 parts) 5 per cent, of saturated
sublimate solution and, when ready to use, 5 per cent, of glacial acetic acid.
Moist spreads are fixed for one to five minute, small pieces of tissue for three
to twelve hours. They are then washed with water or put immediately into
successive alcohols, as given on p. 625.
Staining Solutions:*
1. Giemsa's solution (see p. 624).
2. Loeffler's flagella mordant (see p. 35).
3. Delafield's hsematoxylin (see Lee's Vade-mecum).
4. Carbol-fuchsin (see p. 33).
5. Iron-hflBmatoxylin, Heidenhain (see below).
' 6. Basic fuchsin — saturated alcoholic solution.
7. Methylene blue — saturated alcoholic solution.
8. Eosin, watery solution (10 per cent.).
9. Bordeaux red, weak watery solution.
Heidenhain's iron hsematoxylin stain is as follows:
(a) Mordant and differentiating fluid: Iron oxydammonium sulphate,
2.5 'g.; distilled water, 100 cc (6) Staining fluid: Haematoxylin,
1 g. ; alcohol, 10 cc ; distilled H^O, 90 cc. (To be kept in a red bottle
and allowed to stand for about four weeks before using.) For use
see page 537.
Other fluids used:
Physiological salt solution (0 . 6-0 . 8 per cent.).
Sodium citrate solution (2.5-5 per cent.).
Iodine alcohol (iodine added until color is a clear brown).
Acid alcohol (0. 1 cc HCl in 100 cc. of 70 per cent, alcohol).
Alcohols 60, 70, 95, and 100 per cent.
Xylol for clearing.
Paraffins for embedding (see p. 625).
Cedar oil, or other paraffin solvents.
Canada balsam for mounting.
GENERAL OHARAOTERISTIOS OF PROTOZOA.
Morphology. — Shape. — The shape of protozoa varies so widely that
no general description will fit all types.
Size. — Their size, too, varies within wide limits. Indeed, some forms
appear to be invisible even under the highest magnification known,
while the largest varieties known are two-thirds of an inch long.
The Oytoplasm. — The cytoplasm varies greatly in composition and
structure according to the stage of development and the surrounding
conditions. It consists of a mixture of substances, the most important
of which belong to the proteids. It is more or less fluid, but, because
of differences in the density and solubility of the several parts, it often
* The formulas for most of these stains are given in Part I under staining
methods. The solutions may be obtained ready for use from Griibler, Lieipzig, or
by his agents throughout the world.
CLASSIFICATION AND GENERAL CHARACTERISTICS. 523
presents an alveolar, linear, or granular appearance, which may
come out clearly in fixed and stained specimens, but is usually not well
seen in the living cells. Frequently the protozoan cytoplasm is dif-
ferentiated into a concentrated, viscid, more homogeneous, or hyaline
outer layer called the ectoplasm and a more fluid granular c^tral
portion called the entoplasm. These two portions have different func-
tions. The ectoplasm helps introduce and excrete food and air, there-
fore it becomes modified to help form the various organs of motion,
contraction, and prehension. These organs are pseudopods (false feet),
flagella (whip-like threads), cilia (hair filaments), suctorial tubules
(through which food passes), and myonemes (contractile part of the
ectoplasm found in fusoria, gregarines, and a few flagellates). Other
organs, or organelles as they are called by some, are found in certain
species, such as a definite oral place for the ingestion of food (cytostom),
with sometimes a curved opening leading to the entoplasm; and a
special anal part where the indigestible portion is dejected (cytopyge).
In rare cases definite parts sensitive to light, the so-called pigment
spots (euglena) are developed. The entoplasm digests the food
and contains the nucleus. It may contain various granules which
have been given special names as microsomes, plasmosomes, etc.
These are generally products of food metabolism. The entoplasm
also contains many different-sized vacuoles which serve as food
digestors, and hence contain digestive ferments. The so-called con-
tractile vacuoles which periodically fill and empty themselves may be
considered as excretory organelles.
Further, fibrils of elastic consistency may often be demonstrated
in the cytoplasm. These are probably instrumental in helping motion.
Other substances are seen from time to time in the entoplasm, such as
bacteria, red blood cells, fatty granular pigments, bubbles of gas,
crystals, etc. Some protozoa secrete solid skeletal substances in or
on the ectoplasm, as the chalky shells of Foraminifera, and the silicious
framework of Radiolaria, etc. But these species, as far as is known,
belong to the non-pathogenic protozoa.
The Nucleus. — The second element of a protozoon that is always
present is the nucleus (or the nuclear substance), which varies in size,
number, and structure according to the species and the stage of devel-
opment. The simplest morphologic nucleus is a vesicular body which
is differentiated from the cytoplasm by its essential constituent chro-
matin, so called because it has a strong affinity for certain basic stain-
ing materials. Chromatin consists mostly of nuclein and appears in
the form of smaller or larger granules, masses, or rods. Though always
having the same general staining characteristics, chromatin is com-
posed of many substances having different physiologic as well as chemic
activities.
Generally, the chromatin particles are mixed with a second less
intensely staining substance with more of an affinity for acid stains,
called plastin or paranuclein, similar to the substance from which
the true nucleolus of the metazoan cell seems to be formed. This
524 PATHOGENIC MICRO-ORGANISMS.
substance may appear in one or more distinct rounded bodies. Most
of the chromatic substances of the nucleus in many protozoa are often
massed together in an intensely-staining balMike body called the
karyosome which undergoes various cyclic changes during the growth
and .development of the organism. The centrosome is generally era-
bedded in the karyosome; the latter, indeed, is often simply the centro-
some and attraction sphere. The chromatin and plastin lie embedded
in a third substance in the form of an achromatic network called Hnin
which is closely related to the cytoplasmic network. This network
is filled with the so-called nuclear sap. There may or may not be a
definite nuclear membrane. Sometimes there is no definitely struc-
tured nucleus, but the nuclear substance in the form of small chromatin
masses or granules is distributed throughout the cytoplasm (the so-
called "distributed nucleus")
Somatic and Generative Chromatin, — It has been shown that some
chromatin substances of the cell have physiologic properties different
from others. At times substances which have only vegetative proper-
ties are active, forming the so-called somatic or trophic chromatin;
at other times, substances appear during sexual activities called gen-
erative or sexual or idio-chromatin, and from these the vegetative (so-
matic) chromatin for the new cells is again formed. In the ciliata
both these chromatin elements are present as distinct morphologic
bodies during the entire life of the organism, the somatic form in the
macronucleus and the generative form in the micro-nucleus.
Chromidia (Fig. 182c, p. 584. — ^The chromatin elements, in the form
of granules, small irregular masses, threads, network, etc., which pass
from the nucleus into the cytoplasm, or which at times are, possibly,
formed in the cytoplasm, were named **Chromidien" by R. Hertwig,
who in 1899 first described their appearance. Their function in gen-
erative processes was demonstrated in 1903 by Schaudinn. During
their formation the nucleus may entirely disappear, so that morpho-
logically the cell may be considered non-nuclear. At a certain time
thereafter new typical nuclei may be formed from these chromidial
substances.
Locomotor Nucleus {Kinetic Nucleus), — In flagellates still another
definite physiologic chromatin is seen in the small body called the
kinetic nucleus (Fig. 175, p. 562), which is either apart from or merged
into a smaller body, the blepharoplast forming the root of the flagellum.
The kinetic nucleus is so called because it produces the locomotor
apparatus. Both the kinetic and trophic nuclei may contain somatic
and generative chromatin at the same time.
The Oentrosome. — This is a small body which is always present
in metazoan cells, playing an important part in cell division, but it
has not been demonstrated as a morphologic entity in many varieties
of protozoa; part of the karyosome, however, may take its place, or
there may always be a true centrosome within the karyosome. WTien-
ever a centrosome appears in protozoa, it has its origin in the nucleus,
resembling in this the kinetic nucleus and blepharoplast. All these
CLASSIFICATION AXD GENERAL CHARACTERISTICS. 525
four bodies, therefore, centrosome, blepharoplast, kinetic nucleus, and
karyosome, may be considered as having a similar morphologic origin.
Vital Phenomena. — In common with all other living organisms,
protozoa possess the essential functions of irritability, nutrition, res-
piration, and reproduction.
Irritability or the reaction to external stimuli of nerve response. All
protozoa react in certain characteristic ways toward chemic, mechanic,
and electric stimuli. Many are affected by light, while probably none
react to sound. They manifest the reaction usually by motion of
some sort. When toward the object of irritation, the reaction is said
to be a positive taxis; when away from it, the reaction is called negative
taxis. Most animal parasites, especially the higher forms, exert a
positive taxis for leukocytes, principally for the large mononuclears and
the eosinophiles. This fact is made use of in clinical diagnosis. Ob-
jects suitable for food cause a positive chemotaxis.^
Nutrition in protozoa, as in the higher animals, consists in the
ingestion and digestion of food and the ejection or excretion of waste;
that is, in constructive and destructive activities. Many pro-
tozoa, especially the pathogenic forms, absorb fluid food directly
through the body wall; but the majority take in solid food, such as
small animal or vegetable organisms and organic waste, some through
more or less definite regions of the body, others through any part of
the surface by extending pseudopodia and entirely surrounding the
food object, forming a so-called gastric vacuole.
After the food is digested the waste products are excreted. Where
no known excretory organ exists (as in the sporozoa and some other
forms), the removal of the waste probably takes place by osmosis
through the wall, in the same way that fluid food is taken in. In most
protozoa, however, there are special structures called the contractile
vacuoles which regularly eject fluid substances to the outside of the
organism. In life, this vacuole is a clear spherical area in the ento-
plasm. As it becomes filled with fluid it grows to a certain size and
then suddenly bursts. Vacuoles are generally variable in position
and number. In some forms they move about with the entoplasm,
in others they remain stationary. In these latter there is generally
a more or less definite system of canals leading to the contractile
vacuole which empties its contents into a reservoir, and from this
the waste passes by a definite opening to the outside of the body.
Respiration. — It is supposed that the contractile vacuole has a
respiratory as well as an excretory function. The interchange of
gases is always going on, if not through a contractile vacuole, then
by osmosis through any part of the wall.
Growth and Reproduction. — When the new protoplasm elaborated
by the digestion of food exceeds the waste products formed, growth
results. In this process the nucleus plays the most essential part.
Under favorable conditions, new protoplasm is constructed rapidly,
* See Jennings on Behavior in Lower Organisms. New York: Macmillan & Co..
1906.
526 PATHOGENIC MICRO-ORGANISMS.
and the mass increases faster than the surface. This changed relation
between internal protoplasm and its surface, according to Spencer, in-
itiates cell division. The changes generally appear first in the nucleus.
The simplest variety of reproduction is a two-celled fission which may
be either longitudinal or transverse, either of which may be direct
(amitotic) or indirect (mitotic). A modification of equal fission is
the so-called budding division when a smaller piece breaks off from
a larger. This budding occurs on the surface of the organisms and
may be single or multiple. When growth occurs so that fission is
for a time incomplete, one cytoplasm containing several nuclei which
finally separate into as many daughter organisms, the process is called
multiplicative reproduction, or brood formation. It has also been
called internal budding. In the most extreme cases of multiplicative
reproduction as it occurs among sporozoa the mother cell with its
nucleus separates simultaneously into large numbers of tiny daughter
cells. Such a process when it occurs without conjugation and encyst-
ment is called schizogony and the new cells are called merozoites.
When such a multiplicative division occurs (generally after fertiliza-
tion) within a cyst, it is spoken of as sporogeny and the new cells are
called sporozoites. In this process the entire substance of the body
may take part or there may be a residual portion left which does not
divide. This finally disappears.
Sexual Phenomena. — Sexual phenomena (Syngamy) fundamentally
similar to those seen in metazoa have been observed in all groups of
protozoa studied. The reproduction by the usual division or budding
is interrupted at certain times in the life history of each organism and
individuals come together in such a way that their nuclei fuse after
having undergone characteristic reduction divisions. When the union
is permanent, we speak of it as copulation and liken the process to
that of the fecundation of the ovum by a spermatozoon. When the
union is transient we call it conjugation. Here the two cells fuse
for a time when the nuclei interchange protoplasm and then the celb
separate and each one continues to grow and divide independently.
•W'hen in a partly divided cell or in an apparently single cell, two.
nuclei, after undergoing reduction division, or its like, fuse, the process
is called autogamy. The developmental cycle of a protozoan con-
sists of all the changes which occur in its growth from one act of
fertilization to another (Fig. 186, p. 590). According to Calkins,
such a developmental cycle as a whole should be considered the indi-
vidual and should be made the basis for species rather than any part
or parts of it. Many protozoa carry on the sexual part of their life
cycle in one host and the asexual part in another. It is thought by
some that the so-called intermediate hosts in many instances, if not
all, were the original hosts of the parasites, the change possibly being
due to the fact that as the parasites developed they found soil more
favorable for certain stages in their growth in new hosts.
Oyst Formation. — The function of encystment is a marked char-
acteristic of all protozoa. It is the means developed by these organ-
CLASSIFICATION AND GENERAL CHARACTERISTICS. 527
isms for surviving unsuitable environments. If they do not get
the required amount of water or air or suitable food they cease their
special movements, round out into more or less of a sphere and form
a resisting membrane of chitin within which they may live for a long
time, withstanding periods of desiccation, extreme heat and cold,
and they may be blown about as dust until they find conditions again
favorable for renewed growth when water is absorbed, the cyst is
ruptured and active life begins anew. In parasitic forms encyst-
ment plays an important part in the passage from the old host to the
new. The majority of forms would not be able to exist outside of
the body of the host without having some protective membrane.
The cyst may be formed simply for protection from drought, etc.,
when it is called a hypnocyst, from which the organism may emerge
in about the same form as when it encysted; or the cyst may pre-
cede reproduction by spore formation or simple division, when it
is called a sporocyst. In either case it may consist of a simple wall
or.it may be formed of several walls to enable it to resist prolonged
desiccation, when it is called a resting cyst.
Natural Habitat. — On account of this power to form lasting cysts,
protozoa have a world-wide distribution. They are found in largest
numbers where the climate is warm and moist, but even in Alpine
and Arctic regions a few species which are able to resist long periods
of drought and cold grow freely during the warm season. They are
abundant in both salt and fresh waters. Finally, they are found as
parasites on or in animals and plants.
Oultivation. — ^Protozoa are cultivated en masse in the large aquaria
of the zoological laboratories, where they are mixed with the bac-
teria and the plants and animals usually found in the material taken
to stock such aquaria.
Pure cultures such as are known among the bacteria have not
been obtained with protozoa until recently, when Novy succeeded
in growing certain blood flagellates in the condensation fluid of a
mixture of blood and nutrient agar. Before that it was shown by
Frosch and others that so-called **pure mixed" cultures of certain
protozoa, especially of certain species of amebse, could be obtained by
separating them from other protozoa and feeding them on one or two
varieties of known bacteria.
Though this field is an important one, comparatively little work
has been done in it. Up to the present time zoologists have studied
these organisms as nearly as possible in their natural environment.
They have thought that anything which disturbs the usual surround-
ings might lead to degeneration, or at least to involution, and hence
that wrong interpretations might be drawn from phenomena ob-
served under these circumstances.
The special methods so far used in cultivating protozoa will be
considered under the descriptions of the individual organisms.
Effects of Physic and Chemic Agents.— Some of these have been
already mentioned under irritability. For the physician it is espe-
528 PATHOGENIC MICRO-ORGANISMS.
cially important to know the effects of (1) temperature, (2) elec-
tricity, (3) light, (4) moisture, and the various chemicals used as
(5) disinfectants.
1. Each species of protozoa has an optimum temperature at which
its movements are more rapid and its growth more vigorous than at
other temperatures. With increasing or decreasing temperatures,
movements and growth gradually cease. In intense cold the organ-
isms may continue to live quiescent for a long time, while with a
comparatively moderate amount of heat most of them will die.
2. When a current of electricity is passed through a liquid medium
most active protozoa swim with their long diameters in the direction
of the lines of force to assemble behind the cathode. Most flagellates
and a few ciliates, however, move toward the anode. The direction
of motion has been shown by Dale to vary with the nature and concen-
tration of the medium. This whole question has been little studied.
Slight induction shocks arrest movement, stronger ones cause
contraction, stronger still will kill the protozoa.
3. Most protozoa are greatly influenced by light, some moving
toward the point of greatest luminosity, others away from it. The
light-seeking protozoa have green or yellow chromatophores and
usually, at the anterior end, a red pigment spot. Here, as with other
stimuli, there is different optimum light for different species. The
violet and blue rays are more active than other parts of the spectrum
in determining motion. The effect of x-rays and of radium emana-
tions have been little studied. Most of the colorless protozoa are
negative to light.
4. When protozoa are encysted while drying they will withstand
long periods of desiccation. Most forms when dried quickly, re-
main viable much longer than when dried slowly. A certain amount
of moisture, as we have said, is absolutely essential to renewed
activity.
5. The effects of. the usual chemic disinfectants have been very
little tried on protozoa. In general what is true for bacteria in this
particular is probably true for protozoa.
Chemical Composition. — The chemical composition of the bodies
of animal parasites is an almost unexplored field. The ectoplasm
and the cyst sacs in general are made up principally of a substance
called chitin. Glycogen has been isolated from many forms. Pro-
teolytic enzymes and acid secretion in digestive vacuoles have been
demons.trated. Microchemic reactions have been studied in the in-
dividual organism.
Pathogenesis. — The pathogenic protozoa, indeed the parasitic
forms, are few in numbers compared with the total number of pro-
tozoa. They exert their harmful action mainly mechanically or
by the direct destruction of the special host tissue which they find
suitable for food. That they may produce specific toxic substances
has been demonstrated in only one or two instances, the most marked
of which is that of the poison obtained in the aqueous or glycerin
CLASSIFICATION AND GENERAL CHARACTERISTICS. 529
extracts and the dried powder from mutton sarcosporidia which will
be spoken of later.
Though in general no specific toxins have been shown to exist in
pathogenic forms or to be excreted by them, the fact that there is
spontaneous recovery from various protozoan infections and that a
reinfection does not take place soon after, indicates that some specific
toxins or substances are formed which help to produce immunity.
Rossle has stated that he has obtained immune sera against infusoria;
and antibodies have been demonstrated in animals which have re-
ceived non-lethal doses of trypanosomes and of amcebee.
Infection through protozoa is often accomplished by means of
some of the lower animals either acting as intermediary hosts or as
direct carriers of the virus.
Olassiflcation. — Broadly, protozoa are classified from two principal
standpoints, the physiologic and the morphologic.
Physiologically, they are grouped according to their manner of
living into saprophytic and parasitic forms.
The parasitic protozoa may be further divided into commensal
and pathogenic forms. For our study the former are almost equal
in importance to the latter forms because of their close relationship
to the pathogenic forms and because of the possibility of their becom-
ing pathogenic.
The classification of the protozoa in the strict sense is morphologic
and is based upon variations in the motile organs. It is still in a tran-
sitional stage and it will continue to be so until the relations of the
different groups are better known and until the life histories of the
different species have been more minutely worked out. Hartmann
has just added to the flagellata a new order made up of species taken
both from the flagellates and from the sporozoa. Calkins has also
announced some fundamental rearrangements; so, whatever system
of classification we adopt, we may be sure that the near future will
show us some changes in it. The following grouping is taken, with a
few slight alterations, from the excellent article on Protozoa written
bv Calkins in Osier's Modern Medicine.
«
Classification.
Phylum. Protozoa. — Unicellular animal organisms which reproduce by
division or spore-formation; solitary or united in colonies; free-living
or parasitic.
Sub-phylum I. Sarcodina. — Protozoa with changeable protoplasmic
processes or pseudopodia.
Class I. Rhizopoda. — Sarcodina with pseudopodia in the form of
lobose or reticulose processes, with or without shells.
Sub-class. Amcebida. — Pseudopodia lobose.
Order 1. Gymnamoebida. — Naked amoeboid forms with
lobose pseudopodia. Here are placed a few parasitic
forms belonging to the genera Amceba and Entamoeba.
Order 2. Thecamoebida. — Shell-bearing amoeboid forms
with lobose pseudopodia. One parasitic form, genus
Allogromiaj is placed in this order.
34
530 PATHOGENIC MICRO-ORGANISMS.
Sub-class. Foramini/era, — Divided into 10 orders; the vari-
ous genera are salt water forms for the most part and are
rarely parasitic.
Sub-class. Mycetozoa would be placed here were we to consider
these forms, as protozoa instead of fungi. Here are placed
parasitic forms, such as PUumodiophora, Tetramyxa, Laby-
rinthuUif and NucleoplMga,
Class II. Heliozoa, — ^The genera are confined mainly to fresh
water and are never parasitic. They are subdivided into four
orders according to the nature of the skeleton.
Class III. Radiolaria, — Salt-water forms of protozoa, never para-
sitic.
Sub-phylum II. Mastioophora. — ^Protozoa with flagella.
Class I. Flagellata. — Small forms with from one to several fla-
gella; with a strong tendency to form colonies.
Order 1. Monadida. — Minute forms with from one to three
flagella. There is no definite mouth-opening and nutrition i^
holozoic, saprophytic, or parasitic. The parasites and com-
mensals which belong to this order are species belonging to
the genera CercomanaSj HerjtetomonaSy and TrypanoBoma
(appendix Spirocheia),
Order 2. Choanoflagellida. — With collar-like processes sur-
rounding the base of the flagellum ; not parasitic.
Order 3. Heterommastigida. — With two or more flagella of
dissimilar length ; the genus Bodo is parasitic.
Order 4. Polymastigida. — The flagella are numerous and of
similar or dissimilar size. Here are several ecto- and endo>
parasitic forms belonging to the genera: CoaHa, Teira-
mituSy Trichomonas, Monocercomonas, HexamUus, Lamblia,
Polymaatix, Lophomonaa, Trichonympha, Pyrsonympha, and
Jcmia.
Order 5. Euglenida. — Occasional parasites as Copromonas in
frogs.
Order 6. Phytoflagellida. — Flagellates with coloring matter in
the form of green, yellow, or brown, chromatophores. Fre-
quently colonial. Here belong the most frequent sources of
odors in drinking waters, the following genera being espe-
cially noteworthy: Dinobyron, Synuray and Uroglena, all
colonial forms, with yellow chromatophores.
Order 7. Silicoflagellida. — A single genus of salt water ma»-
tigophora with latticed skeleton. DistephanuSy parasitic on
radiolaria.
Class II. DinoflagellcUa. — Never parasitic.
Class III. CystoflageUcUa. — Two genera of characteristic form.
One, Noctilucay is remarkable for the vivid phosphorescence which
it causes.
SuB-pHYLUM III. Infusoria. — Protozoa with cilia.
Class I. Ciliata. — Cilia present at all times.
Order 1. Holotrichida. — The cilia are distributed over the sur-
face, and there is no specialized (tral apparatus known as
the '*adoral zone" consisting of cilia fused into **mem-
branelles/' Here are found some parasites belonging to the
genera Ichthiophthirius, BUtschlia, Anophrys, Isothrica,
DasytrichOj Opalina.
Order 2. Heterotrichida. — With cilia distributed over the gen-
eral surface, and, in addition, a specialized zone in the mouth-
region. Here are several well-known parasitic form^ be-
longing to the genera Nyctotherus, Balantidium, Entodinium,
Ophryoscolex and Cycloposthium.
CLASSIFICATION AND GENERAL CHARACTERISTICS. 531
Order 3. Hypotrichida. — The cilia are limited to the ventral
surface, and are frequently fused into specialized organs of
motion and touch, the cirri. There are no strictly parasitic
forms.
Order 4. Peritrichida. — ^The cilia are greatly reduced, in some
cases to the adoral zone, but additional rings may be present.
Several ectoparasites belong here, especially the genera
SpirochonOf kerUrochana, Lichnophora, Cyclochceta and Tri-
chodina.
Class II. Suctoria. — Infusoria with suctorial tentacles in the place
of cilia except in the young phases. They are frequently ecto-
parasites and the young of some genera, e. ^., Sphoerophyra,
are internal parasites in other infusoria.
Sub-phylum IV. Sporozoa. — Protozoa without motile organs; repro-
duction by sporulation; always parasites.
Gass I. Teloaporidia. — Sporozoa in which the act of reproduction
ends the individual's life, the entire protoplasm bemg used in
forming spores.
Order 1. Gregarinida. — The young stages alone are cell para-
sites, the adult organisms living in fluids within the cavities
of animal hosts. There are no human parasites.
Order 2. Ck>ccidia. — Intracellular parasites, mainly in the
epithelial cells of vertebrate and invertebrate hosts. Human
parasites have been traced mainly to the genus Coccidium.
Order 3. HsBmosporidia. — Sporozoa of small size living in the
blood corpuscles of vertebrates. Human parasites belong to
the genera PUtsmodium and Babesia,
Class II. Neosporidia, — Sporozoa in which the entire cell is not
used at one time in forming spores, the latter developing
while ordinary vegetative processes are carried on.
Order 4. Myxosporidia. — Neosporidia with spores containing
polar capsules and anchoring threads. Here belong several
genera of note, in that serious epidemics of lower animals
are caused by them, e. g.y Nosema — causing p^brine diseases
in silkworm, MyxoholuSy Myxidium^ etc.
Order 5. Sarcosporidia. — Neosporidia in which the initial
stages are passed in muscle cells of vertebrates. Cysts are
formed with double membranes in which kidney-shaped re-
productive elements are produced. The one genus occasion-
ally parasitic in man is Sarcocystis.
Bibliography.
1. Braun, Animal Parasites of Man. Trans., 3d edition, 1906.
2. Calkins. The Protosoa. First edition, New York, 1901. Also article
entitled "The Protozoa" in Osier's Modern Medicine. Philadelphia, 1907. Vol.
I. Also Protoiodlogy, New York and Phila., 1909.
3. Doflein. Lehrbuch der Protosoenkunde. Jena, 1909.
4. Doflein und Prowazek. "Die pathogenen Protozoen" in Handbuch der
pathogenen Mikro-organismen. KoUe und Wasserman. First edition, Jena,
1903.
5. Hertog. "The Protozoa " in The Cambridge Natural History. First edition.
Cambridge, 1906. Vol. I.
6. KisskaU und Hartmann. Praktikum der Bakteriologie und Protozoologie .
First edition, Jena, 1907.
7. Lang. "Protozoa" in Vergleichende Anatomie der wierbellosen Thiere.
New edition, 1909.
8. Moore. The Pathology of Infectious Diseases of Animals. First edition,
Ithaca, 1902.
9. Lankester^s "Treatise on Zodlogy. " First edition, London. Part I. First
and second fascicles, 1909.
CHAPTER XL.
GYMNAM(EBIDA. MYCETOZOA.
GYMNAMCEBIDA.
Introduction. — Under gymnamoebida (syn., ainebae) we include
forms composed of naked, simply constructed protoplasm having the
power of producing lobose pseudopodia which are used as organs of
motion and of nutrition.
The pseudopodia are protoplasmic processes which are projected
in irregular succession from different parts of the surface of the cell,
producing in this way an irregular motion. The form of the pseudopo-
dia varies considerably in the different species. For instance, there
are broad, blunt processes or narrow, less blunted ones, and each may
be short or long, single or slightly branched. The entoplasm may or
may not take a share in their formation. The forms of course varv
within limits according to the condition of the medium in which the
amebee are living. Movements are always called forth by some physic
or chemic excitant. When such an excitant is desirable for food the
pseudopods flow aroun^ it, and it is subsequently absorbed in the cyto-
plasm of the organism.
The members of this group may possess one nucleus or several.
Amceba hinucleaia has two nuclei in the young adult stage, and
Pelomyxa pcUtistris, living in the bottom ooze of ponds, has an enor-
mous number of nuclei. A marked feature of the nuclear apparatus
is the formation of chromidia which, as has already been noted, mav
play such an important part in sexual reproduction. Generally each
ameba has one contractile vacuole, but occasionally some are seen
with several or with none.
Saprophytic forms belonging to this order are common. They
may be found wherever there are moisture and decaying vegetable
matter. The pathogenic forms are not so frequent. Because of the
possibility of the still unknown causes of certain diseases (see rabies and
smallpox) being organisms related to this order, it is especially impor-
tant to study both saprophytic and pathogenic varieties, since a
knowledge of the former which are more easily studied may help us
understand obscure points in the life history of the latter.
Notwithstanding the common occurrence of saprophytic forms, the
full life history of few of them has been worked out, and until the full
cycle of development of any so-called ameba is known it is impossible
to say whether that particular form belongs among rhizopoda or
whether it is a developmental form of another group, as amel>oid forms
may occur at some time in the life history of all groups. It is quite
532
GYMNAMCEBIDA. 533
possible that some of the organisms described as belonging to this order
are really members of entirely different orders. For instance, it is
known that the flagellate Trichomonas loses its flagella before copula-
tion and crawls about by means of short blunt pseudopods as a typical
ameba.
Gymnamabida reproduce by simple fission, by budding, and by
brood formation. In the last case the reproduction is usually pre-
ceded bv encvstment. Schaudinn has worked out in several rhizo-
poda a complex life cycle, part of which is sexual and part non-sexual
in character. Calkins has worked out a complete life history of
Amoeba proteus, in which secondary nuclei form within the primary
ones and conjugate after leaving the latter.
Oymnamoebse in Human Beings. — Several authors have reported
the finding of amebae in man, especially in so-called tropical, ulcerative,
or amebic dysentery, but as the first descriptions were incomplete and
the laws of nomenclature were not strictly followed there resulted
many synonyms for the same species and many species bearing the
same name.
Only four out of all those mentioned have been described with enough
minuteness to be considered as distinct species. These are Entameba
histolytica, the form described by Schaudinn from tropical dysentery
and considered by him the cause of that disease; (2) Entameba coli,
the kind found in normal human intestines by Schaudinn and thought
by him to be harmless; (3) Entameba buccalis, found by Prowazek
in tartar of teeth and considered harmless; (4) Entameba tetragena, a
form found by Viereck in certain cases of tropical dysentery.
The chief differential characteristics of these amebae, as reported by
various investigators, are given in the table on p. 534.
Historical Note. — Stiles has given a detailed history of the generic name
Amceba and of the specific one Am^ba proteus, and, finally, of the naming of
the intestinal amebae. He shows why the name Entamoeba should be given to
the genus described by Lamdl and Losch.
This article illustrates very forcibly the absurdity of bringing forth new
names for organisms only half studied and of claiming that such organisms
belong to new genera.
The first report on intestinal amebse of man was made by Lamdl in 1860 who
announced the presence of ameboid forms in the intestinal mucus of a child
who had died from enteritis. Supposedly the same forms were more fully
described by Losch in 1875 under the name Amoeba coli; Losch found his
organisms in stools of a patient suffering from chronic dysentery and he
succeeded by rectal injections in producing superficial ulceration in the large
intestines of dogs. He therefore claimed that this organism is the cause
of dysentery. His work was corroborated by many observers. In the
meantime, amebse were found in diseases other than dysentery, and Grassi
in 1879 reported them in the healthy intestines. The work of Kartulis,
however (1886), helped largely to establish the fact that amebse play an
important part in the etiology of dysentery in Egypt. He was the first
to find the organism in abscess of the fiver in tropical dysentery. In our
own countr>' among the most important workers in this field are Councilman
and Lafieur (1891). They conclude that amebic dysenter\' should be regarded
etiologically, clinically, and anatomically as a distinct disease. They disap-
534
PATHOGENIC MICRO-ORGANISMS.
55
<
<
n
9
•<
OD
H
O
u
H
b.
O
s
n
H
H
H
H
H
o
H
h
H
s
o
o
>
o
u
•J
PQ
H
S
o
1
U3
a
o
in
9
g
IT
a
8
0,
S
s
o
3
a
J
a
^a
o S
« E
1 ♦* ' Jrf 5
J3
as
QjQ «:s
o
:> « QjQ «au a
i-l
9 O e^ ^jQ ^
Sa
ggos
« • B O ''S c
a
a
a .
o u
hi
■s
a
O
a as
e8
o a
08
Sg-I
■Pf€
.1. o
tf S w^
1.2
jj Si5 • S «
a^
On
1?
-= 2 5*
.2 z: o g «
32
.5-2 "^i a E^
— "ja " tf g p
** d'^ ^ * 1 «;
e8 2 ^ d
a o.S aSM«»-S
jS gil-s-g i a
§1 ^iM a>
o 3
13*0 S S'l-S 9 S
l^*iilii|ii
aT
•S-: d
0*3.2^3
^
jy
-1^
:^
It
1^
4^
I
a-^
15
aa.s^>>d
- 55 $ o a 32
c
.3
GYMNAMCEBIDA. 535
prove, however, of the name Amoeba coli and propose the name Anuxba dyaen-
teri(B for the pathogenic form ; but as they do not show in any way than by
its pathogenesis that the species they describe is a new one, their name, ac-
cording to the rules of zoological nomenclature, cannot be accepted. Harris's
work, too, is important in showing an etiologic relationship between amebse and
a certain form of dysentery, but neither did he describe the morphology of his
organism minutely enough to identify it with Schaudinn's histolytica which
is described below. Casagrandi and Barbagallo in 1897 were the first to claim
that the amebee so far described in man show differences enough from the
fresh-water amebee to belong to a new genus< They therefore created the
genus Eniamceba and gave the specific name EntanuBba homini^ to ameb®
of the Amoeba coli type. Schaudinn and Stiles agree with them as to the
generic name, but consider that the correct specific name is complicated by
the fact that there are different species in this group. Many observers
(Kartulis, Councilman and Lafleur, Quincke and Roos, Knise and Pasquale),
have considered that there are different varieties in the human intestines,
but they have given no morphologic differences distinct enough to classify
such varieties. Schaudinn is the only one who seems to have shown quite
clearly (1903) that at least one species amon^ them is pathogenic and one non-
pathogenic. The latter, which he found m normal human intestines, he
considers resembles those already described as Amoeba coli; therefore he gives
it the name Entomceba coli; while the former, which he found exclusively in
ulcerative tropical dysentery, he calls Entamoeba histolytica.
The different views upon the relationship to disease of amebse found in the
human intestine^ may be summarized as follows:
1. That the amebse in man have no pathogenic properties, hence are not
the cause of amebic dysentery. (Cunningham, Grassi, Celli and Fiocca,
Casagrandi and Barbagallo, and others.)
2. That any intestinal ameba may become pathogenic and cause the
specific malady known as amebic dysentery. (Musgrave, Clegg, and others.)
3. That' amebse are able to keep up a pre-existing inflammation. This
was the original view advanced by Losch when he described the most com-
monly cited form. Amoeba coli, and several authors have followed Losch in
this opinion.
4. That more than one species of amebee are found in man, at least one
pathogenic, and one non-pathogenic. (Kartulis, Councilman and Lafleur,
Quincke and Roos, Strong, Schaudinn, Craig, and othiers.)
The study of bacillary dysentery by Shiga, Kruse, Flexner, and others
(see under bacillary dysentery) has demonstrated that there are at least
two forms of dysentery, one produced by amebse and the other by bacilli,
and from the work on the former it now seems certain that it is produced by
a specific form of amebse.
Because of the incomplete earlier descriptions, however, we cannot yet
decide in what percentage of cases Schaudinn's non-virulent form is found and
in what percentage his virulent. The csises described by Councilman and
Lafleur and by Harris in our country were probably all due to the histoly-
tictty especially as they mention the distinct eoto- and entoplsism of their
organisms; but as they do not go into detail of its life history we cannot be
absolutely sure. Schaudinn speaks of finding the coli accompanying the
histolytica in cases of true amebic dysentery and of finding them increased in
numbers in cases of simple diarrhoea, but does not mention their presence
in liver abscesses. The whole work of Schaudinn needs more corrobora-
tion, but until that time, his classification and descriptions must be provi-
sionally accepted. Lesage, Craig, and Wenyon claim to have corroborated
more or less of Schaudinn' s work.
AmebsB have been reported in teeth cement and in carious teeth sis well as
in abscesses of the jaw. Flexner in 1892 described an amebic organism in the
latter condition, and considered it identical with the organism described by
Losch, Councilman and Lafleur as Am<jeba coli. In the same year Kartulis
536 PATHOGENIC MICRO-ORGANISMS.
described similar organisms found in similar lesions. Gross and 8ternl>er?j
found them in tartar of teeth. Prowazek (1904), however, is the first to have
separated a mouth ameba as a distinct morphologic species under the name
Entamoeba huccalis.
Comparatively recently, successful cultures have been made of amebep
obtained from the intestines of man and other animals, as well as from
certain fruits and vegetables.
Musgrave and Clegg (1904) studied amebse in the Philippines by the cul-
ture method and came to the conclusion that forms obtained from various
sources were probably all a single species.
Kartulis (1885) reported growing pure cultures of the amebfle from a
bacteria-free liver abscess in dysentery. He used straw decoction as a
medium. In 1895 Celli and Fiocca claimed to have obtained a pure groiJi'th
of amebae from an abscess of the liver, free from bacteria, upon an alkaline
medium containing Fucus crispus. But the organism did not reproduce in
transplants. In 1898 Tsujitani reported the pure development of encysted
cultures of amebae. He took old cultures of a favorable symbiotic organism,
and heated them for an hour at 60° C. (to kill organism). These dead oi*gan-
isms were then inoculated with encysted amebse, and development occurred,
though not so luxuriantly as with a living organism.
Walker made an extensive cultural study of 40 strains of amebse obtained
from human dysentery and from other sources and agreed with Musgrave
and Clegg in considering the pathogenic and the non-pathogenic human
forms as single species.
The results from cultural work alone, however, cannot be accepted a>
disproving Schaudinn's work. The organisms must be studied comparatively
in their human habitat and in cultures before judgment can be passed upon
his work.
Sites of AmebsB in the Human Body. — Intestines and neighboring tis-
sues; abdominal cavity; abscess of liver, lung, pleura, and mouth;
necrosis of jaw-bone; urine; tartar of teeth.
Material and Methods for Study. — ^Fresh material containing the patho-
genic amebae is so seldom on hand in the northern part of this country that
it cannot be counted on. The Entamoeba buccalis may be found sometimes
in the tartar collections about human teeth, but the demonstration is often
unsatisfactory. If found it must be examined immediately on a warm sta^
in order to detect motion. The non-pathogenic form in human intestines
might be obtained after administration of a saline cathartic, but generally
one must depend upon saprophytic forms for work with students, or upon
cultures obtained from cases of amoebic dysentery. Material rich in sapro-
phytic forms may be obtained from an infusion in water of lettuce, cabbage,
potato skins, or other vegetable material. Such an infusion should be made
a week or two before it is needed, when it will be found that the pellicle
which forms contains many varieties of protozoa and bacteria, among which
are generally large numbers of ameboid forms. Often one may get good
material from the faeces of many of the lower animals, such as the lizard,
toad, or guinea-pig.
If one has material containing human intestinal amebae, cats may be fe<l
with the cysts in order to obtain a new supply. The amebse should be ex-
amined in both the fresh and fixed condition. Cultures mav also be made
as described below.
Examination of the Fresh Material. — The study of the living amebse is ex-
tremely important. This may be done by making a hanging drop or hanging
mass (p. 41) from fluid containing amebae. The size, kind of motion, fre-
quency of pulsation of contractile vacuole, and as much of the cell contents
as possible should be noted.
G YMSA M (EBIDA . 537
»
The stools should be examined on the warm stage as soon as possible after
their passage (not later than two hours), and should be kept at blood heat
until examined. A platinum loopful of material should be taken from the
slimy masses in the thinner part of the fseces, diluted with physiologic
salt solution, covered with a cover-glass, and examined under moderate
magnification.
Harris found that a drop of a watery solution of toluidine blue added to a
small particle of the faeces stains the entoplasm of the amebse at once and the
ectoplasm a few minutes later. The amebse seem to be quickly killed and
often when natural forms are beautifully preserved the coverslips, after being
washed in water and mounted in Farrant's medium, may be preserved for
months, but after a time the stain completely fades.
Permanent Preparations. — Thin films are made on glass slides or cover-
glasses, and immediately, before they are allowed to dry, they are placed in
the fixing solution. Cover-glasses may float, film down, on the surface of
the fixative. Among the best fixatives are the following:
1. Hot sublimate alcohol (50° C.) (Schaudinn), or saturate sublimate, to
which 5 per cent, glacial acetic acid may be added. The preparation should
remain in it a few seconds, then should be washed for one-half hour in 60 per
cent, iodine-alcohol, and then placed in 70 per cent, alcohol. They may re-
main here for an indefinite time, until they are to be stained, when they are
rinsed in distilled water and then placed in the staining fluid.
2. Zenker's fluid (p. 522). See p. 625, for u.se.
3. Hot (50** C.) Hermann's fluid (see p. 522) for a few seconds, washed in
distilled water for ten minutes, in 60 per cent, alcohol, and then in 70 per
cent, alcohol, from which they may be stained at any time.
4. Methyl alcohol for a few seconds.
Stains. — Among the many good staining methods the following may be
mentioned :
1. Thin Delafield's luematoxylin from one-half to several hours, then
washed in water. (If over-stained, the preparation may be differentiated
in acid alcohol, controlling under the microscope, then washed in water.)
The film or section is then passed successively through 70 to 95 per cent,
and 100 per cent, alcohol, absolute alcohol+ xylol, xylol, cedar oil, or Canada
balsam.
2. Heidenhain's iron hematoxylin (see p. 522). The smear is put from
distilled water into the iron-alum mordant for 4 to 12 hours, or overnight;
well washed in distilled water; in stain from 2 to 24 hours, excess washed out
in the iron mordant, controlled under the microscope (as decolorization occurs
very (juickly), until the nucleus is sharply differentiated; the chromatin of the
nucleus must be a deep blue-black, and the cytoplasm' a 'light gray; then a
thorough washing in tap water and passage through the alcohols and xylol,
and in Canada balsam, or cedar oil for mounting.
3. After fixation in methyl alcohol one may use Giemsa's staining method
(see p. 624), or a modification of the method suggested by Van Gieson for
staimng the Negri bodies in smears (see p. 624).
4. Mallory's eosin methylene blue method (see p. 625).
Masses containing amebae, as mucous flakes or portions of the intestinal or
liver abscess wall in amebic dysentery, or pieces of decaying vegetable may
be fixed in toto in hot sublimate alcohol for one-half hour, washed in iodine-
alcohol for 24 hours, passed through the different strength alcohols and em-
bedded in paraffin (see p. 625) for section-cutting if desired.
Mallory and Wright recommend the following method for tissues:
1. Harden in alcohol. 2. Stain sections in a saturated aqueous solution
of thionin three to ^ve minutes. 3. Differentiate in a 2 per cent, aqueous
solution of oxalic acid for one-half to one minute. 4. Wash in water. 5.
Dehydrate in alcohol. 6. Clear in oleum origani cretici. 7. Wa.sh off with
xvlol. 8. Xvlol balsam.
538 PATHOGENIC MICRO-ORGANISMS.
Mallory's eosin methylene-blue method \a alao very good for sections.
Onltures of Amebe may be made Id the following way: From the material
containing amelMe a small loopful is removed with a platinum wire and
isolated spots are touched over the surface of the following media poured in
sterile Petri dishes: Agar 1.0, tap water 90.0, ordinary nutrient broth 10.0,
mixed and sterilized in the regular way. It should be slightly alkaline to
phenol phthatein (1 per cent.). If necessary, ftecee contents may be thinned
with physiologic salt solution before planting. In one to several days at
25° C. the amebte with the accompanying bacteria may overgrow the entire
plate. We have found that amebte will grow as well upon nutrient agar—
better with certain bacteria— as on the special media just mentioned.
Klat^ch preparations may be made of these cultures, or small pieces of agar
and culture may be embedded entire. Prom such a culture the "pure
mixed" cultures of Prosch may be made as follows: The amebie which have
crept out to the periphery of the growth are taken out with their accom-
panying bacteria and transplanted. Usually one or two organisms favor-
able for the growth of the amebs accompany them and in this way one may
finally get the amebte growing with one definite bacterium. We have isolated
from a culture a single ameba unaccompanied by bacteria by the following
simple method: Under the low-power lens with a fine platinum loop an iso-
lated ameba is drawn to the edge of the agar plate. When it is well sepa-
rated a disc of agar containing it is cut out following the margin of the ob-
jective and is transferred to a fresh agar plate. A very small quantity of a
desired bactenim is now added to the disk near the ameba, and a "pure
mixed " culture results.
ComparatiTS Characteristics of Amebaa from Haman Sonices.—
The chief properties of these organisms so far described may be
learned by studying the table on p. 534.
Morphology. — -The morphologic characteristics of the ameboid
stage, as described by various observers, seem not to have been mi-
nutely enough studied to be depended upon in differentiating the species.
Enlamaia hitlalvtlca (Scbauitinnl frum the stool of b dyHDU'ry patient. The uue iodividu
'^{^^"'l^nkt^ell'suo': 1. Afle'r j'^^eui (from Kuskslt sad Hartmsna"?
Moreover, descriptions have differed markedly. While Schaudinn
and others, especially Craig of the more recent writers, say that it is
easy to differentiate between the ameboid forms of histolytica and coli,
Musgrave and Clegg, Strong, and others say the points of difTerencf
are not marked; but there ha.s not been enough work done to disprove
Sfhaudinn's work; hence hi.s observations and those of his followers
GYMNAMCEBIDA. 539
must be provisionally accepted until further study of each species
under varying conditions shows whether or not these characteristics
hold.
The observations of Schaudinn and others may be summarized
as follows: (1) Ent, coli is, on the whole, smaller than ErU, histolytica;
(2) its ectoplasm is so small in amount and so slightly differentiated
that it is only seen when the organism puts forth pseudopods, while
the cortical zone of the Ent. histolytica is wider and is distinctly differ-
entiated from the entoplasm; (3) the pseudopods of the former are
small, rounded, delicate, and not highly refractive, those of the latter are
larger, finger-shaped, firmer, and more highly refractive, thus indicating
the power of the organism to penetrate its host's tissues; (4) the nucleus
of Ent, coli is very distinct in life as well as in stained spreads, due to a
definite membrane, a more distinct karyosome, and much chromatin
which is distributed throughout the nucleus with more of a collection
about the periphery; the nucleus of Ent. histolytica on the other hand,
is seen with difficulty during life, and stains faintly, owing to its delicate
membrane, its small amount of chromatin, and small karyosome, the
chromatin is collected about the karyosome and the periphery of the
nucleus, the nucleus, moreover, is much more variable in shape, in the
active organism than is that of the Ent. coli; (5) the entoplasm of
Ent. coli is less granular and vacuolated and contains fewer red blood
cells than that of Ent. histolytica which sometimes shows immense
numbers of these blood cells.
The above points of difference cited for organisms in the ame-
boid stage may hold for forms living in the human intestines; but we
have found that organisms from widely different sources (e. g., intes-
tines of guinea-pigs and of dogs from New York and of humans from
the Philippines) when grown with a favorable bacterium in the ther-
mostat at body temperature niay show appearances similar to each
other and similar also to those described by Schaudinn for Ent. histo-
lytica. As already said, therefore, more corroborative work seems to
be needed before we accept the above observations as being the whole
truth.
Reproduction. — ^The most important point of difference between
these organisms, according to Schaudinn, and the one upon which he
rightly bases his classification into different species is the manner of
propagation.
In the vegetative stage probably all these forms divide by a primi-
tive mitosis, though Schaudinn, Craig, and others saw only amitosis.
All of our culture forms divide by mitosis and many observers have
recently reported similar division in related forms. Ent. coli in the
vegetative stage may also divide by breaking up (schizogony),
into, at the most, eight daughter cells. In the latter instance the
nucleus undergoes a somewhat complicated process of division. At
first it increases in size and then the chromatin gathers together about
its periphery into eight particles, the nuclear membrane disappears, and
the chromatin masses lie in the cytoplasm which separates into eight
540 PATHOGENIC MICRO-ORGANISMS.
parts about each nuclear mass, forming the eight daughter amebie
which creep away.
The vegetative stage of each intestinal organism takes place in the
upper part of the intestines; as the fseces become thicker, most of the
vegetative forms die off, while some pass on to permanent cyst for-
mation. As with many coccidia, parasitic amebae may pass throu^
a long period of vegetative life before entering upon a sexual phase
wherein forms are produced capable of infecting a new host. The
length of this period depends upon a number of circumstances.
Under conditions favorable for the growth of the ameba, as in cases of
diarrhoea, the vegetative phase is considerably lengthened, while in
healthy intestines, as the amebse pass down with the thickening
faeces, the infecting cysts are more or less quickly formed.
Ent, histolytica during the vegetative stage may multiply by budding
as well as by binary fission, but never by multiple division, as doe>
Enl. colt.
Sexual Phenomena.— The most marked difference between the two
forms, according to Schaudinn, is seen in their cyst formation and
accompanying sexual phenomena. Schaudinn has described the
process as follows.
Within the cyst of Ent. coli the special form of conjugation kno^n
as autogamy (Fig. 167) takes place. The cell becomes rounded, rids
itself of all foreign matter, and forms a mucous wall about itself. Then
the nucleus divides by mitosis into two daughter nuclei, which sepa-
rate one from the other and between them appears a lens-shaped hole,
as if two not fully divided cells were forming. These are the gam-
etes. The nuclei give out most of their chromatic substance as chro-
midia into the cystoplasm and then gradually break down and become
absorbed. The chromidium is generative and from it two new nuclei
are formed, the sexual nuclei. These divide mitotically, forming
two reduction nuclei, which gradually disappear. By this time
a firm cyst membrane is produced. The central clear space in the
cytoplasm disappears and the two reduced nuclei divide by a primi-
tive mitosis with the two spindles lying parallel, and each half of
one nucleus unites with the corresponding half of the other, one
remaining stationary as a female nucleus and the other moving over
to it as a male nucleus — thus two fructified nuclei are formed. After
this autogamous phase there follows a mitotic division until eight
nuclei are formed. The cyst containing these nuclei is characteristic
for this species, and is not seen, according to Schaudinn, in any other
intestinal parasites.
In the beginning of the large intestine of the new host the cvst
wall is dissolved, the cyst contents divides into eight young amelw
(sporogony), and the cycle begins anew.
The permanent cysts of Ent. histolytica, on the contrary are formed in
an entirely different manner. The nucleus of the ameboid form en-
larges, the chromatin increases, and most of it passes out as chromidia
into the cytoplasm which is finally filled, while the nucleus degenerates.
GVMS'AAKBBIDA. 541
On the surface of the cytoplasm are now formed small rounded bodies
of 3 to 7ji diameter. These balls, which can be seen forming in the
hanging drop, produce about themselves a double-contoured membrane
which after several hours takes a clear brownish-yellow color, and be-
comes very refractive. At this stage no structure can Iw made out within
the ball. The rest of the ameba finally degenerates. The stained prep-
divisioD. S ft
. cyit. T fini
.D of the gai
t and 14 opomKCDv njttiin the r
1»u tynrnrin, 14 UntlDK o[ tt
aration shows that the chromidiuni passes to the periphery of the en-
toplasm and then into the ectoplasm where it forni.s a thick network.
As the small spherical bodies develop they are seen to be filled with this
networkof chromidia, but after the refractive brownish membrane forms
about them no structure can be made out even in the stained prepara-
tions. So the further minute changes in the life cycle at this period are
not known. But Schaudinn showed, by experiment, that with these
small spherical bodies the infection of the new host is probably brought
about. He exariiine<l nianv slides of fffces from one of his amebic
542 PATHOGENIC MICRO-ORGANISMS,
dysentery cases from China, and after finding that they contained
no cysts of Ent, coli, but only those of Ent. histoltftica^ he
washed off the dried faeces with water and fed a certain amount to
a young cat whose faeces had been found to contain no amebie or
their cysts. On the third day the cat had bloody diarrhoea! fnces
containing many forms of the typical Ent. hiMolytica. Twenty-four
hours later the cat died, and the autopsy showed typical ulcerative
dysentery with the penetration of the amebee into the epithelia, as
Jurgens, and Councilman and Lafleur showed. A cat fed with material
from these stools containing only the vegetative forms of the amebie
remained healthy; but the same cat fed some weeks later on material
containing many of the small spores came down with typical dysentery
six days later and died in two weeks with all the typical symptoms of
the disease. This case shows that the cysts and not the vegetative
forms in all probability produce the new infection.
The discovery of Viereck and of Hartmann of an apparently new
species of ameba causing amebic dysentery in Africa {Ent. tdragena)
is interesting and makes one realize that the diagnoses of these forms
made in their natural habitat should be made with the greatest care.
Hartmann's description of the cyst formation and the sexual phe-
nomena of this new form, resemble more the descriptions of these
phases in Ent. coli than of those in Ent. histolytica.
Viability. — The pathogenic amebee are apt to lose their motility
very quickly above or below body heat, while the saprophytic forms
remain motile at higher or lower degrees. Though the former lose
their motility, they are not all killed by cold. They may still be infect-
ive after freezing. Musgrave kept an encysted culture from a dysen-
teric stool at — 12^ for 45 days and found it still viable at the end of
that time.
A temperature of 60° C. for one hour usually kills encysted cul-
tures of amebee, according to Strong, but considerable variation has
been noted in the degree of temperature necessary to destroy differ-
ent strains.*
Enemata of quinine sulphate and saturated solution of boric acid
do not affect amebse in the intestinal canal, though ^^ quinine
sulphate added to the stools invariably kills them in ten minutes.
They are also destroyed in stools by weak solution of hydrogen
dioxide, potassium permanganate, toloidine blue, and dilute acids.
Luttle found that i^rSinr hydrochloric acid or ^-^ silver
nitrate check motility, but do not destroy parasites except after pro-
longed contact. Musgrave and Clegg found that in cultures treated
with 1 : 2500 solution of quinine hydrochlorate the parasite quickly
encysts, and in from five to eight minutes may break up and disap-
pear; ten minutes later cultures made produced no growth of amebee,
while the bacteria grew well.
• An air-dried agar plate culture of ^^ Amceha coli" given us by Dr. Calkins
who obtained it from the Philippines was viable after three years at room
temperature.
GYMNAMCEBIDA, 543
Onltnres. — We have found that cultures of certain species may be
grown with ease on ordinary nutrient agar, as well as upon numerous
other nutrient culture media (see p. 536).
Pathogenesis. — Lower Animals. — Just how pathogenic Ent coli is
for lower animals cannot be determined, as we have before stated,
until a more minute study of the intestinal amebee is made.
In regard to the amebee from tropical dysentery (presumably Ent.
histolytica), it has been shown to be pathogenic to young cats, dogs, and
monkeys. The infection may take place in two ways: (1) By feeding
material containing the cysts; (2) by rectal inoculations of the vege-
tative forms. The best work done on dogs is by Harris in 1901,
who found that puppies were particularly susceptible after rectal
injections of fresh material from human dysentery cases. Morphine
was administered before the injection in order to retard peristalsis.
The disease developed in two or three days and lasted from four to
sixteen days.
The chief symptoms were a bloody diarrhoea and progressive emaciation.
The lesions observed in the intestines on post-mortem examinations were a
swollen and congested mucosa, over whicn were scattered numerous small
ulcers. In two cases there were liver abscesses.
Microscopically, the mucosa first showed slight exudative and productive
inflammation, followed by necrosis and desquamation of the epithelial cells
and their basement membrane. At the same time the interglandujar tissues
beneath became swollen and small hemorrhages occurred. Great numbers
of macrophages collected. Ulceration proceeded from above downward.
Many amebsB were first seen in and between the epithelial cells, then in the
connective tissue at the base or sides of the ulcers. Necrotic and suppura-
tive processes producing varying degrees of suppurative inflammation may
complicate the lesions.
The abscesses which form in the liver contain degenerated liver cells, poly-
nuclear leukocytes, red blood cells, and groups of small amebse.
As controls Harris tried rectal injections of various bacteria, in-
cluding the Shiga bacillus. All gave negative results, however, and
he considered that the amebee showed their specific action very plainly.
Though he did not describe the morphology of the organism from
his cases with enough minuteness to identify it with Schaudinn's
histolytica, he gave enough points to make the inference strong that
it is the same species. Whether Entamoeba coli would produce similar
dysentery in young dogs is yet to be proved. As stated above, Schau-
dinn found that he could produce the typical disease by feeding young
cats with cysts of Ent, histolytica, but could not get the same results
by feeding the vegetative forms.
Musgrave and Clegg injected "pure mixed cultures" of material
from cases of clinical amebic dysentery as well as similar cultures
of amebee from various sources into monkeys and produced dysen-
teryv Musgrave fed monkeys with encysted amebae in bacterial cul-
tures and obtained, in a small percentage of the cases, dysenteric
stools and ulcerations in which amebae were found without their
accompanying bacteria. Kartulis, Kruse and Pasquale and Strong
544 PATHOGENIC MICRO-ORGANISMS,
injected into the rectum the contents of liver abscesses containing
apparently only the amebse and produced typical dysentery, with
lesions similar to those seen in man.
Strong states that the lower monkey and the orang-outang in the
Philippines contract the disease naturally.
In Man. — According to Craig, about 50 per cent, of human beings
harbor harmless amebee in their intestines. Schaudinn states that he
found this form of ameba in one-half the cases examined in East
Prussia, one-fifth of those in Berlin, and 256 times in 385 examinations
in Austria. In order to obtain fresh material for study he infected
young cats as did Casagrandi and Barbagallo. He infected himself
for a like reason and found that the amebse remained in his intestines
about two months. They remain in the upper and middle parts of
the colon where the reaction is alkaline and they produced no patho-
genic symptdms.
The disease produced by pathogenic amebse in man is known as
amebic dysentery (amebic colitis, amebic enteritis, amebiasis).
Incidence. — The disease occurs endemically in tropical countries.
It is particularly prevalent in Egypt, India, and the Philippine Islands.
It occurs frequently in parts of South America and southern United
States. In northern United States few cases are reported, though
Patterson, who in 1909 described three cases (without a description of
the amebffi present), and who calls attention to fifteen cases reported
as endemic in New York City, since 1893, states that this disease is
probably more widespread than is generally thought, and that if it
were searched for more carefully more cases would be recognized.
Patterson adds to his report a bibliography of cases reported a5 orig-
inating in North America. Sporadic cases are found in Russia, Ger-
many, Austria, Italy, and Greece. An occasional small epidemic
may occur in the milder climates, A^Tiere it is endemic, the largest
number of cases occur after the heavy rains have begun in early
summer. Males are more frequently attacked, because more exposed
to infection. It may occur at all ages, but young adults seem most
susceptible. The foreign white race seems to be more susceptible than
natives. Unhygienic surroundings are generally a predisposing
factor, but in the Philippines all classes are likely to be attacked who
do not take continuous and extraordinary precautions in regard to
their drinking water.
Symptoms. — The symptoms may be mild or severe. The disease usually
runs an irregular course marked by periods of intermission and exacerbation.
It may begin acutely with slight fever, griping, tenesmus, and frequent stools.
Occasionally, however, the outset is gradual, lasting from a few days to sev-
eral weeks. The disease is generally chronic, extending over a period of a
few weeks or of many years. In the mild form which is usual in children,
the general condition may be remarkably good, the only symptoms worth
mentioning being the increased number of stools — 2 to 6 in twenty-four
hours, which contain few to many amebae. In the severe forms there is a
loss of appetite, great emaciation, some fever, acceleration of the pulse,
sweating, abdominal pains, and a decided increase of the number of stools
GYMXAMCEBIDA. 545
— 6 to 20 daily. The stools are more fluid and slimy and may be bloody.
They contain amebse in varying numbers. In very severe forms the stools
are watery, filled with blood, mucus, and sometimes sloughs. They vary in
numbers from 20 to 50 in twenty-four hours and may contain many amebae.
The milder forms may change suddenly to the severest, and the severest
may suddenly become better and completely recover.
Tissue Changes. — The lesions are chiefly in the large intestines. The
walls are thickened in chronic cases, especially the submucosa. There are
raised hemispheric areas of hemorrhagic catarrh and of ulceration. The
whole of the large intestines may be affected or only more or less circum-
scribed areas. The ameb® pass between the epithelial cells, generally
through small erosions, and they finally reach the submucosa by the lymph
channels. Here reproduction takes place and the irritation to the tissue
causes oedema and infiltration of small spheroidal cells. This produces small
elevations into the lumen of the intestines. The epithelium over these
raised areas is finally eroded and then bacteria and intestinal contents help
form the succeeding ulcers. The erosions or ulcerations have congested under-
mined margins, and yellowish-red bases. They vary in size from 2 mm.
to about 2 cm. They are round, oval, or irregular in outline. The ulceration
usually extends only to the submucosa, but may expose the peritoneum, and
large sloughs may be cast off into the lumen of the intestines. Generally
the slow inflammatory process in the submucosa leads to great thickening of
the intestinal wall.
The processes may be modified in various ways by the action of other
microorganisms, especially the bacteria in the faeces. Healing takes place
by the formation of connective tissue in the floors and by a gradual cover-
ing over with epithehum. In extensive lesions, scars may form.
Peritonitis may occur with the production of an opaque gelatinous fibrinous
fluid in which the amebce may be found.
Abscesses may form in the liver (about 20 per cent, of all cases), less often
in the lungs, and only occasionally in the brain and spleen. Amebae may
reach the liver through lymph channels, portal vein, and peritoneal cavity.
The other organs are only slightly changed.
Source of Amebse. — Nothing can yet be said about the exact
source of Schaudinn's pathogenic variety, as so few have identified
the organism. Strong states that in Manila the greatest source of in-
fection from amebae is the water supply, that amebae were cultivated
from the water in large numbers in 1902, but no attempt was made
to demonstrate their pathogenicity. In 1904, however, Musgrave
produced dysentery in a monkey with a culture of a water ameba,
though in a few experiments he w'as unable to infect cats from the
amebae obtained from this monkey. Practically, it is proved that
people in Manila avoid being infected with amebae if they do not
drink local water, unless^ sterilized. Fresh vegetables as well as
certain fruits may be sources of infection.
As dilute acids quickly kill the motile amebae, it is probable that
many of those ingested in this form are destroyed in the stomach.
Immunity to the disease may exist. It is supposed that the
amebae as they die produce toxic substances which call forth antibodies,
but this has not yet been determined. The necrosis produced in the
liver abscesses when bacteria are absent is an indication of the pro-
duction of necrogenic substances (D. Wills).
Prognosis. — ^^Fhe percentage of deaths in the severe cases is quite
35
546 PATHOGENIC MICRO-ORGANISMS.
large, especially if accompanied by abscess of the liver. Probably
25 per cent, of all cases are fatal. When treatment is begun early the
prognosis is better.
Treatment. — ^^Fhere is no specific curative treatment. Besides rest
and diet, high enemata of bisulphate of quinine have been recom-
mended. Harris has gotten good results from hydrogen dioxide ene-
mata diluted from 4 to 8 times with water. About a quart is injected
twice daily for a week, then the amount is gradually decreased. Ipe-
cacuanha is highly recommended by Manson, Dock, and othere,
especially since the introduction of salol-coated pills which allow the
remedy to reach the intestines before it is absorbed, so that large
doses may be given, without inducing marked nausea and vomiting.
Points in IMagnosis of Ameb» Found in Man. — Examination of
stools should be made as quickly as possible after they have been
passed and they should be free from urine. The amebse must be
seen motile because, after encystment or death, it is often difficult to
distinguish them from other intestinal contents. Bloody mucus or
small pieces of necrotic tissue should be examined first as they often
contain large numbers of amebse.
If the movements are solid a dose of salts should be given and the
fluid part of the resulting stools examined.
For a differentiation between Ent. coli and Ent, histolytica, Lesage
has recommended the addition of a dilute watery solution of iodine
to fluid stools. This causes the characteristic cvsts of either form to
become noticeable in a few minutes.
Craig differentiates, living pathogenic forms from non-pathogenic
varieties by the former's: (1) larger size, (2) greenish color, (3) dis-
tinct hyaline, refractive ectoplasm, (4) faint nucleus, (5) manv
vacuoles and red blood cells, (6) marked motility. His differentia-
tion of the stained specimens is given in the table, p. 534.
An absolute diagnosis of liver abscesses can often only be made by an
exploratory puncture and the finding of the amebse. If this is done,
the surgeon should be at hand to operate if necessary.
Ent. buccalis is usually found in the thick group of leukocytes and
microorganisms collected between the teeth. The amebse are distin-
guished from the leukocytes and cell detritus by (1) their large size,
(2) their light, highly refractive greenish appearance, (3) their glisten-
ing red color in contrast to the yellow-red of the leukocytes when
hanging drops are stained with enough of a concentrated solution of
neutral red to make them appear pink.
Differential Diagnosis between Amebic and Bacillary Dysen-
tery.— In amebic dysentery (1) the disease is generally chronic;
(2) dysentery bacilli are usually not found in fseces; (3) no severe
toxic symptoms present; (4) abscess of liver frequent sequela; (•)'
lesion is in caecum and descending colon, not in small intesdhes.
In bacillary dysentery, the finding of the bacilli, and a positive
agglutination test, together with the clinical symptoms of intoxication
make a certain diagnosis.
GYMNAMtEBlDA. . 547
AHBBX IN OTHXB DISEASES.
Baelz found a very large ameba in the bloody urine and in the vagina
of a twenty-three-year-old Japanese who was suffering from tuber-
culosis of the lung. Jurgens, Kartulis, and Posner also reported
finding similar amebee in cases of cystitis and bloody urine.
In the ascitic fluid of a man who had carcinoma of the stomach
T^eyden found motile cellular elements which Schaudinn first pro-
nounced independent organisms belonging to the rhizopoda and
named by himLeydenia gemmipara (Fig. 168). Similar organisms were
foun<l ill the ascitic fluid of a girl who had an al)dominal tumor. The
Leydmia frnniiiipara (n ptuue a[ chlunydopbrys Btercorvn. A. Bintle amebs; B. Plasmodia anil
buddina: n, nuclmu^ n', nmUma divldioc: cr. contractjle vacuole: v, vaeuole; tr, nd bkiod cell;
Kn. buda: Ka. ameba developed from bud.
organisms remained motile in the ascitic fluid seven days after removal.
The organism possesses a pulsating vacuole and one vesicular nucleus;
it divides directly and by budding. The individuals seem readily to
fuse (plastogamy). Schaudinn later (1903) considered this organism
a phase in the rhizopod chlamydophrys, and decided that it had no
pathogenic action.
Amebte occurring in the mouth have already been noted.
MTOETOZOA.
Introdaction. — There is some confusion in regard to placing this
group, due to the fact that in it are put many more or less indefinrte
forms which are difficult to classify. Some forms have both distinct
rhizopod and flagellate phases, and they produce simple cysts for
reproduction, while others have more plant-like characteristics.
Among the former is placed the Plasmodiophora hrassiciE, Waro-
nin, of historic interest in medicine because of the claims made from
time to time that it or forms related may produce human tumors.
l^E Plasmodiopuora brassic.£ is an intracellular parasite of
members of the Cruciferse, producing large tumors in their roots
548 PA THOGESIC MICRO-ORGAXIS.VS.
("fingers and toes," "cliib-foot"). When inoculated into experi-
mental animals it produces small granulomata, which finally disappear.
The spores taken in by the macrophages under these conditions resem-
ble some of the cell inclusions seen in the human malignant tumors,
hence the reason for the belief that under certain conditions they may
have an eliologic relationship. At present the idea is abandoned.
The study of the Plasmodiopkora, however, may be helpful to u^ in
coming to an understanding of the nature of some of the pathc^nic
protozoa, since it is so closely related to the rhizopoda. Material niav
be more or less easily obtaine<l, and a certain amount of developmeni
may l)e observeil in the hanging drop. Much of the life cycle may Iw
satisfactorily demonstrated from sections of cabbage seedlings and ihf
older plants. There are many points, however, in the life history which
still need explanation or corroboration.
The Organism. — The roots are supposed to l>e infected by the fiagel-
lated ameboid sporozoites which leave the spore cysts in the nioi?t
earth and enter the young rootlets of the seedlings. Here they po*
and divide by cell hipartition and by a multiple 'increase of the nucleus
through a primitive karyokinesis. As these forms increase in numhers
they are supposwl to fuse into a Plasmodium due to overcrowding
GYMSAM(EBIDA. 549
(Fig. 169 A). Following this fusion there is a simultaneous nuclear
division by definite karyokinesis (Fig. 169 B) until the whole host cell
i.s filled with an indefinite mass containing many tiny nuclei, which,
according to Prowazek, are sexual nuclei, gametes, that fuse two by
two, forming a copula around which a spore wall is produced. Thus,
Two c«\h ineitiated with sporeg of (he Platmodiaphora braiiara. (UoSeiD.)
fertilization by endogamy {sexual union between decendants of the
same cell) is accomplished. These small spores fill the dead cell of the
host (Fig. 170), and are contained in the soil where they remain until
favorable conditions allow the infection of a new host.
BlBLItlGRAPHV.
I'litkins. "Fertilization of Amoelja proteUB." Biological Builetin, IHOT,
XIII, 2HI.
^"The Pathoeenic Rhizopoda" in ''ProtoioOlogy." New York and Phil.,
mo9.
Craig. Studiea upon the -AmelME in the Intestines of Man. Jour, of Inf, Di».,
iilOS. V. 324.
Councilman and Lafieur. Johns Hopkinf
Dock. The Journ. of the .\m. Med. .\sboc ,
Harri*, "On the .\lterations Produced in the LarRe Intestines of Dogs by the
AmtBbie coli." etc., Philadelphia, 1901. Also, " Amcehic Dysentery," .\m. Journ.
of Med. Sciencea. 1905.
KarluliK, in Kolle and Wassermann's " Handbuch d. path. Mikroorg." Erg&nE-
ungshand, lat Hft., 1II06,
Mutgrare and Clfgg. ". Amelias; Their Cultivation and Etiological Sig-
nificance." Manila, Bureau of Public Printing, 1904.
Patteraon, H. S. Endemic Dysentery in New York, with a Review of its Dis-
trihution in North America. Am. Jour. Med. Sci., 1!)09, CXXXVIII, 198.
pTowcaek. " PlaBmodiuphora Brassies. " Arbeiten a. d. Kainerl. Gesundh.-
amtp, 1903, XXII, 3»6.
Sckaudinn, " Unt«rHUchunRen Uber die FortpRanziinic einiger RhiBopoden."
Arbeiten a, d. Kaiserl. Gesundh.-amte. 190;), XIX. .j47.
Slila. Report of the I'ommittee on the Relation of Protozoa (o Diseaxe. ' Am.
Pub. Health Assoc., 1904.
Strong. ".\m(pbic Dy.-entery," in Osier's Modern Meilicinc, Philadelphia.
Vol. 1, 1907.
CHAPTER XLI.
FLAGELLATA.
General Characteristics. — ^Flagellata are protozoa which move,
and in some forms feed, by one to several flagella or whip-hke proc-
esses. If pseudopodia develop, they are transitory.
Generally the flagella arise from the anterior part of the organism,
and in motion the larger ones (primary flagella) are directed for-
ward, while smaller ones (secondary flagella) are directed back-
ward, acting as rudders. Certain flagella ta possess a modification
of their bodies in what is called the undulating mejnbrane, which
consists of a fluted protoplasmic process attached along one side of
the organism, the free edge of which is prolonged as the flagellum.
It has been shown that flagella are not simple protoplasmic processes,
but that they have more or less of a framework of elastic fibres is
well, hence their power in locomotion can be better understood.
Except with special stains, which bring out these fibres, they appear
homogeneous.
The flagella arise from some definite place in the cytoplasm, some-
times from a distinctly differentiated chromatic body which has been
given various names, such as blepharoplast, centrosome, basal granule,
microsome, diplosome, or flagellum root; sometimes directly from the
nucleus. The basal granules seems to be derived primarily from the
nucleus, and from a physiologic standpoint may be considered as
a part of the motor nuclei.
The body of the flagellates is generally more or less elongated and,
except in most primitive ones, is fixed in its outline. The latter
characteristic is chiefly due to the fact that the organisms usually
possess a definite though delicate membrane containing elastic fibrils
The cytoplasm is usually not differentiated into an ento- and ecto-
plasm. It often contains one to several contractile vacuoles, as well
as food vacuoles, and there is frequently a definite opening or c\lo-
stom for the entrance of food. There are usuallv manv crannies
and inclusions of various kinds scattered throughout the cytoplasm,
and myoneme striations are seen in some forms. The nucleus, as a
rule situated anteriorly, varies much according to different species and
to different stages of development.
The flagellata multiply either in the free motile condition or after
encystment. In the first case, as a general thing, they divide longi-
tudinally. The basal granules and flagella divide with the nuclei.
Multiple division is also observed. In the second case, they mayor
may not conjugate before they encyst. Then division forms occur
in the cyst by a process similar to that in the amebee.
5.50
FLAGELLATA,
551
The sexual cycle varies much in diflFerent species. (See Fig. 171.)
Isogamy has been noticed between fully grown individuals as well as
between smaller forms. The union of diflFerent-sized forms, or ani-
sogamy, has also been observed. Also autogamy is not infrequent.
Schaudinn claims that certain of the flagellates pathogenic in man
require a second host for the development of their sexual cycle.
Natural Habitat. — ^Flagellates are numerous in foul and stag-
nant water, along the banks of ponds, lakes, and rivers, in the ocean,
in the intestinal contents of various animals, fish especially, and a
few in the body fluids of higher animals.
Fig. 171
Diagram of variations in life cycle of flagellates: 1, a young flagellate; 2, adult flagellate;
3, longitudinal division of adult free form; 4, daughter flagellate; 5, encystation; 678, division
into isogamet^; x and z, division into macrogametes and microgametes, characteristic for some
forms; 9, conjugation of the isogametes; y, conjugation of the macrogametes and microgametes;
10, resting-stage — aygote; 11-12, division into young. (.Aiter Doflein.)
Classification. — Following the classification we have adopted,
the flagellates parasitic in man are from three orders, the Monadida,
the Heteromasiigida, and the Polymasiigida, The chief differences
l>etween these orders are those of size and number of flagella.* Under
the Monadida are placed the genera Cercomonas, Ilerpeiomonas,
and Trypanosoma f with Spirccheia as an appendix; Bodo is put with the
Heteromastigida ; and Trichomonas and Lambli a are classed with the
Polymastigida.
Hartmann puts the Trypanosomaia, with other blood parasites, in
a new order, the Binncleata, and makes the Spirochefa an appendix
of this order. According to this arrangement the Ilemosporidia are
taken from the Sporozoa and placed with the Trypanosoinaia in this
order, the malarial organisms supposedly lose through their parasitism
many of the characteristics ascribed to this order.
• See classification, p. 529.
552 PATHOGENIC MICRO-ORGANISMS,
Material and Methods for Study.— A number of flagellates (Bodo, for
instance, see p. 584) are found in the large intestine of the lower animals.
The toad, the grass lizard, and the guinea-pig may contain some interesting
forms. As these forms are easily obtained and remain alive a long time out-
side of the body, they are well fitted for class study.
The fffices are obtained by pressing lightly over the anus of the animal, or
if the whole intestinal tract is to be examined, by sacrificing the animal and
dissecting out the parts wanted. The material is placed in a clean watch-
glass and thinned if necessary with physiologic salt solution. Hanging
drops may be made in physiologic salt solution or in such a solution made a
little thick by the addition of gelatin in order to retard the motion of the
flagellates somewhat so they may be better studied.
Permanent preparations may be made according to directions given on
p. 537. As most of the pathogenic members of this group may be difficult
to obtain in the living condition at any stated time, they must be studied by
students principally in stained smears and sections.
If one can obtain rats infected with Tr. Lewisiy others with one or more
pathogenic forms; still others with Sjdrocheta Obermeieri, the infecting
organisms can be kept alive by frequent reinoculation of the heart's blood,
subcutaneously or intraperitoneally into the fresh animal, or cultures maybe
carried on (see below). But this is an expensive and tiresome work in those
laboratories where such work is not being carried on, and generally one must
rely on the permanent preparation. In the development in the second host
one must also study the stained specimens in the great majority of instances.
The fresh specimens of blood are obtained from the tail tip of the rat. or
the ear of the dog ; they may be examined after dilution with physiologic salt
solution in the hanging drop, or in a drop spread under a cover-glass and
ringed with vaselin. For permanent preparations films of the blood are
spread, fixed, and stained in the usual way; Giemsa's method of staining
(p. 624) is very satisfactory.
For section work of the various organs the fixatives and methods given on
page 521 may be used. Special methods are given under each organism.
Artificial Ooltores of Blood flagellates. — These, according to Now
and MacNeal, may be made on a culture medium consisting of a mix-
ture of ordinary nutrient agar with variable amounts of fresh defibri-
nated rabbit or rat blood. The best all around results are obtained
with equal parts of blood and agar. The agar is melted and cooled
to 50° C., then the blood is added and thoroughly mixed. The tubes
are inclined until the medium stiflFens, when they should be inoculated
at once with blood or other infected material containing living trr-
panosomes. The surface of the medium should be very moist, so
water of condensation may form, (yenerally evidence of growth mav
he observed in three or four days.
0ER00M0NA8.
The members of this genus are round or oval flagellates with a long
anterior flagellum and a more or less pointed posterior one which is
sometimes ameboid. The vesicular nucleus is situated anteriorly,
and lying near it are one or two contractile vacuoles. Divison into two
daughter forms has been observed.
A number of cercomonada, none of them well studied, have been
observed in different animals as well as in man.
Cerconwnas hominis (I)avaine, 1S54) was observed in the dejections
of a cholera patient by Davaine. The body is 10/( to 12/i long and
FLAGELLATA. 553
pear-shaped, pointed posteriorly. The flagellum is twice as long
as the body. The nucleus is difficult to see. Davaine also reported
a smaller form in the stools of a typhoid patient. Other observers
have noticed similar forms in human stools, some associated with
**Amwba coli.*' Similar forms have been seen also in an echinococcus
cyst of the liver, in the sputum from a case of lung gangrene, in the
exudate of a hydropneumothorax, and a few times in the urine. They
are all probably harmless invaders.
HERPET0M0NA8 AND OBITHIDIA.
Certain flagellates found in the digestive tract of mosquitoes, flies, and
other insects are very similar to the trypanosomata. Among them
two distinct types have been recognized, Herpeiomonas and Crithidia,
the main differences between them being (1) the large size of the adult
monadian form of the former as compared with the latter, and (2) a rudi-
mentary undulating membrane in the latter. The distinctions between
these two genera and the genus Trypanosoma which have been recog-
nized are: (1) the former contain no undulating membrane or only a
rudimentary one, and (2) their centrosome or blepharoplast usually
lies at the side of, or anterior to, the nucleus instead of posterior to it, as in
Trypanosoma.
These distinctions, Novy claims, disappear in the cultural forms
of the three genera, when all show a rudimentary undulating mem-
brane and an anterior blepharoplast; he therefore considers them all
one genus, Trypanosoma, But most protozoologists do not agree
with him. His caution, however, in regard to confusing these insect
flagellates with developmental stages of vertebrate blood para-
sites should be remembered.
From the recent work done on the Leishman-Donovan bodies found
in kala-azar it seems probable that they are closely related to this
group of flagellates. Indeed, the latest conclusion reached in regard
to the classification of these organisms is that they belong to the genus
Herpetomonas. Babesia (Piroplasma), on the other hand, which was
thought earlier to be closely related to these genera, now, on account
of lack of corroborative studies, is put as it originallv was under the
h»mosporidia.
HERPETOMONAS DONOVANI (LEI8HBIANIA DONOVANI,
LEISHMAN-DONOVAN BODIES) AND ALLIES.
Certain fevers of severe malarial-like types known in different sec-
tions of the tropics by different names (dura-dum fever, cachexial
malaria, kala-azar) have recently been shown to have a causal relation-
ship by the finding of similar protozoon-like bodies in the lesions.
These bodies were first minutely described by Irishman in 1903 as
being present in certain cells in the spleen of cases called by him dum-
dum fever, occurring in India. He considered them as possibly trypano-
somes, but did not name them. Later in the same vear Donovan de-
scribed similar bodies in cases of what he called malarial cachexia. The
554 PATHOGEXJC MICRO-ORGA.MSMS.
bodies were first called the Irishman-Donovan bodies, then Laveran
and Mesnil who examined Donovan's preparations and considered
the organisms similar to those causing Texas fever in cattle, called them
Pirophsma Donovani. Ross, however, thought thej constituted a dis-
tinct genus and named them LeUkmania donovani, by which name thev
are still known. But there is little doubt, through the work of Rogers
and of Patton, of their belonging to the genus Herpetomonas. They
have since been found in different parts of India, in China, Tunis.
Algiers, Arabia, and Egypt, and Wright in this country has reported
in a case of Delhi boil from an Armenian immigrant, bodies which,
according to his excellent photographs and description, must be identi-
Pmloion in n coic of tropical ul»r. .Smnir [irpparatinn (rom the leaion ■uinnl wilh Wruibr<
KomuiowKky blood-!! iHiains Quid. The rini-Uke bodies, wilh nhiw rrntiBl ponioiu und rantam-
m:ite5?.'VAf^rWnght.)''"'^ """" " ""™*"'"°"'" " " 0 I ■■ ".on, X , .pprou-
cal with, or very closely related to, Leishman's bodie.s. On account
of the different pathologic conditions in which they are found, thev
are considered by some a different species, Ilerpelomonnx iropira.
Wright. The form found in infantile splenomegaly may I>e consid-
ered another species, with the name Herpetomonas infanlile,unu\ further
study. Darling has recently described an organism resembling that
of kala-azar found in a fatal disease of tropical America. Though
(he organi.sni, he .says, resembles //. Donovani, he thinks it has enough
points of difference to be placed in a different genus; therefore he
gives it the name Hlxtoplasma cap»ulatvm, and calls the disease his-
toplasmosis. He says it differs from H. Donovani in the form and
arrangement of its ihrimiatin iincleus and in not possessing a chn)-
FLAGELLATA. 555
matin rod. It has a refractile achromatic capsule. As he has not
yet grown the organism or made studies on its intermediary host, it is
too early to determine whether his generic name will hold.
The bodies have been found in large endothelioid cells in the spleen,
liver, mesenteric glands, bone-marrow, kidney, lungs, testes, skin, ulcers
in intestines, and in the polynuclear leukocytes in the peripheral blocHl.
In this last situation they are only Found in appreciable numbers in
advanced cases.
556 PA THOGENIC MICRO-ORGAMSMS,
Morphology. — The bodies are circular to elliptical in shape, from
2fi to 4/jt in diameter, and contain a double nucleus, a large oval one
at one part of the periphery and a small circular or rod-shaped one
near or at the opposite part of the periphery. This smaller body
stains more intensely than the larger one, while the cytoplasm of the
parasite stains very dimly, sometimes showing only a faint i>eriph-
eral rim. Any nuclear and cytoplasmic staining methods will bring
out these points in Zenker-fixed material. Smears stain well by
Wright or the Nocht-Romanowsky methods. The large cells contain-
ing the parasites are supposed by Christophers to be the endothelial
cells from the finest capillaries. Donovan states that he found small
forms in the red blood cells in the peripheral circulation when the
temperature was above 103°, and Rogers has grown abundant pure
cultures of the bodies in a slightly acid citrated blood medium at
20° to 22° C. Nicolle and later Novy have shown that H. infantum
is pathogenic for dogs and that cultures may be obtained with ease
from the infected animals (Fig. 173).
Rogers and Patton have shown that the bedbug transmits the dis-
ease, and Patton has demonstrated the development of the organism
up to the fully flagellated stage in the gut of this insect.
Effect on Human Host. — The symptoms, in the cases of general infection
are: (1) very much enlarged spleen and less enlarged liver; (2) progre^ve
ansemia with peculiar earthy pallor of skin, progressive emaciation, and mu.*-
cular atrophy; (3) long-continued, irregularly remittent and intermittent
fever (97° to 104°) ; (4) hemorrhages, such as epistaxis, bleeding from gums
into subcutaneous tissue, producing purpuric eruption; (5) transitory
oedemas of various regions. There are often complications, such as congestion
of lungs, dysentery, and cancrum oris. The blood count shows practically
no loss of hsemoglobin, but there is a decrease in the leukocytes, principally
polynuclears, giving a relative increase of mononuclears.
Negative points which help in the diagnosis are: absence of malaria, no
typhoid or Malta fever reaction, resistance to medication, quinine, as a rule
having no effect, though in early cases, and with large continued doses a few
good results have been reported. Splenic puncture with the finding of Leish-
man-Donovan bodies makes the diagnosis certain.
The duration of the disease is from a few months to several years. The
percentage of deaths is great ; in some forms of the disease, at the height of an
epidemic, it may reach 98 per cent.
Segregation and perfect cleanliness, especially in regard to bedbugs,
are recommended as the best means of eradicating the disease.
Bibliography.
S. T. Darling. The Morphology of the Parasite (Histoplasma capsulatum),
etc. Journ. of Exper. med., 1909, XI, 515.
Nicolle, Le kala-azar infantile. Ann. Inst. Pasteur, 1909, XXIII, pp. 361
and 441.
W. S. Patton. Scientific Memoirs by Officers of Med. and San. Depots of Gov
of India. New series No. 31.
Rogers. The Milroy lectures on Kala-azar. Brit. Med. Journ., 1907.
Wright. Jour. Med. Research, 1903.
CHAPTER XLII.
TRYPANOSOMA.
Introduction. — The name trypanosoma (boring animal) was given
by Gruby in 1843 to certain free-swimming hsemoflagellates found by
him in the blood of frogs. Much later similar flagellates were found
in the blood-plasma of many different species of vertebrates and in the
intestinal tract of several blood-sucking invertebrates.
Typical trypanosomes are characterized (Fig. 175, p. 562) by a
comparatively long, spirally-twisted body, along one side of which is
attached an undulating membrane having a cord-like edge that is
continued forward as a free whip (flagellum). The flagellum arises
near the posterior end of the organism in a small granule, called the
blepharoplast which lies near, or may be merged with, a larger chroma-
tin mass, called the kineto-nucleus because of its control over the motor
apparatus.
The nuclear apparatus consists of a tropho-nucleus with an intra-
nuclear centrosome, and of the above-mentioned kineto-nucleus and
blepharoplast which last functions as a centrosome for the kineto-
nucleus. The tropho-nucleus is usually situated nearer the flagellar
end; it is granular, thick, and egg-shaped, but varies somewhat in
size and shape.
The cytoplasm is faintly alveolar or granular, varying with age,
environment, and possibly species. Reproduction occurs usually by
longitudinal, occasionally by multiple division. The life cycle is not
well known. Though transmission occurs through the bites of various
invertebrates, notably flies, no definite sexual changes have been
proved to take place in the intestines of these intermediate hosts.
That an intermediate host is not necessary for the continued life of
at least one species of trypanosome seems to be proved by the fact of
direct transmission of T, equiperdum from horse to horse through
coitus.
Pathogenic Forms.— About 60 species of trypanosoma have been
described, but of these only a few are reported as distinctly pathogenic,
and two of these are known to be pathogenic for man. All these
are found in tropical countries. The accompanying table gives a list
of the better-known pathogenic forms with their hosts and with the
diseases produced by them. Though only slightly pathogenic, T.kwisi
is included among them, because of its similarity to the more patho-
genic forms and because of the ease with which it may be obtained
and studied.
557
PATHOGEMC MICRO-ORGANISMS.
I '=
i it
i It
If; 11 1
|fl
ll
HI
Is
III
11 = i i i
ii 1 I
ill I |t|
■ 4 A
fl
11
III i I
iHf lli'l i
i I
'11 fl° i i
"Eli I^E I I
TRYPANOSOMA. 559
Historical Note. — The first species of trypanosome studied with any degree
of fullness is the comparatively non-virulent T. lewisi. It was probably
first seen in the blood of the rat in 1845, but was not well described until
1879, when Lewis studied it more fully. Since then it has been studied by
many observers, especially by Kempner and Rabinowitsch, Wasielewski
and Senn, Jiirgens, Laveran and Mesnil, No\'y and MacNeal, Prowazek and
Moore, Breinl and Hindle.
The first of the more pathogenic trypanosomes was discovered by Evans
in the blood of East Indian horses suffering from surra, but it was not well
studied until 1893, when Lingard's important work on surra led, in a way, to
all the subsequent work on diseases caused by trypanosomes. The next year
a trypanosome was discovered by Bruce in the blood of horses and cattle
suffenng from nagana in Zululand and other parts of Africa. Bruce further
demonstrated the important fact that the disease was transmitted by the
bites of flies, the tsetse flies {Gloasina morsilans). Announcements of other
pathogenic trypanosomes in different parts of the tropics quickly followed. In
1896 Rouget found that dourine, a disease of equines in Algiers and South
Africa, was caused by a trypanosome (T. equiperdum). Then the South
African disease of horses, called mal de Caderas, was shown by Voges to be
due to a similar flagellate, while in 1902, Theiler found a variety of trypanosome
in the blood of cattle in the Transvaal suffering from the disease called
galziekte, or gall-sickness. The number of trypanosomes found in the tropics
is constantly increasing — both pathogenic and non-pathogenic forms.
Man was thought to be immune to trypanosomes until recently. Then
a few isolated cases of infection were reported before the important discovery
was made that trypanosomes are the specific cause of a definite disease
known as sleeping sickness, which occurs chiefly in the African negro.
In 1898 Nepvieu reported having found trypanosomes in the blood of
6 out of more than 200 cases of human beings examined for malarial
organisms and in a seventh case which was apparently in good health.
The eighth case is reported by Dutton in 1901. This case was a European
who had lived some years in West Africa. The principal symptoms were
gradual wasting and weakness; irregular temperature, never very high and
of a relapsing type; local oedemas, congested areas of the skin, enlargenaent
of the spleen, and constant increased frequency of pulse and respiration.
It ended fatally after one year and eight months. The chronic character
was repeated in animals. Some white rats were refractory; others died in
two to three months. In monkeys {Macacua rhesus) it was fatal in about two
months. Dogs were unaffected. This trypanosome was distinctly smaller
than species described in lower animals, and there was little doubt of its being
a distinct species. Dutton also found tr>T)anosomes in the blood of 1 out of
150 apparently healthy Gambian children examined by him.
The tenth case is published by Manson in 1902. This was a missionary's
wife who had resided on the upper Ck)ngo for a year. She presented the
same group of symptoms as Dutton's case, and after repeated examinations
trypanosomes were found in her blood. Manson soon after published a
similar case. Broeden published two more cases, and Baker three.
In 1904 Castellani stated that the cause of sleeping sickness of the negro
is a trypanosome. He found trypanosomes in the centrifugalized cerebro-
spinal fluid of 20 out of 34 cases of this disease. His work has been cor-
roborated by Bruce, Nabarro, Greig, and others. Bruce found trypanosomes
in the fluid obtained by lumbar puncture in all of the 38 cases examined and
in 12 out of 13 cases in the blood. The trypanosomes found in these cases
resemble those already found in other human beings, and probably belong to
the same species; they are, therefore, included under the saine name, Try-
panosoma gambiense, Dutton.
Chagas, in 1909, states that a trypanosome which he had discovered in a
small monkey (CcUhihrix hapalepenecellata) and called T. cmzi, is the cause
5(i0 PATHOCEXIC MICRO-OROAXISMS.
of human infection in Rio de Janeiro. It ia [tarried by a bemi|>tera, genui',
I'onorrhinua. The flagellate is small with a large blepharoplast (kineto-
iiucleus). It grows on blood agar readily and inferta laboratory' ani-
mals easily, Chagas reports developmental forms in the monkey's lung and
in the gut of the fly.
Oomparative Characteristics of the Different Species.— The form
changes of the same species in the same host are so varied that few
have been found absolutely characteristic of a single species, and, as
physiologic properties are not used in species classification, we cannot
l>e sure that all of the organisms in this group described as separate
species are so until more of the complete life histories are known ; until
ARCIufio'tioD o' Trgpaaomma Lrmiri. (Lavcmnand Menil.)
this time each new form found with distinct physiologic properties,
though apparently morphologically similar to others, may expediently
be considered a new species.
Morphology. — Si«e. — The variations recorded in the dimen^oM of
the eight species we are considering may l>e seen by glancing at the
above table. The trypanosome pathogenic for man (T. gambifnsr)
has the smallest average size of the group. With the exception of
T. theileri, which is much larger than any other of these eight forms,
the variations in size of the different species are not so marked as they
are on the same species under different conditions.
Shape. — In shape, though all follow the type, each species varies
greatly according to conditions of growth and multiplication. At times
they may be slender and worm-like, at others they may be so short and
thick as to be almost round. T. Uwisi has the posterior (aflagellar)
end often thinner and more pointed than the other species. T. evensi
PLATE II
••-•/
8
/o
//
A^
/J
t '.
b -':
\ •
Various Spirochetes (after Muhlins) and BJood Parasites. (All
Giemsa stain except 7, which is by Levaditi's method.)
1. Trypanosoma Lewisi. 2, Spirocheta balbianii. 3. Spirocheta from mouth: a. Spirocheta
buccalis; 6, middle form; c, Spirocheta dentium. 4. Spirorlieta dentium, pure culture. 5. Spirochete
with Vincent's fusiform bacillus. 6. Spirocheta pallida frt»ra ulcus dunira. 7, Spirocheta pallida,
from liver section. 8. Spirocheta Kallinurura. 9. Spirocheta from esophji^us carcinoma. 10. Spirocheta
(7) or Spirilla (?) in mouse blood. 11. Spirocheta from \nn«, ji-unRrene 12 I'istivo-autumnal parasite:
o, ring form; 6. macrogametocyte. 13. Quartan panisite: a, half-(n*own organism: b, full-grown
organism. 14. Prioplaama bigeminum, showing varioiLs stages in division within red blood cell.
i
TRYPA\OSOMA. 501
is generally a little longer and thinner than T. lewisi, while T, hrucei
has a more rounded aflagellar end than either, and is generally wider.
The Cj^oplasm diflFers slightly in the different forms. T. lewisi is
relatively free from chromatoid granules, while T. hrucei has usually
many. Myoneme fibrils have been demonstrated in some species and
probably all contain them. An oval vacuole has been seen in some
species.
The nucleax apparatus is essentially similar in all forms. The two
nuclei (tropho- and kinetonucleus) vary somewhat in position and
size in the different species and at different stages in the same species.
In r. theileri and in young forms of T. lewisi, both nuclei lie close
together near the centre of the organism. In T, lewisi the tropho-
nucleus is situated more anteriorly than in the other species.
Many variations from the type forms are seen. Some are no doubt
degeneration and involution forms. Three forms, however, which are
more or less constantly seen in all the species have been interpreted as
definite phases in the life cvcle. These forms were first described bv
Schaudinn in T. noctuae, and were interpreted by him and since then
by others as male, female, and indifferent form. The male cells are
smaller, more hyaline, and more free from granules than the female.
The nucleus of each sexual cell rids itself of male and female chromatin,
respectively. The indifferent cell, on the contrary, has a complete
nucleus. Opinions differ as to Schaudinn's interpretation being the
correct one. ^lore research is needed before we can arrive at a definite
conclusion.
Motility. — The first thing noticed on examining a fresh hanging
drop of blood at a magnification of 100 to 300 diameters is active
movements of the red blood corpuscles in certain areas, and, on care-
fully focusing over one of these areas, the rapidly wriggling worm-like
organism may be seen. As the movements become slower, the flagellum
mav be seen swaving from side to side and the wave-like movements
of the undulating membrane are quite discernible. Movement is
two-fold: (1) progression with an auger-like motion effected by the
undulating membrane assisted by the flagellum; (2) contractions of
the body assisted no doubt by myoneme-like structures. Relatively,
T, lewisi is most active and T, hrucei least. Motilitv soon ceases
outside of the body, continuing longer if the organism has been kept
in the ice-box than at higher temperatures.
Reproduction. — ^The usual and probably the universal method of mul-
tiplication is binary longitudinal fission (Fig. 1 75). In T, lewisi a rosette-
like segmentation has also been observed. Longitudinal fission begins
usually with division of the kineto-nucleus, then of the flagellum, and
last of the tropho-nucleus and cytoplasm; but this order of division
seems to be quite variable. The flagellum often appears to be dividing
first, and probably division always starts with the centrosome-like basal
granule of the flagellum. In some cases a new flagellum seems to be
formed instead of division of the old one. The details of division
have not been frecjuently studied, but it is probable that both nuclei
36
5(i2
PATHOGENIC MICRO-ORGANISMS.
divide by a primitive mitosis. During division the kineto-nucleus gen-
eraltv- moves near the tropho-nucleus. The cytoplasm di\'ide3 last,
beginning usually at the flagellar end. Generally this fission is equal,
but occa.sionally the daughter trypanosomes may be quite unequal in
size. This is notably the case in division of T. lewisi where the cyto-
plasm may divide so unequally that the process may be compared to
budding. The resulting small parasites have at first no undulating
membrane, hence they resemble somewhat Herpetomonas. These
young forms may divide several times in succession, producing smaller
and smaller fusiform parasites. As a result, some forms are so small
that they can only be seen when agglomerated or in motion (Schaudinn).
The Life Cycle of Trypanosomes. — The question as to whether try-
panosomes undergo phases of development in their invertebrate hosts
has i>een widely studied, especially since Schaudinn's observations on
Ilamoproteus noctua indicated a complex life cycle of the haemo-
flagellates and their close relationship to the heemosporidia. Schau-
dinn's work still holds, according to many observers, but some, notably
Novy and his co-workers, insist that Schaudinn did not give evidence of
having .sufficiently guarde<l against the error of mixing up the hfe his-
tories of distinct protozoa. Schaudinn claimed that the intracellular
hcemoproteiis of the owl classed with. the hemosporidia is only a staf^
in the development of Trypanosoma (trypanomorpha) nochtce, which is
transmitted to owls by the mosquito Culex pipiens, in whose gut il
undergoes sexual changes, Xovy and others claim that Schaudinn's
mosquito phases are forms of mosquito flagellates and not of the bird
trypanosome.
TRYPANOSOMA. 563
Encysted forms of some species have been seen by certain observers
in the fly carriers (Minchin, Laveran and Mesnil, and others). Other
species, however, seem to undergo no important change in the
fly, so the whole question is waiting for further research.
That the different forms of trypanosomiasis (with the single excep-
tion of dourine) are transmitted by the bites of flies is a fact. Bruce
(1894) first showed that T. brucei was conveyed by the fly Gloasina
morsitans. Since then other varieties of flies also have been shown
to spread the disease. Ghssina palpalis (see Fig. 176) is supposed to
be the chief agent in transmitting human trypanosomiasis. These
flies bite by day and in full moonlight. The infectivity of the insects
lasts for about forty-eight hours after they have bitten a sick animal.
Bruce found living trypanosomes in the proboscides of the flies at
the end of that time. Up to one hundred and eighteen hours they
were found in the flies' stomachs, but after one hundred and forty
hours the stomachs were empty, and what appeared to be dead
parasites were found in the excreta.
Cultivation. — Novy and MacNeal were the first (1903) to cultivate
trypanosomes in the test-tube. They have grown T, levrisi through
about 100 culture generations extending over several years. At the
end of this time the parasites were as virulent as at the beginning.
The culture medium used in their work was ordinary nutrient agar
containing variable amounts of fresh defibrinated or laked rabbit or
rat blood. The best results were obtained with a mixture of equal
parts of blood and agar. At room temperature the growth is slower but
surer than in the thermostat. A culture at room temperature retains
its vitality for months; thus in one case the trypanosomes were alive
after three hundred and six days. Novy and MacNeal also cultivated
in vitro T. brucei, T, evansi, and various bird trypanosomes. The
latter they found especially easy to cultivate, while the former are much
more exacting in their requirements than is the T. lewisi. These
investigators state that the cultural characteristics are such as
to enable perfect differentiation between T, Brucei and T, Levrisi,
For in cultures T. brucei has characteristic granules, T. levrisi has
none; the T, brucei shows little variation in size (15/i to 17// in length),
T. lewisi varies so much (l/i to 60/i long) that there are forms small
enough to pass a Berkefeld filter; T, brucei has a slow, wriggling
motion, T, lewisi moves with great rapidity and in an almost straight
line; and finally T. brucei forms small, irregular colonies, while T. lewisi
forms large, symmetrical ones.
The great majority of trypanosomes experimented with have been
found bv various workers to be cultivatable, with more or less ease.
T, gambiense, however, the cause of human trypanosomiasis, has so
far resisted artificial culture methods.
Effect on Vertebrate Host (Pathogenesis). — Lower Animals. —
Many of the lower vertebrates have become, through mutual toleration,
natural hosts of the trypanosome. It is probable that each pathogenic
trypanosome has an indigenous wild animal as natural host and that
564 PATHOGENIC MICRO-ORGANISMS.
in this way the supply to strange mammals coming into the vicinity
is kept up. These strange animals, being unaccustomed to the native
trypanosomes, succumb to the infection.
In general the descriptions given of the symptomatology of trj'panosomia^is
m various animals show a great similarity, though there is much variation in
individual cases. The average clinical picture, according to Musgrave and
Clegg, is as follows: After an incubation period which varies in the same
class of animals and in those of different species, as well as with the condi-
tions of infection, and during which the animal remains perfectly well, the
first symptom to be noticed is a rise of temperature, for some days a remit-
tent or intermittent fever may be the only evidence of illness. Later on the
animal becomes somewhat stupid; watery, catarrhal discharges from the
nose and eyes appear; the hair becomes roughened and falls out in places and
the peripheral lymph nodes are enlarged. Finally the catarrhal discharge?
become more profuse and the secretions more tenacious and even purulent;
marked emaciation develops; oedema of the genitals and dependent parts ap-
pears; a staggering gait, particularly of the hind parts, comes on, in some
forms passing on to paralysis. This is followed by death. There may \»
various ecchymoses and skin eruptions. Parasites are found in the blood
more or less regularly after the appearance of the fever. They are often more
numerous in the enlarged lymph nodes and in the bloody oedematous areas
than in the general circulation.
The autopsy generally shows anaemia, an enlarged spleen with hj'pertro-
phied folUcles, more or less gelatinous material in the adipose tissue, the liver
slightly enlarged, a small amount of serous exudate in serous cavities, oedem-
atous condition, and small hemorrhages in various tissues.
The duration varies from a few days to many months. The prognosis
seems to be influenced to a certain extent by the species of host. It is prob-
ably always fatal in horses. Some cattle recover. The cause of death is
possibly a toxic substance, though no definite toxin has been isolated. Me-
chanical disturbances (emboli, etc.) also probably play a part in producing
death.
Man. — Sleeping sickness, or human trypanosomiasis , is a di.sease of
the negro, endemic in certain regions of equatorial Africa. Neither
age nor sex are predisposing factors, but occupation and social position
seem to have a marked influence, the great majority of cases occurring
among very poor field workers. As these workers are all negroes, the
question of the influence of race cannot be determined. The white
race, however, is not immune, as has been shown by the cases quoted
above.
In places where most of the cases occur, a fly belonging to the spe-
cies glossina {Glossi7ia palpalis, see Fig. 176) is very abundant; in places
where this fly is not found no cases occur. Hence, it is highly probable
that, as in the trypanosomiasis of the lower animals, the contagion i>
spread by a biting insect.
Sjrmptoms. — The course of the disease is very insidious, as the trypano-
somes may exist in the blood for a long time before entering and growing
in the cerebrospinal fluid and causing the characteristic symptoms of sleep-
ing sickness. Therefore, the symptoms may be divided into two .stages. In
the first stage there is only an irregular fever with enlargement of the per-
ipheral lymph nodes. In the second stage the fever becomes hectic, the
TRYPANOSOMA.
565
pulse is ronstantly increaned; there are neuralgic pains, partial crdemas and
erythemoH, trembling of the muscles, gradually increasing weakness, eniaria-
tion. and lethargy. The somnolence increases until a comatose condition is
developeil and death occurs. In the second stage trypanosomes are always
found in the cerebrospinal fluid. Throughout the disea.te they are usually
found in small numbers in the blood.
Dnntioii. — The first stage may last for several years; the second, from
four to eight months. The percentage of deaths in rases reaching the second
stage is 100. Whether some in the first stage recover is not yet certain.
T. gambiense, the chief trypanosome pathogenic for human beings, is irreg-
ularly pathogenic for some monkeys {Slacacux rhemix and others), for dogs,
cats, and rats. Tt is less pathogenic for mice, guinea-pigs, rabbits, and horses.
Cattle and swine seem to be refractory.
Patbological OhuigeB.— Congestion of the meninges; increa-seil quantity
of cerebrospinal fluid; hypertrophy of spleen, liver, and lymphatic ganglia;
diminished hiemoglobin and number of red cells; number of leukocytes about
normal, but a relative increase of eosinophiles, mast cells, and lymphocytes.
Enlargement of the superficial lymph nodes has been noted as an early
symptom and has thus been made use of in diagnosis, Dutton and Todd
found that 91 per cent, of natives in the Congo Free State, who had posterior
cervical glands enlarged, showed trypanosomes in the puncturecl gland juice.
Diagnosis of Trypanosomiasia in Oeneral.— 'I'bis should be made
as early as possible in order to prevent the .spread of the di.sease. An
early positive (lia^nosi.i can only l)e made by the determination of
the peripheral infection. Thi.s is done in two ways: first, by the micro-
566 PATHOGENIC MICRO-ORGANISMS.
scopic examination of a hanging drop of freshly drawn blood, or tissue
from enlarged peripheral lymph nodes; second, by animal inoculation
of the blood or other tissue. In the microscopic examination it may
be necessary to examine the blood of the suspected animal for sev-
eral days in succession. The parasites are rarely absent in the early
stages in domestic animals for more than a few days at a time, while
in man the time may be much longer.
Methods of Examinatioii. — Blood. — If the direct examination of
the blood is negative, 10 c.c. should be withdrawn from the vein, and
after adding a tenth of its volume of citrate of sodium it should be
centrifuged for ten minutes, and the sediment examined in hanging
drop and in smear. The great majority of the parasites will be found
collected with the white cells in the thin white layers which may easily
removed with a fine pipette. If only a small amount of blood can be
obtained, the tiny tubes recommended by Wright in his opsonin work
(p. 183) may be used.
Cerebrospinal Fluid. — Ten c.c. of the fluid withdrawn by lumbar
puncture should be centrifuged for fifteen minutes and the deposit
should be examined under 150 to 200 diameter magnification.
The Inoculation Test. — If the trypanosomes cannot be found by
the above methods, animal experiment should always be made.
Monkeys, if possible, should be used, or if monkeys cannot be ob-
tained, dogs or rats may be used. A few drops to 1 c.c. of the blood
or other tissue from the suspected animal should be inoculated intra-
peritoneally or subcutaneously.
Blood smears may be stained by any modification of the Roman-
owsky method. Giemsa's method (p. 624) gives good results.
Prophj^azis. — ^The disease is readily controlled by preventive meas-
ures. There should be strict quarantine regulations governing the
importation of animals. When the disease has once appeared, the
following general measures should be taken: 1. Suspected animals
should be isolated. 2. All infected animals should be destroyed.
3. As far as possible, all biting insects should be destroyed. 4. The
bodies of infected animals should be protected from biting insect^
for at least twenty-four hours after death. 5. Susceptible animals
should, if possible, be made immune.
Treatment. — The whole question of treatment is still in the experi-
mental stage. The chronic course of the disease with relapses often
after long intervals makes it impossible, especially in cases of human
trypanosomiasis, to come quickly to a conclusion in regard to the effi-
cacy of any drug. Many drugs have been found to possess trypano-
cidal properties. They may be classified according to Breinl and
Nierenstein into three groups: 1. Compounds containing arsenic
in inorganic form, as sodium arsenate. 2. Those containing organic
radicals, as the amine group in atoxyl and allied compounds and in
certain coloring substances of the diazo type (trypanred, parafuchsin.
tryparosan, etc.) introduced by Ehrlich. 3. Antimony in form of
sodium antimonvl tartrate and isomers.
TR YPA NOSOMA . 567
Atoxyl (p-amino-phenyl-arsenic acid,
/OH
AS = <^0
^ON.
NH,
introduced by Thomas (1905) and used first by Thomas and Breinl
in treatment of experimental trypanosomiasis, has proved to be the
drug above all others to have a beneficial effect in the different forms
of this disease. Much experimental work has been done on the dif-
ferent phases of treatment by atoxyl and its allies, in the course of which
some very interesting facts relating to chemo-therapeutics have been
demonstrated. Ehrlich has added to his '* side-chain theory," while
others have advanced quite different views in regard to the action of
this group. The chief facts are the following:
1. Atoxyl does not act in vitro unless mixed with an oxidizing
substance.
2. After the first few treatments with any of these drugs trypano-
somes may become resistant to the drug. This resistance is more or
less specific for all members of the group to which the drug used
belongs. There are a few exceptions, e. g., an atoxyl-resisting strain
may still be influenced by arseno-phenyl-glycine or by orsudan.
This acquired resistance lasts for some time in the species of animal
used, but may be quickly lost if the resisting trypanosomes are inocu-
lated into another species.
3. The time of the reappearance of parasites after a discontinuation
of treatment is more or less regular. With T, gambiense, in rats
and monkeys, the period is generally 50 to 60 days. With T, brucei,
in rats, guinea-pigs, and dogs, the time is only 11 to 25 days.
4. In order to pronounce an animal cured a long period must elapse,
since relapses may occur at a very late date (226 days in rats infected
with T. brucei and treated with atoxyl).
In human trypanosomiasis favorable reports from atoxyl treatment,
still continue to come, though the percentage of cures claimed by Koch
is probably not reached. Just now good reports are being received
from the use of another arsenic compound, introduced by Ehrlich,
namely, arseno-phenyl-glycine.
Serum Therapy. — ^Various normal sera from different animals
have been tried with practically no success. A few have prolonged
life. Thus Laveran and Mesnil state that human serum injected in
sufficient quantities shows manifest action on the disease, and that
sometimes cure results in mice and rats. Further, by alternating
human serum with arsenic they obtained better results still. Kant-
hack, Durham, and Blandford showed that animals recovering from
trypanosoma infection were immune to further infection. Rabino-
witsch and Kempner have made a. very careful study of immune
serum produced by T. lewisi. They have shown that an animal
568 PATHOGENIC MICRO-ORGANISMS.
may be hyperimmunized and that then its serum, in cx>mpara-
tively large doses, inoculated into mice at the same time as the try-
panosomes, or twenty-four hours before or after, allows no develop-
ment of the organisms. Laveran and Mesnil state that the serum
causes the rapid destruction of the organisms by the leukocytes,
though MacNeal, on the other hand, states that the trypanosomes are
destroyed by a cytolytic action of the serum. This immune serum
also has a similar action on the trypanosoma of dourine. The serum
of animals hyperimmunized against other varieties of trj'panosoma is
not as active as that obtained by the inoculation of T, Uwisi.
Koch suggested that an immunity might be established by the inocu-
lation of attenuated parasites, and Novy and MacNeal have suc-
ceeded in attenuating cultures of T, brucei, and have obtained some
success in protecting experimental animals against virulent cultures.
Bibliography.
Breinl and Nierenstein. Ann. of Trop. Med. and Parasit., 1909, III, 395.
Bruce. "Trypanosomiasis" in Osier's Modern Medicine, 1907, I, 460.
Ehrlich. Uber Partial funktionen der Zelle. Miinch med. Woch., 1909, V, 217.
Laveran et Mesnil. Trypanosomes and Trypanosomiases, Trans, by Xabarro.
London, 1907.
MacNeal and Novy, in Contrib. to Med. Research. Vaughan Anniv., 1903,
p. 645.
Macneal and Novy, Trypanosomes of Mosquitoes, Journ. of Infect. Diseases,
1907, IV, 223.
Mesnil et Kerandel. Sur Taction i>r6ventive et curative de I'ars^nopWnyl-
glycine dans les trypanosomiases experimentales et en particulier daas les infec-
tions h T. gambiense. Bull. d. 1. Soc. d. path, exot., 1909, II, 402.
Musgrave and Clegg. Trypanosoma and Trjrpanosomiasis, etc., Manila, Bureau
of Public Printing, 1903.
Nocfit u. Mayer,, in KoUe and Wassermann's Handbuch der Pathogenic Blikro-
organismen Erg&nzungsband, Ist Hft., 1906.
Navy and MacNeal, Trypanosomes of Birds, etc., Journ. of Infect. Diseases,
1904, I, 1905, II, 256.
Woodcock. The Haemoflagellates and Allied Forms in Lankester's "A Treatise
on Zoology," London, 1909. Part I, first fascicle, p. 193.
CHAPTER XLIII.
SPIROCH^TA AND ALLIES.
The genus Spirochcpfa was introduced by Ehrenberg in 1838, who
differentiated it from spirillum by its flexibility. Schaudinn in 1905,
thought he saw an undulating membrane in spirocheia refringenSy so he
added this characteristic to the genus and considers that thus its
relationship to the flagellated protozoa, genus trypanosoma, is
indicated.
Since the appearance of the work of Schaudinn (1905) on the etiology
of syphilis, the Spirochetce have been brought into great prominence.
Numerous spirochetes and spiral organisms have been described,
some associated with Treponema pallidum in syphilis, some in other
lesions or in the normal secretions of both man and the lower animals;
and still the question as to their classification is unsettled. The major-
ity of observers, however, are willing to admit that the structure of
many of the varieties classed with this group is more complicated than
that of bacteria and that hence the group may be an intermediate one
between protozoa and bacteria. For this reason it is still retained
in the section on protozoa.
The chief reasons given for considering them protozoa are: (1) their
flexibility and the indications in many of longitudinal division and of undulat-
ing membrane; (2) the demonstration of forms intermediate between the
trypanosomes and the spirochetes {Sp. bcUbianii) ; (3) the spirochetal forms
of certain trypanosomes (Tr. nociucp). In favor of the bacterial nature of
spirochetes are: (1) the rigidity of some forms, the lack of undulating mem-
brane in most and of definite nuclear apparatus in all, and the evidence of
transverse division in all and of flagella arising from the periplast in some;
(2) the cultivation of certain forms (e. g.^ Sp. refringenSy by Levaditi; Sp.
Obermeieri, by Novy) for many generations without development of trypano-
some forms.
So far the studies on this group show that the spirochetes and allies probably
occupy a position intermediate between the protozoa and the bacteria. We
study them here because of the claims that they are closely related to the
trypanosomes.
It may be well to note briefly the chief characteristics of the more
familiar non-pathogenic ones in order better to understand the relation-
ships between them and the Treponemo pallidum and other pathogenic
forms.
Material and Methods for Study. — The large Spirocheia balbianii is found
in the stomach of oysters fresh from salt water. Smaller spirochetes are
frecjuently found in human mouths. When fresh syphilitic or relapsing fever
material can be obtained this should be examined. The Treponema palli-
dum (the spiral organism of syphilis), because of its low refractive index, is
seen when alive with difficulty by the ordinary microscope, but with the dark-
569
570 PATHOGENIC MICRO-ORGANISMS.
stage illumination, especially if a drop of distilled water is added to the aenim
containing the organisms, it is seen- distinctly and its motion and structure
may be more easily studied. The fluid containing the organisms shoidd be
dropped on an ordinary glass slide, covered with a thin cover-glass and well
sealed with vaselin as most spirochetes are anaerobic. Material may be
obtained from syphilitic lesions as follows: The lesion is first thoroughly
washed and dried with distilled water and sterile gauze. Part of the base and
margin is then scraped with a curette until the superficial tissue is removed
and blood appears. The blood is wiped away with sterile gauze until clear
serum begins to ooze. A drop of this serum is used for examination.
Smears should be made as thin as possible and may be stained (1) by
GiEMSA according to the method on page 624 (Tr. pallidum stains reddish.
See pi. II, Fig. 6); a modification of Giemsa, used by Schereschewsky (see
bibliography) has been highly recommended by various workers; (2) by
Goldhorn's method as follows :
Dye. Water, 200 cm. Lithium carbonate, 2 grams. Methylene blue, 2
grams. (Merck's medicinal or a similar preparation.) This mixture is
heated in a rice boiler with a moderate amount of heat until a rich poly-
chrome has formed. This is determined by examining a sample against
artificial light and noting the appearance of a distinctly red color. The
solution is allowed to cool and the residue is removed by filtering through
cotton. To one-half of this filtrate 5 per cent, acetic acid is gradually added
until a strip of litmus-paper shows above the line of discoloration a distinct
acid reaction. The remaining half of the dye is now added, so as to earr>'
the reaction back to a low degree of alkalinity. A one-half per cent. French
eosin solution is now added gradually, while the mixture is being stirred
until a filtered sample shows a pale bluish color with slight fluorescence.
The mixture is allowed to stand for one day and filtered. The precipitate is
collected on a double filter-paper and dried at a temperature not exceeding
40° C. It is then removed from the filter-paper and dissolved in commercial
wood alcohol. It is allowed to stand for one day in an open vessel and then
filtered.
To use the stain on smears sufficient dye to cover the smear is dropped on
an unfixed preparation and allowed to remain for three or four seconds; the
excess is then poured off. The slide is now introduced slowly into clean
water with the film side down, is held there for four or five seconds and is
then shaken in the water to wash off the excess of dye. It is then allowed to
dry and is ready for examination. The pallidum stains violet.
Until recently the demonstration in smears of the syphilis spirochete by
(3) the silver impregncUion method ^ so successfully used by Levaditi in sections
has been unsatisfactory. Stern, however, and Flexner corroborating him,
have gotten beautiful results by the following simple method :
1. Air-dried into 37° incubator for some hours.
2. Ten per cent, aqueous silver nitrate for some hours (Flexner thinks three
to four days' exposure better) in diffuse daylight.
3. When the brownish co^or reaches a certain tone (easily recognized after
experience) and when a metallic sheen develops the slide is washed well in
water, dried and mounted.
The blood cells are well preserved, they have a delicate dark brown contour,
and contain ^ne light brown granules. The spirochetes are deep black on a
pale brown and in places a colorless background.
Other spirochetal organisms may be silvered by this method, but as they
may be differentiated with greater difficulty than with Giemsa's stain, the
latter should always be used as well.
(4) These organisms may also be demonstrated by the India ink method
(see p. 47).
The flagella are brought out by Loeffler's method or by the stain recom-
mended by Goldhorn.
SPIROCHMTA AND ALLIES. 571
Sections are prepared by the silver impregnation method of levaditi
as follows: Fix small pieces of tissue one-half mm. in thickness for twenty-
four to forty-eight hours in formalin, 10 per cent. Wash in 95 per cent,
alcohol twelve to sixteen hours. Wash in distilled water till the pieces sink.
Impregnate two to three hours at room temperalure and four to six hours
at 50° C. in the following fluid: Nitrate of silver, 1; pyridine, 10 (added
just before using); aq. dist., 100. Wash rapidly in 10 per cent, pyridine.
Reduce the silver by placing in the following mixture for several hours:
Pjrrogallic acid, 4; acetone, 10 (added just before using); pyridine, 15; aq.
dist., 100. Harden in alcohol; xylol; paraffin. Levaditi's first method
is longer but more reliable. Fix small pieces in formalin, 10 per cent. Harden
in 95 per cent, alcohol. Wash in distilled water several minutes. Im-
pregnate three to five days at 37° C. in 1.5 per cent, solution silver nitrate.
Reduce twenty-four hours in: Pyrogallic acid, 4; formalin, 5; water, 100.
Imbed in paraffin. By these methods the spirochetes appear densely black.
OoltareB. — ^Pure cultures have been obtained of the Spirocheta deniium
in the following manner: Poured serum agar plates are made of various
dilutions of material from the mouth containing these spirochetes. After
being kept in the thermostat at 37° C. under anaerobic conditions for nine
to twelve days the spirochetal colonies are finished and planted in agar tubes
as stick cultures.
Pure cultures of Spirocheta Obermeieri by Novy and of Spirocheta refrin-
gens by Levaditi have been obtained by growing in collodion sacs. (For other
culture experiments see below.)
Spirocheta balbianii, Certes (Plate II, Fig. 2).— This great form,
next largest known to the Spirocheta plicatilis Ehrenberg, may be
found in the stomach of the oyster. It is important because it is appar-
ently a transitional form. In fact, it is considered a trypanosome
by Perrin and others. Muhlens gives its characteristics as follows:
Length 26// to 120/i, width ^/i to 3/i. The body is flattened and pos-
sesses an undulating membrane which* is visible during life on some
individuals. It has 4 to 8 flat, wide spiral coils. Its movements are
lively, similar to those of trypanosomes, but more corkscrew-like.
During motion its form is apparently easily changed. The rim of
the undulating membrane does not end in a free flagellum, but one
end of it seems to be attached to a triangular mass of chromatin
(basal granule, blepharoplast ?) which is a part of the central chro-
matin material. The nuclear material is arranged in a more or less
spiral line along the entire center of the organism.
Before division this nuclear line, after passing through chroma-
some-like changes, breaks up into pairs, and division takes place
longitudinally between them. Division is often incomplete for a
time, the two ends remaining attached.
Spirocheta balanitidis. — This is a spirochete found by Simon in
Balanitis circinata and regarded by some as the specific cause of this
disease. Hoffmann and Prowazek describe it as a rather strongly re-
fractive, actively motile band-shaped organism, shorter and thicker
than Spirocheta pallida, with 6 to 10 coils, staining bluish-red with
Giemsa's method and exhibiting an undulating membrane and at
either end a periplastic ciliam.
Muhlens thinks this may be identical with Spirocheta rejringens.
572 PATHOGENIC MICRO-ORGAXISMS.
Levaditi has recently reported cultivating it (see below under Trepo-
nema pallidium).
The Mouth Spirochetes. — Three varieties of non-pathogenic forms
are commonly found in normal mouths.
1. Spirocheta buccalis, Cohn (Plate II, Fig. 3a). — Length, I0fito20n;
thickness, J// to §/£. It has 3 to 10 irregular flat coils. Xo true cilia
have been demonstrated, but Schaudinn, Hoffman, and Prowazek
say it has an undulating membrane. It stains violet with Giemsa.
2. Spirocheta dentiom, Koch (Plate II, Fig. 3c), — This is much
smaller than the previous form. It is as thin as the pallidum and is
somewhat similar to it in refraction, staining qualities, and in the
fixity of its coils during motion. It is somewhat smaller and stains a
little more easily with LoflSer's flagella stain, and flagella have been
demonstrated. Neither definite undulating membrane nor nuclear
material have been seen. It is 4/i to 12// long, and has 4 to 20 regular
spirals of about the same appearance as those of the paUidum. Pure
cultures have been made from this spirochete as described above.
3. A Bfiddle Form (Plate II, Fig. 36) between these two has been
found in the mouth. This also is somewhat similar to the pallidum, but
it is larger and has less regular spirals; moreover, it stains more in-
tensely with the blue of Giemsa, only in poorly prepared specimens
does it appear red.
Spirocheta refringens (Fig. 177) is also found in the mouth, but it
is especially interesting from the fact that it is so often found asso-
ciated with the Treponema pallidum in the various lesions of syphilis.
It is not in such large numbers as the pallidum and probably bears
the relation of a restricted secondary invader. It is generally longer
than the pallidum (10// to 30/?) and much thicker (J/i to |/i). In life
it is much more refractive. It has 3 to 15 irregular wide, flat spirals
which change their shape during motion. Its movements are much
more lively than those of pallidum. With Giemsa it stains quickly
and easily, a blue to a blue-violet tone, according to the length of
staining. Schaudinn states that it possesses an undulating mem-
brane. Levaditi claims to have demonstrated terminal cilia for
this organism and to have cultivated it in collodion sacs in the rabbit's
peritoneum.
Spirocheta Vincenti (Plate II, Fig. 5).— Accompanying the fusi-
form bacilli in Vincent's angina (see p. 230) are many spirochetes
similar to the ''middle form" found in the mouth. WTiether thev
are identical with these spirochetes or whether they are a special
variety (or, as some think, a second form of the fusiform bacillus) still
remains to be determined. Their relationship to the disease is also
uncertain.
Miscellaneous Spirochetse.— Besides the spirochetes found in
syphilis, in framboesia, in certain tumors of mice and human beings,
and the spiral organisms causing African and European relapsing
fevers, all of which will be described below, spirochetes have been
found (1) in the normal intestinal tract of mosquitoes and human
SPIHOCH.ETA AND ALLIES. 573
beings as well as in the diarrhoeal stools of the latter; (2) in the blood
of mice, fowls (Sp. gallinartim, causing relapsing fever in fowls), and
geese; (3) in various ulcerative and gangrenous processes of man.
Most of these have l>een very little studied.
Treponema pallidi'm (Spirocheta pallida}.— This organism is
found in large numbers in .typhilit, an infectious disease of human
l)eings, characterized by its long course and by the definite stages of its
clinical manifestations.
HiBtoiiul Not*. — Notwithstanding the fact that syphilis is one of the
oldest diseases known and studied, only recently has definite light been throvn
U|>on its cause in the discovery of the Treponema pallidum (Scnaudinn).
Before this it was thought that the tMicillua described by Lustgarten and
others as occurring in small numbers in the lesions of syphilis bore an etiologic
relationship to the disease, but there were no evidences to support this view.
.Many other bacteria have been erroneously regarded as the |)robable cause of
From time to time \'arious observers have described protozoan- like bodies
in syphilitic lesions, but their observations have not l(cen confirmed.
Schaudinn announced early in 1905 that working with Hoffman he found
in the fresh exudates of chancre a spiral organism possessing charaetcr-
istics similar to those of the spirochetes and he named it Spimchela pallida.
Later he concluded that this organism was indi-
vidual enough (that is, it showed no undulating ^'"^ '^^
membrane, but possessed a flagellum) to be placetl
in a separate genus, so he called it Treponema
pallidum. He thought that the organism was the
cause of the disease. Since then there have been
extensive studies on human syphilis and on ex-
perimental syphilis in lower animals with the
result that the work of Schaudinn and Hoffman
has been abundantly corroborated and many new
facts have been brought out.
The Organism (Fig. 1 77 and Plate 11, Figs. 6
and 7).— The Treponema pallidum is a very
delicate structure closely resembling in mor-
phology and staining reaction.s the Spirocheta
deniium. It is somewhat longer, 4fi to 'M/t
long (average lOu), and thinner, ^/< to ^/t in
diameter. It has four to twenty sharp, deep
spirals. The relationship between the length
and the depth of the -spirals is different in
the two species; in Treponema pallidum vfat^. anTr'^^dul^ibribm
length is to depth as 1 is to 1-1 .. 5 (l/* long and dLnnTn^'HoSiaS™' "'"''"'"
1/1 to 1 .5/1 deep), while in Hpirocheta deniium
the average relationship is 1:0,.'), the spirals Iwing more shallow. The
angle of the spiral turn is very sharp in both forms (more than 90°).
Flagel la-like anterior and posterior prolongations are often seen in
the pallidum. The double flagella occurring rarely at one en<l are
interpreted by Schaudinn as beginning longitudinal division. Schau-
dinn states that the division oc'curs very quickly (hence the reason
why so few dividing forms are seen in stained preparations) and that
574 PATHOGENIC MICRO-ORGANISMS.
it may be followed only by the most experienced observers during life.
In the living condition the organism is not very refractive, so it is seen
at first with difficulty. Its characteristic movements are rotation on itv
long axis, quivering movements up and down the.spi'ral which is com-
paratively rigid, slight forward and backward motion and bendin^r
of the entire body. By the use of the ultramicroscope the motility
of the organism is clearly seen (Fig. 178).
It stains red as does Spirocheta dcntiutn by Giemsa's method, while
most other spirochetes stain blue in properly prepared specimens.
Onltivatioa. — Up to 1909 numerous attempts had been made to
cidtivate this organism in artificial media, without success. In May,
1909, Schereschewsky reported that he had obtained a ciilture of a
Treponema palLiluDi Bppearina u bright refractive body on * dark Geld u ■bown
by India inlf or ultraimcroscopa.
.spirochete from syphilitic lesions and blood in the following culture
me<lium: horse serum sterilized by heat (58' to 60" C. [ ?] until it is of
jelly-like consistency, and afterward autolysed at 37° for three days. A
piece of tissue excised from the lesion (e. g., base of a papule or part of
a lymph node) is inoculated into this medium, and grown at 37° C.
The culture begins in three days, but the optimum is reached in 5 to 12
<lays. Such a culture is always impure and, moreover, it has so far
given negative results with specific serum and on animal inoculation.
Miihlens reported in July, 1909, that he had also obtained » culture
of a pallidum-tike spirochete from syphilitic lymph nodes, grown at
first in Schereschewsky's medium and afterward transplanted to broth
and grown anaerobically. Animal experiments arc being made.
I.«vaditi and Stanesco about the same time reported growing two
species of spirochetes from a case of balanitis. One, a new one, which
they found very like pallidum, but nonpathogenic for monkeys; ami
SPIROCHJUTA AND ALLIES. 575
which they named Sp, gracilis; the other Sp. balanitidis. They em-
ployed as media (1) collodion sacs in tubes of fluid horse serum;
(2) horse or human serum heated to 75® C. These spirochetes were
never obtained in pure culture, but in ** pure-mixed'* cultures, similar
to those required by amebas (see p. 536).
Pathogenesis. — So far as is known, syphilis in nature appears
only in man. Since 1879, when Klebs stated that he had produced
syphilis in monkeys by the inoculation of human virus, various experi-
menters have reported its transmissibility to these animals by direct
inoculation. Most of the earlier reports did not state the exact identity
of the animals employed nor did they give details of methods and
results.
Metchnikoff and Roux in 1903 produced a typical chancre on the genital
mucosa of the young chimpanzee twenty-six days after inoculation. The
essential lesion was followed by inguinal adenitis, and thirty days later
by a generalized papular eruption. The virus was transferred in this case
to lower monkeys. Most monkeys developed a primary lesion only, but some
had abundant secondaries.
Since the discovery of the Tr. pallidumy experiments on monkeys have
been more numerous and have been followed by more helpful results. More
has been learned about the course of the infection in man, the evidence in
favor of the Tr. pallidum being the cause of the disease has been strengthened,
and many interesting investigations in regard to immunity have been made.
Important features in regard to course of the infection have been sum-
marized by Ewing as follows: "If the virus is applied to the broken epithe-
lium, a chancre develops, but if similar virus is inoculated into the subcu-
taneous tissue an initial lesion does not follow, immunity does not develop,
and the animals remain susceptible to subsequent inoculation of the epithe-
lium. Yet in several instances Neisser was unable to produce chancres in
monkeys which had previously received subcutaneous injections of syphilitic
material, indicating that immunity may sometimes appear after such sub-
cutaneous injections. Possibly the leukocytes of the subcutaneous tissue
destroy the virus before it can begin to multiply. Hence, small superficial
wounds may be more dangerous in man than deep ones. Nevertheless,
it is recorded by Jullien that two French surgeons accidentally inoculated by
deep needle punctures developed pronounced signs of constitutional syphilis,
as attested by Fournier, but failed at any time to show signs of a chancre
at the point of inoculation. It remains to be seen whether the observations
of the clinicians or those of the experimental pathologists represent the true
laws of infection in syphilis.
**In monkeys the virus exhibits a certain choice of epithelium for its entry.
The abdominal skin resists the entry, the eyebrows and genitals are most
readily inoculable in apes, and the palpebral borders in catharinians. The
period of incubation varies from thirty days, on the average, in the chim-
panzee, to twenty-three days in lower monkeys, but the shorter the incuba-
tion, the shorter and less severe the subsecjuent disease.
"That the virus circulates in the blood m certain stages of syphilis has been
clearly shown experimentally. Although Neisser inoculated human subjects
with the blood of florid syphilis without effect, a result which is now ex-
plicable, Hoffmann, in two of four experiments, produced syphilis in monkey
{Macacus rhesus) by inoculating the skin with human blood drawn forty
days and six months after the appearance of the chancre. The resulting
primary lesions were typical, appearing after the usual incubation and show-
ing a characteristic histological structure and the presence of Tr. pallidum.
576 PATHOGENIC MICRO-ORGANISMS.
" Syphilographers are agreed that tertiary lesions are not contagious. Ex-
perimental studies have shown, however, that some tertiary lesions are
capable of transmitting the disease. Salmon had negative results with an
ulcerated gumma in the eighth year of the disease. Yet Neisser produced
chancres and secondaries in a gibbon and in a macacus with the material
from a non-ulcerated gumma (duration unknown), but the periods of incuba-
tion were very long, fifty-one and sixty-eight days. All tertiary lesions do
not seem to contain the virus, as Neisser found the material from tul>ero-
serpigenous lesions non-infectious. It appears also that secondary infec-
tion and ulceration of tertiary lesions reduces their infectivnty. None of
these observations invalidates the clinical experience that tertiary lesions are
practically harmless for the patient's neighbors, but they suggest greater
caution in deahng with tertiary lesions.
"According to CoUes' law, a mother who gives birth to a syphilitic infant
may not herself contract the disease, but thereafter remains immune to in-
oculation. This law may be explained by the infection by the embr\'o or
ovum, and the transference of immunity to the mother by the blood or by
some other method. The probable mode of origin of the maternal immunity
is suggested by an observation of Buschke and Fischer who found spiro-
chffites in the inguinal lymph nodes of such a case which remained entirely
free from the symptoms of the disease. The observation, taken with the
failure of subcutaneous and intraperitoneal inoculation to infect monkeys,
may explain the workings of CoUes' law. Levaditi and Sauvage claim to
have shown that Tr. pallidum is capable of invading the ovum. Finger and
Landsteiner found the semen in one case of secondary lues infectious for
apes, but in other cases their results were negative. It is, therefore, only
necessary to suppose an occasional escape from the genital tract in order to
complete the necessary conditions for the infection of the embr>-o with im-
munity in the mother.
"Neisser endeavored to determine the degree and duration of the infec-
tivity of the organs of monkeys and found that the virus persists especially
in the blood-forming organs, spleen, lymph nodes, and marrow, while in the
testicle also the virus is long preserved in active form. The other organs
gave entirely negative results."
Syphilis in the Rabbit. — Bertarelli and others have inoculated syphilitic
virus into the cornea and anterior chamber of rabbits' eyes and they have
obtained ulceration and increased numbers of spirochetes. After several
generations of passage, Bertarelli successfully inoculated a monkey from
such a cornea.
S3rphilis in Man. — The course of the disease is divided into three stages,
primary, secondary, and tertiary. The general character of the
lesions in these stages is a more or less circumscribed formation of
new tissue which is largely made up of small spheroidal cells alone
or accompanied by fewer polyhedral cells, and occasional giant cells.
The initial or primary lesion occurs in the form of a papule which de-
velops into the so-called chancre, an ulcer with hardened base. Following
this there is hyperplasia of the nearest lymph nodes. These lesions subside
and six or seven weeks later the secondary lesions appear in various general
eruptions on skin and mucous membranes and in other constitutional dis-
turbances. The tertiary lesions which consist principally of the ma.'^ses of
new tissue called gummata are found throughout the viscera and in the
periosteum.
Schaudinn's spirochetes have been demonstrated in practically all
the lesions of syphilis (they are most easily demonstrated in the pri-
mary and secondary lesions), including the congenital types, in Mich
SPIROCH^TA AND ALLIES, 577
numbers and position as to make the majority of workers in this field
look upon them as the almost certain cause of syphilis.
The technic first used failed to bring them out as well as that later
employed. But the method most recommended, that of silver im-
pregnation, is the one most assailed by the opponents to the view of
the organismal nature of the spiral bodies.
The adverse criticism made by Saling, Schultze, and a few others is based
upon the fact that silver nitrate impregnates nerve endings and elastic fibrils
so that both appear as spiral organisms; even the Giemsa and other stains
used, they claim, may stain fibrils of certain tissue in such a way that they
look like spirochetes. This criticism has been weakened very lately by the
fact that the pallidum may be brought out in smears by the silver impreg-
nation method as well as by Giemsa and in section by Giemsa's stain as well
as by the silver impregnation (see technique) . The use of the ultramicroscope
has made certain the fact of the Treponema being a living organism.
Immunity. — Natural immunity in syphilis is very peculiar. After
the development of the primary lesions, man is usually insusceptible
to reinoculation during the active stage of the disease, but during all
stages both man and monkey can, in some cases, be reinoculated.
Reinoculation in the tertiary stage gives precocious lesions of the
tertiary type, gummata and tubercles. Neisser found reinoculation
from twenty-four to one hundred and four days after primary in-
oculation in monkeys sometimes effective, more often negative.
During the stage when the skin is refractory to inoculation secon-
daries develop, showing that there is no complete immunity of the
skin to the virus, since the Treponema is abundantly present in the
lesions. Neisser suggests that cutaneous secondaries develop at
periods of relative deficiency of immunity. He has shown that
failure to reinoculation is not due to immunity to foreign infection
and susceptibility to auto-infection, since the patient's own virus in
both man and monkev is ineffective.
Attenuated Virus. — Efforts to secure an attenuated virus to be used
for inoculation have been unsuccessful. Fresh material loses its
virulence in six hours, and the results of inoculation with such virus
in all types of monkeys have been entirely negative. Passage through
monkeys does not attenuate the virus, and the absence of secondaries
in lower monkeys is apparently no indication of a change in the quality
of the virus, but only in the reaction in the host.
Passiye Immunization. — Injection of large quantities of serum of
syphilitics into chimpanzees has failed to produce definite immunity,
although some of Neisser's animals after such treatment failed to take
sj'philis. The serum of a monkey cured of syphilis and subsequently
injected over a period of fifteen months with the blood of syphilitic
subject in roseolar stage was without therapeutic effect. How-
ever, this serum dried and powdered prevented the chancre when
placed on the site of inoculation one hour after the virus.
The WasBermann Reaction. — Wassermann, Neisser, and Bruck were
the first to apply the Bordet-Gengou phenomenon (see Part I) to the
37
578
PATHOGENIC MICRO-ORGANISMS.
Fio. 179
Lipoid
Reagin
diagnosis of syphilis. According to many workers, enough work
has been done since then to establish its value as a diagnostic test,
but a few still think it not dependable. Some interesting points have
been brought out in connection with this study. In the first place,
it has been shown that the antigen helping to bring about the reaction
is not specific since alcoholic extracts from normal organs (liver,
kidney) react as well as those from syphilitic organs. With further
study it has been found that certain lipoids (lecithin) possess great
antigenic power, though not quite as great as the total extracts. Swift
(see bibliography) represents the "luetic system'' as shown in Fig. 179.
The Explaiiation and Technique of the Wassermann Test for SyphOiB. —
Five components are present in this test: Antigen, antibody, comple-
ment, hemolytic amboceptor, and blood cells.
As originally employed the antigen consisted of a watery extract of
the liver of a syphilitic foetus, and this is still preferred by some workers,
but extracts of normal liver, either waterv
or alcoholic, and also of many other
Syphilitic virus organs, especially guinea-pig hearts, are
considered by most authorities to be
equally effective. For the watery ex-
tracts the finely divided tissues are
mixed with normal salt solution in the
proportion of 1 to 4, agitated at room
temperature for twenty-four hours, cen-
trifuged and the supernatant fluid drawn
off into sterile vessels and kept in the ice-
box till needed. Alcoholic extracts are
also made from the fresh minced or crushed tissue in 96 per cent
alcohol.
The antibody is contained in the patient's serum; the blood l>eing
drawn from a vein, is allowed to coagulate or is centrifugalized, and
the serum is pipetted off and inactivated by heating in a water-bath
for one-half hour at 56®.
The complement used is that in fresh guinea-pig's serum.
The hemolytic amboceptor is obtained by inoculating an animal
with the washed blood cells of another species (as a rule, rabbits
inoculated with sheep cells). The blood cells, after washing four to
five times in salt solution to free them from serum, are suspended in
fresh salt solution and inoculated either subcutaneously or intraperitone-
ally at interv^als of five to ten days for from four to five injections; and
nine to ten days after the last inoculation the blood of the rabbit is
drawn, centrifugalized, the serum pipetted off, inactivated at 50 degrees
for one-half hour and stored in the ice-box.
The blood cells used in the test (generally those of sheep), after being
freed from serum by repeated washing in salt solution, are suspended
in fresh salt solution in proportion of 5 per cent.
Antiseptic precautions to avoid bacterial contamination are used
Complement
SPIROCHMTA AND ALLIES. 579
«
throughout, and all glassware must be thoroughly cleansed and made
neutral in reaction.
The amount of hemolytic amboceptor necessary to dissolve the
blood cells, and the optimum proportion of antigen and antibody to
fix complement must be determined by preliminary titrations in each
case, after which, as originally described by Wassermann, the
materials for the test are each diluted with salt solution in such pro-
portion that 1 c.c. will contain the desired amount, making 5 c.c. in
each tube in the completed test, the amboceptor being used in double
the hemolytic dose, and the deficiency in the contents of the control
tubes, from which one or more factors have been omitted, being supplied
by added salt solution. A specimen of normal serum and one of proved
syphilitic serum must always be examined at the same time with the
serum which is being tested for syphilis, and in addition to this a full
series of control tubes must be used in each case.
In making the test the antigen antibody and complement are meas-
ured into the tubes and these placed in the incubator for one hour at
37 degrees to allow time for the fixation of complement. The hemolytic
amboceptor and blood are then added and the tubes returned to the
incubator for two hours, after which they are shaken, and the results
read after further standing at room temperature or in ice-box.
This test has been very widely used and positive results have been
obtained in an immense number of cases. On account of the difficult
technique involved it can only be of use in the hands of experienced
workers. Positive results have been reported in a number of other
diseases as well as syphilis, but in many cases these results have not
l)een generally accepted, and in other cases the diseases showing posi-
tive reaction as yaws, leprosy, dourine, etc., have been as a rule con-
fined to the tropical countries, Or else the positive reaction has been
found only during a limited stage of the disease, as in scarlet fever or
the differential diagnosis is otherwise marked, as with tuberculosis.
The general opinion at the present time seems to be that while theoret-
ically the test is open to criticism, as not strictly specific, it is, never-
theless of great practical value in the majority of cases of syphilis.
Many investigations of the test have been proposed; but, with the
exception of the Noguchi method, these have not found extensive
acceptance. In this method human blood cells are used together
with antihuman hemolytic amboceptor from a rabbit, thus eliminating
the source of error due to the occasional presence of hemolytic ambocep-
tors for sheep blood in human serum. Antigen, antibody and guinea-
pig complement are prepared in the form of reagent papers which
remain stable if kept perfectly dry, thus avoiding many of the difficulties
of the Wassermann method. As to the comparative accuracy of the
two methods, opinions are still divided. According to a recent writer
by a combination of the two methods correct interpretations were
obtained in 98.2 per cent, of 1,400 cases.
The attempted explanation of the nature of the reaction has given
rise to much discussion. It has been found that syphilitic serum is
580 PATHOGENIC MICRO-ORGANISMS.
able to cause complement binding not only with organ extracts, but
also as noted above with numerous inert and apparently unrelated
substances, such as lecithin, cholesterine, vaseline, etc., and it is, there-
fore, no longer possible to regard the reaction as due to the action of
an antigen with its specific amboceptor in the patient's serum, and the
term amboceptor is therefore without significance in this connection,
although still used as a matter of convenience. Bordet's absorption,
therefore, seems to many a rational use, and many 'observers are
inclined toward a simpler chemical explanation of the reaction. On
the assumption of some precipitation as the underlying factor it has
been proposed to use precipitation tests of various kinds such as those
of Fornet, Porges and Meier, Dansner, Sachs, and Altmann and
Noguchi in place of the complement binding test, but the latter has been
found by most observers to be more accurate than the proposed substi-
tutes. Beyond the evidence as to the liquid character of the active
substance furnished by the fact of its solubility in alcohol nothing is
definitely known at the present time as to the true nature of the reaction
giving rise to complement binding.
Spirochetes in Framboesia tropica (Yaws). — Castellani in 1906
announced that he had found in yaws a spiral organism which he
called Spirocheta pertenuis. He determined that monkeys are sus-
ceptible to inoculations with material from yaws patients apparently
containing only this spirochete. Such material filtered is inert.
Monkeys successfully inoculated with yaws do not become immune
for syphilis, neither do those having had syphilis become immune
for yaws. Further specific characteristics between the two diseases
are brought out by means of the Bordet-Gengou reaction. The
spirochete, however, is morphologically similar to the Treponema
pallidum, and should therefore be called Treponema pertenuis. His
work has been corroborated by several observers.
Spirochetes in Tumors (see Plate II, Figs. 9 and 10). — Loewen-
thal, Borrel, and others found spirochetes in small numbers in certain
mouse tumors. Ewing and Beebe found a few in some dog tumors
and others have reported their occasional presence in both ulceratin^f
and non-ulcerating human tumors, but apparently never in sufficient
numbers to account for the tissue reaction. Gaylord, however,
found that in repeated transplants of a mouse tumor, as the inoculated
material became more virulent the number of spirochetes greatly in-
creased. Calkins studied the morphology of Gaylord's spirochete
and decided that it is a distinct species. He has also found this
species in primary as well as in transplanted tumors. It is much
shorter and thicker than the pallida, and has blunt ends. It closely
resembles the spirochetes found comparatively frequently by Tizzer
and others in apparently normal mice, though the possibility of in-
fection in these cases was not ruled out.
Spirocheta Obermeieri (Sp. recurrentis) and Allies. — ^These or-
ganisms are classed with the spirochetes as protozoa by Schaudinn,
Hartmann, Mtihlens, and others, but by Norris, Novy, and others they
SPIROCH.ETA ASD ALLIES. 581
are still placed with the bacteria. N'ovy and Knapp have made
extensive studies of Sp. Obermeieri, the cause of relapsing fever in
Europe) as well as of Sp. Dvitoni (the cause of tick fever), spirochetes
from American relapsing fever, and Sf. gallinarum (fowl spirochete)
and considers that he has demonstrated their bacterial nature and
that many, if not all, spirochetes should be placed in this group.
Spirocheta Obenneieri was first observed bv OI>ermeier in 1873 in
the blood of persons suffering from relapsing fever. It was found in
large numbers during the height of the fever, it disappeared about the
time of the crisis, and reappeared during the relapses. It was not
found in other diseases. Obermeier considered it the cause of the
disease, and his views were shown
to be correct by the production of
the disease in man and ape through
experimental inoculation.
Morphology. — The organisms are
long, slender, flexible, spiral or
wavy filaments, with pointed ends,
from 16;f to 40/i in length and from
one-<|uarter to one-third the thick-
ness of the cholera spirillum (J/i
to J/i). They stain somewhat
faintly with watery solutions of
the basic aniline dyes, better with
LoefBer's or Kuhne's methylene-
blue solutions, or with carbol-
fuchsin; best with the Ronianowsky ''iSSSf^iElo.*'' x1«S"7Af'JSiNS5;T
method or its modifications. They
are negative to Gram. Xovy has demonstrated a terminal flagellum
(Fig. 178). There are three to twelve wide, irregular spirals.
Biologic Obarsetars. — In fresh preparations from the blood the
spirochetes exhibit acitve progressive movements, accompanied by
very rapid rotation in the long axis of the spiral filaments or by undulat-
ing movements. They are found only in the blood or blood organs,
never in the secretions, and only during the fever, not in the intermis-
sions, or at most singly at the beginning of, or for a short time after, an
attack.
When kept in blood serum, or a O.fi per.cent, solution of sodium
chloride, they continue to exhibit active movements for a considerable
time. They may be preserved alive and active for many days in .sealed
tubes. They are killed quickly at ()0° C. but they remain alive for
some time at 0° C. I'nsuecessful efforts to cultivate them in artificial
culture media have been made from time to time. Koch has observed
an increase in the length of the sprilla and the formation of a tangled
mass of filaments. N'ovy has finally succeeded in cultivating them in
celloidin capsules placed in the peritoneum of rats.
Patbogenosls. — In man, whether the disease is acquired naturally or
by artificial inoculation, the organism causes the following symptoms:
582 PATHOGENIC MICRO-ORGANISMS.
After a short period of incubation the temperature rises rapidlv, re-
mains high for five to seven days, and then returns to normal bv crisis.
About seven days later there is another sudden rise of temperature,
but this time the crisis occurs sooner. A second or third relapse may
occur. The organisms increase in numbers rapidly in the blood from
the beginning of the fever, large numbers often being found in even.-
microscopic field. They began to disappear a short time before the
crisis, and immediately after the crisis it is practically impossible to find
them in the circulating blood. The mortality varies in different
epidemics from 2 to 10 per cent. When monkeys are inoculated
with human blood containing the
spirilla, they become sick about three
and a half days later, but show only
the initial febrile attack or, at the
most, an occasional short relap^.
The organisms are found to have the
same relation to the pyrexial periods
as in man. Blood from one animal
taken during the fever induces a
similar febrile paroxysm when inoc-
ulated into another animal.
Metchnikaif showed that during the
intermissions when the spirilla disap-
spinchna obtrmnrri blood .moBt. pearcd from the Circulating blood they
Md Nkmiumir" *"""■ '*""■"''""'" accumulated in the spleen and were
ingested in large numbers by certain
phagocytes and finally were destroyed.
According to Lamb, a certain amount of immunity is conferred upon
monkeys (Macacua radialus) soon after an attack, but it di.^appears
quickly. If the serum is removed during this time it is found to have
some protective action when mixed with the blood containing spirilla,
and also to cause agglutination of the organisms. Novy (1906)
showed that a powerful specific germicidal body exists in the blood of
rats during and after recovery, notably in the blood of hyperimmunized
rats. An immunizing body probably distinct from this is also present.
He also showed that passive immunity can be imparted by injections
of rec'overcd or hyperimmunized blood, that both active and passive
immunity may last for months, and that the serum has both a preven-
tive and a curative action.
Infection probably occurs through the bite of blood-sucking
insects.
Splrocheta Dattoni.— The organism .shown by Dutton (19(l.i) to
be the cause of African tick fever is very similar morphologically to
S, Obermeieri, but Novy Franckel, and others have shown slight differ-
ences which make them believe that it is another variety, if not another
.species of this group. Dutton demonstrated that this organism can
be transferred to monkeys by the bites of young ticks {OmUkodonit
mouhata) at their first feed after hatching from infected parents. He
SPIROCH^TA AND ALLIES. 583
accidentally demonstrated the fact that the disease can be inoculated
into human beings through a cut surface, for after a wound received
at autopsy he developed the disease which eventually caused his death.
Spirocheta Oarteri. — This spirochete was described by Carter in
1877 as causing relapsing fever in Bombay. Monkeys were inoculated
by Carter successfully with the human blood containing this spirochete
Spirochetes from Relapsing Fever in America. — Recently Darling
has reported a study of the relapsing fever of Panama. He isolated
the organisms in two cases and studied their characteristics. He
finds they agree with those reported by Carlisle, Norris, and Novy
for the organisms isolated by Norris, but they can only be diflfer-
entiated from the other relapsing fever spirochetes by animal inocu-
lations and by the disease in humans. Moreover, he finds that in all
probability a polyvalent serum may be necessary for cure, since the
serum from one strain did not protect against the other strain.
Bibliography.
BertarelH. Centralbl. f. Bakt., 1906-1907, XLI, p. 320, p. 639; XLIII, p.
167, p. 238.
Ccukins. Journ. of Infect. Diseases, 1907, IV.
CasUllani. Journ. Hygiene, 1907, VII, 558.
Darling. Arch, of Int. Med., 1909, IV, 150.
Ewing. N. Y. State Journ. of Med., 1907, VII, 177. (With good bibliography.)
FUxner. Medical News, 1905, LXXXVII, 1105.
Muhlens. Zeitschr. f. Hygiene, etc., 1907, VII, 405.
Noguchi. The Journ. of Exp. Med., 1909, XI, 84 and 392.
NorriSf Papvenheimer and Floumoy. Journ. of Infect. Diseases, 1906, p. 527.
Novy and iCnapp. Journ. of Infect. Diseases, 1906, III, 291.
Pemn. Arch, fttr Protist., 1906, VII, 131.
Schaitdinn u. Hoffmann. Arbeit a. d. Kaiserl. Gesundh., 1905, XXII.
Schereschewsky. Centralbl. f. Bakt.. etc. Orig. Abt. I, 1908, XLV, 91.
Swift. The Journ. of Cutaneous Dis., 1909, July, and the Arch, of Int. Med.,
1909, IV, 376 and 494.
CHAPTER XLIV,
BODO. POLYMASTIGIDA. CILIATA. SPOROZOA.
BODO LAOBRTA (GRASBI).
Bodo lacerttt is frequently found in the intestinal contents of most
of the higher animals, hence it is easily obtained for class study. A
species of the Bodo has been observed in human urine (Bodo urin-
carius), but it is probably a harmless invader.
It is lancet- or wedge-shaped, the posterior part of the body being
turned a half to a whole spiral on itself. It possesses two character-
istic fla^ella, equal in thickness but unequal in length. In motion the
<L0Ul7Tll! °Afl"f*v. Prewniekl" f ram ^kalt "and'' ttarUnann.) ™' '""
longer one is directed forward, while the shorter is carried backward,
functioning as a rudder, or a towing flagellum. Both fiagella spring
from basal granules which are well demonstrated by the iron hfema-
toxylin stain. They are situated in the extreme anterior part of the
body and are attached to the nucleus by a delicate fibril (Fig, 1.S2,
b, c). The movement of the organism is characteristic, it consists in
584
BODO. POLYMASTIGIDA. CILIATA. SPOROZOA. 585
a rapid irregular swimming in various directions with the anterior
flagellum moving from side to side. The body itself shows a slightly
sinuous motion.
There are two types of nuclei seen. First, the typical vesicular
nucleus most frequently seen among the flagellates. This is round
and has a definite membrane about which chromatin is arranged in
irregular masses. In the center, or eccentrically placed, is a compact
karyosome. Iron hsematoxylin preparations bring out an achromatic
network between the chromatin masses and the karyosome. In the
living condition the nucleus appears as a greenish glistening refractive
vesicle (Fig. 182, a and 6).
The second type of nuclear apparatus is seen in smaller organisms.
This is a similar nucleus except that it is smaller and more compact;
posterior to this is another nuclear-like body, varying much in shape
and arrangement of chromatin (Fig. 182, c). This is the sexual
chromidia.
The cytoplasm appears in iron hflematoxylin stained specimens as
finely reticular. It contains many deeply stained granules. There is
no mouth opening. Food is taken in by osmosis.
In propagation, the two types just described develop diflFerently.
The first or ordinary type forms round division cysts. The flagella
disappear and a delicate cyst membrane is formed. The increase in
the size of the nucleus and the subsequent division may be followed
in life. It lasts about twenty minutes. After a single or, more seldom,
a double division of the cell, the daughter cells, while still within the
cyst, form their flagella, become very motile, finally break the cyst wall
and swim out.
The second type increases, in the free living condition by longi-
tudinal division. The basal granules divide, the principal nucleus
divides by mitotic division, the chromidia by amitosis. This all can
be seen in haematoxylin preparations. Sexual division in this species
occurs in cysts by autogamy. It is not easily followed in life be-
cause of the high refraction of the cyst. The changes must therefore
be studied in specimens stained with iron hsematoxylin.
They are shortly as follows: The nucleus becomes larger and about
its membrane appear small spheres of chromatin which finally leave
the nucleus and gather together, forming the so-called chromidial or
sexual nucleus, while the original or somatic nucleus gradually degene-
rates. The new nucleus divides amitotically into two daughter nuclei,
from these two smaller parts are then separated, as reduction nuclei,
which also degenerate. The remaining parts of the two nuclei increase
in size and then fuse to form- a new nucleus. The organism may then
leave the cvst or the cvst may become a lasting cvst and serve to
infect a new host.
Besides this method of fructification by autogamy in a cyst, is seen,
though seldom, a copulation between two individuals of different sizes
which afterward become encvsted and divide into two to sixteen
daughter flagellates.
LJ
586 PATHOGENIC MICRO-ORGA.VISMS.
FOLTHABTiaiDA.
The order polymastigida consists of flagellates having several
flagella projecting from different parts of the body. The majority
of the forms known are parasitic in certain lish.
Trichomonas Vaginaiis^ — Donn^ in 1837 described a form which he
found in the human vagina, and which he therefore called Tricho-
monas vaginalis. It has been found by other obser^■ers to be a fre-
quent habitant of .the vagina at all ages. It has also been found
a few times in the acid urine of males.' The mode of infection of
the female- is unknown. The body of the parasite at rest is pear-
shaped, but during action its amceboid movements cause it to as.sunie
various shapes. The size varies from l2/i to 25fx long and Hp to
15/< wide. The protoplasm is finely granular, excepting for two
rows of larger granules which begin on either side of the nucleus and
converge posteriorly. From the anterior part project three to four
flagella, which seem to begin at a basal thickening near to^ot con-
nected with, the more or less oval, indistinctly vesicular nucleus.
From the origin of the flagella an undulating membrane extends
backward. The body also seems to possess a certain linear structure
connected with the membrane. Contractile vacuoles have not been
seen.
Trichomoiuui bominis Davaine. — This form, found frequendy in the
human alimentary canal, is very similar to the Trickomonae vaginalis.
but it is smaller and. more pear-shaped. It has I>een found often in
acute diarrhoeas, hut no causal relation between it and the pathologic
process has been shown.
A similar form has been seen a few times in lung gangrene, as-
piration pneumonia, and bronchiectases.
Lamblia int«8tinalis (Lunibl, 1859), a flagellate belonging to this
group, parasitic in the small intestines of mice, rats, rabbits, dogs,
cats, and sheep, has also l>een found occasionally in the human in-
' It hns been found by us in the slightly acid urine of a colored woman, ajce 4 j,
sufferioK from acule nephritis. .None were found in the vagina in this case.
BODO. POLYMASTIGIDA. CILIATA. SPOROZOA. 587
testines. It is beet-shaped, bilaterally symmetrical, 10/t to 2I/i long
and 5/1 to \2fi wide, possessing flagella 9/( to 14ju long. Anteriorly,
this species has a characteristic concavity, the rim of which seems to
W contractile, forming a sucking apparatus. The eight flagella of
the organism are arranged in pairs: one anteriorly, two laterally,
and one posteriorly. The nucleus is situated anteriorly and has a
central constriction. The protoplasm of the body is thick and hya-
line. Contractile vacuoles have not been seen. Schaudinn has
recendy observed encystment, copulation, and complicated nuclear
change.^ in this organism.
Infection follows the ingestion of the cysts with unclean food.
The parasites fasten themselves to the free surfaces of the epithelial
cells by their sucking apparatus, but seem to exert no harmful influ-
ence on their hosts. They have been found most frequently in poor
children who play often in dirt containing the cysts. Repeated
small doses of calomel will cause their disappearance from the fwces.
OILUTA.
The Ciliata (Fig. 185) belong to the most complex of the pro-
tozoa. They possess a definite entoplasm containing nuclei and food
vacuoles, and a definite ectoplasm containing basal granules from
which arise the cilia which give the group its name. They have
BalaMidiam coti: I. 2, (Ufea of divuiou; 3. conjufatian. (After Leucknrl.)
organoid structures which receive the food, some have definite
mouth openings, indeed, and definite places for excreting wa.sie prod-
ucts. The food vacuoles may contain acid or alkaline digestive
products. The nuclear material is differentiated into two forms, a
large macronucleus and a much smaller micronucleus. The function
of the macronucleus is supposed to be vegetative, and that of the
micronucleus reproductive. The macronucleus varies in size and
shape and is completely filled with an alveolar chromatin. The micro-
nucleus also varies in size and shape, but unless in reproductive pliases
588 PATHOGENIC MICRO-ORGANISMS.
is generally vesicular in structure, with the chromatin heaped in one
mass. Division of the nuclei takes place by mitosis in the case of
the micronuclei, and by amitosis, as a rule, in the case of the macro-
nuclei. Under conditions unfavorable for growth the ciliata may
encyst.
Conjugation is necessary to the life activity of these organisms.
The phenomena of conjugation in the ciliata has been well worked
out. The micronuclei play the most important part, whereas the
macronuclei simply break up and disappear in the protoplasm.
According to the arrangement of the cilia, the ciliata are divided
into the four orders given in the general classification. Among these,
the second, the order of the Heterotricha, interests us. In the Hetero-
tricha the cilia are uniform over most of the body, while a specialized
set fused into a series of firm vibratory plates are found about the
mouth.
Only one genus, Balantidium, has been observed in man.
Balantidium coli (Malmst, 1857). The body of this infusorium
is egg-shaped, with a funnel-shaped mouth opening. The surface
of the body is covered with a pellicula, under which is a distinct
ectoplasmatic sheath containing rows of basal granules from which
the short, fine cilia arise.
The cloudy entoplasm contains fat and starch granules and may
contain many red blood cells and other food particles from the host.
Two contractile vacuoles have been seen. Posteriorly there is a
small prominence marking the place where excreta are expelled. The
chromatic macronucleus is bean-shaped, and the vesicular micronu-
cleus is nearly spherical.
Division is transverse, the macronucleus dividing by simple constric-
tion and the micronucleus by mitosis. Conjugation has been observed.
Spherical cysts surrounded by a thick membrane are formed.
Balantidium coli has been found in the large intestines of hu-
man beings and of swine — probably two distinct varieties. The
variety occurring in human beings has been found in about 60 cases,
principally in Sweden, but also in Russia, Scandinavia, Finland,
China, Italy, Germany, and the United States. Most of these cases
were suffering from severe chronic intestinal catarrh, often accom-
panied by bloody diarrhoea. A number of observers (Strong, Brooks,
and others) think the balantidium the primary cause of the catarrh,
while others believe it to be a harmless inhabitant of the intestines, or
at least only a secondary excitant (Opie, Mahnsten, Doflein,and others U
Schaudinn has described two additional species of balantidium
found in the human intestines, which he has called, respectively.
Balantidium minutum and Nyciooiherus jaba, probably both non-
pathogenic.
THE SPOBOZOA.
The Sporozoa are a group of exclusively parasitic protozoa of very
widespread occurrence, living in the cells, tissues, and cavities of animals
BODO, POLYMASTIGIDA. CI LI AT A. SPOROZOA. 589
of every class. Generally they are harmless, but some varieties may
produce pathologic changes and even fatal diseases severely epidemic.
As their name indicates, they are all characterized by reproduction
through spore formation, but they exhibit the utmost diversity of
structural and developmental characteristics. As a rule, each species
is parasitic on one kind of tissue of a particular species of host. They
are generally taken into the system in the spore stage either (1) with
the food of the host, (2) by the bites of insects, or (3) by inhalation.
ITie spore membranes are dissolved by the fluids of the host, and thus
one or more germs or sporozoites are set free to bore into the special
cells of the host. Here they grow, some remaining permanently in-
tracellular, others only in the young stages. The latter either pass
different phases of their more or less complicated life history in differ-
ent parts of the body of one and the same host or they pass some
phases of their life cycle in the cells of an intermediate host.
The sporozoa vary widely in size as well as in other characteristics.
From the smallest, several of which can be contained in a single blood
cell, there are all gradations in size up to those that may be seen by
the naked eye {Porospora gigantea, 16 mm.).
Besides being characterized by the power to produce more or less
resisting spores, the sporozoa are also characterized by the fact that
as a class they possess none of the special organs found in other pro-
toza for ingesting or digesting solids. Many develop flagella during
sexual phases or show amoeboid movement during certain stages of
their life cycle, but the flagella and pseudopodia are organs of loco-
motion and not of nutrition. Food vacuoles or contractile vacuoles
have not been found.
The life cycle of a tvpical sporozoan is represented after Schaudinn
in Fig. 186.
A somewhat similar cycle may be followed in the study of the Coc-
cidium cuniculi of the rabbit, a description of which is given below.
The other forms in this group, which are parasitic in man, or which
are of some medical interest, are, besides a number of not fully studied
Coccidia, Nosema, Sarcocystisy Babesia and Plasmodium Tnalaricp and
its allies.
OOOOIDIUM OUNIOULI (RIVOLTA, 1678).
The Coccidium cuniculi is a sporozoan parasite of the rabbit. Young
rabbits are especially susceptible, and extensive epidemics may occur
in breeding houses.
Material and Methods for Study. — Rabbits infected with Coccidium
cuniculi are often found, and the whole course of the infection may be fol-
lowed with more or less ease.
A certain amount of development may be watched in hanging drops of salt
solution emulsions. Sections and smears are prepared as described on p. 537.
The cysts are stained with difficulty. It is recommended that a thin solu-
tion of Delafield*s or Grenacher's hflematoxylin be used for twenty-four
hours followed by eosin. Heidenhain's iron hcematoxylin stain (p. 537) fol-
lowed by Bordeaux red is especially good for sections.
590 PATHOGENIC MICRO-ORGANISMS.
Description of Fig. 186. (After Schaudinn.)
The life cycle of Eimeria schvbergi. I to VII represent the asexual reproductioii or achisosooj:.
commencing with infection of an epithelial cell by a merosoite or a sporoioite; the meroaoite after
stage VII may start again at stage //, as indicated by the arrows, or it may go on to the fonaatun
of gametocytes {IX to XII). IX to XIV represent the sexual generation, the line of dereftopoMsit
becoming split into two lines — male o^ and female 9 — culminating in the highly differentiated
gametes, which conjugate and become again a single line, shown in XI V and X V, The zysote thai
formed goes on to the production of spores, XVI to XX. I to IV represent epithelial cells showmg
penetration of a merozoite or a sporoioite and its change into a schizont. F, the nudens of thr
schizont dividing. VI, numerous daughter-nuclei in the schizont. VII, segmentation of the acha-
ont into numerous merozoites, about a central mass of residual protoplasm, which in this figuR
is hidden by the merozoites. VIII, merozoite passing to reinfect host cell and repeat the procem
of schizogony. IX, X, merozoites to be differentiated into male and feihale gametocytes. XU,
and XI la, the two gametocytes within a host cell; the microgametocyte (,/^) has finegrmnolatioM:
the macrogametocyte (9) has coarse granulations. Xlb, an immature female gametocyte witha
a host cell. XIc, a female gametocyte undeisoing maturation, still in the host celL XI It, mature
mocrogamete, freed from the host cell, and sending a cone of reception toward an. approachaif
microgamete. Xllh, a full-grown microgametocyi« within a host oelL In XIIc Uke nuekeus of tbe
microgametocyte has divided up to form a great number of daughter-nuclei. In Xlld the
nuclei of the last stage have become microgametes, each with two flagella. Xtlt, reptv-
sents the free microgametes, swimming to find a macrogamete. XIV, the zygote (feitiiLsad
macrogamete), surrounded by a tough membrane or o6cyst, which allows no more inicroc>UBetet
to enter, and containing the female chromatin, which is taking the form of a spindle, and the male
chromatin in a compact lump. XV, the chromatin from these two sources united and no longer
distinguishable as male and female. XVI, the nucleus of the zygote dividing. In XVII four
daughter-nuclei are formed — the nuclei of tiie sporoblasts. In XVIII the four sporoblasts beeoose
distinct, leaving a small quantity of residual protoplasm; eafch sporoblast has formed a membrane.
the sporocyst. In XIX within each sporocyst two sporosoites have been formed about a spor^
residuum. In XX, the sporozoites, becoming free by bursting the sporocysts, pan out throoi^
an aperture, in the wall of the odcyst, and are ready to enter the epitiielial cells of the host. (Fi
Lang.)
BODO. FOLYMASTJGIDA. CILIATA. SPOROZOA. 591
592
PATHOGENIC MICRO-ORGAMSMS.
The symptoms of the disease are fever, diarrhcea. yellowish
mucous discharge from the nose and mouth, and progressive wasting.
The liver is much enlarged and shows throughout its substance vari-
ously sized gray-white tubercles, generally surrounded by a capsule, and
containing a slimy mass of degenerated host cells, in which are em-
bedded the parasites. The parasites are also found in the f»ces and in
the epithelial cells of the intestines, gall-ducts, and liver. The acute
stage of the disease lasts about three weeks. The contents of the
coccidial tumors in animals that have withstood the infection may later
be emptied, leaving only a mass of cicatricial tissue. In such animak
Btioa in Coccufiun eunicuJi from the li
celli of the s&lMuots (the i "
'*ie protopU --'-■-■
L spberioat Vonn; /, i
implete apona in
- -nd ■ ^n qui
(After B^bii
the oocysts may remain for a long time in the gall-bladder and intes-
tines, and by passing out gradually with the fteces may provide a source
of infection for other animals. The infection is carried by food soiled
with cyst-containing fseces. The cysts pass with the food into the
stomach, where the cyst wall and the spore sac are destroyed and the
sporozoites are set free. The motile sporozoites pass through the
ductus choiedochus into the liver, some probably passing into the
intestines and infecting the cells directly, a later infection of the in-
testines occurring from forms developed in the liver. The organism
develops within the epithelial cells of the liver and gall-ducts until
the cells are finally broken down and tissue cysts are formed, within
which, after more or less complicated changes, cysts of the parasite
are again formed.
A few cases of human infection of the liver with the CoccidiuiK
cunicndi have been reported. The Coccidium hominis Rivolta, found
a few times in the human intestines, as well as similar coccidia, found
in the intestines of lower animals, may belong to the same species.
. biffeminium (Stiles) is found in the f«ces of dogs, cats.
i possibly human beings. The organism is characterized
ion of the odcyst into two united cysts, containing four
e size is 8/( to i6/i. The life cycle is not well known.
idium kinealiji is the name given by Minchin and Fan-
) to a probable sporozoan found in the nasal mucous
BODO. FOLYAtASTIGIDA. CILIATA. SPOROZOA. 593
nieiiibrane of certain cases from India that were troubled with hemor-
rhagic nasal polyps. Xais reported four similar cases and Beattie
another in 19Ut>.
HTXOSFORIDU.
The Myxosporidla belong to one of the most populous and abundant
groups of the sporozoa, showing great structural variation as well as
divergence in mode of hfe. Nevertheless the members have, as a
group, the following well-marked characteristics: The trophozoite is
ameboid; spore formation begins at an early period and proceeds con-
tinuously during the growth of the trophozoite; the spores are pro-
duced endogenously — i. e., within the protoplasm of the trophozoite,
and each spore always possesses one or more very distinctive struc-
tures, "the polar capsules" (Fig 188, c, d).
The myxosporidia are habitants of fishes, reptiles, arthropods,
and some other classes of animals. They infest especially arthropods,
causing often most virulent epidemics. The most interesting mem-
ber of this group is Nonema bombi/cis, the cause of silkworm disease
(P^hrine), The organism forms many small spores each with one
polar capsule. The spores which are carried by the food into the in-
testinal canal of the caterpillar, pass through the walls of the intes-
tines, and infect all organs. Spores found in the ovary may be carried
over to the newly hatched silkworms, thus causing a further dissemi-
nation of the disease.
The other member of this group, of interest here, is Nosetna lophii
DoHein. Its interest lies in the fact that it has been found to in-
fect only the ganglion cells of the sea-devil, thus apparently resem-
bling in its parasitic nature the organism causing hydrophobia.
594 PA THOGEXIC MICRO-ORGANISMS.
8ABC0SP0KIDIA'
This order is very little knonn but, considering the fact that through eating
uncooked infected meat, it may be found in man, though rarely. Us chid
characteriatica ahould be not«d here.
The Sarcoaporidia are parasites of the striped muscles or connective tiaeue
of some of the warm-blooded vertebratea (\arioua birda and mammals) . They
are found in the adult state in elongated sacs known as "Rainev's" or
'■ Miescher's Tubes." (fig. 189.)
The trophozoite la a motionless elongated body, limit«d by a cuticle grow-
ing into a complicated structure. Spore-formation begina at an early stage
and proceeds during the growth of the trophozoite (Nmsporidia) which may
become very large. The spores, which are many, are minute sickle-shaped
»ot
Sanocvait miackrri. a. hhibLI cella from a «U crDup. b. hwaeiiina at Uie pnlopbn froB
the cell wall. c. d. sickle-shaped bodies (sporaioit«) formed from the nniM celk. (From Wwe-
or spindle-shaped mononucleate bodies with a delicate envelope and at one
pole an oval striated body which represents the polar capsule found in the
myxosporidia. (Fig. 190.)
In some cases the cyst wall calcifies and the contenta of the cyst degen-
erate, with apparently no harm to the host; in other cases the cysts burst
and their contents spread into the surrounding tissue, producing abscesst^
and tumors as with many myxosporidia and sometimes causing the death <rf
the host.
Thp avmntoma of sarcosporidioais in the pig are paralysis of the hind
a skin eruption, and general systemic symptoms, as iitcreaaed
and pulse.
specially, the disease often causes fatal epidemics. In the mouse.
luris is a deadly parasite. Theobald Smith showed that gray and
may becoine infected with Sar. muris by eating infected mouse
ling motile sporozoiles.
BODO. POLYMASTIGIDA. CI LI AT A. SPOROZOA. 595
Laveran and Mesnil claim to Jiave extracted a toxin (Sarcocystin) by
means of glycerin or salt solution, which they have found extremely toxic
for experimental animals. (0.0001 gm. kills 1 kgm. of rabbit.) The dried
and powdered extracts are also virulent. These extracts will remain virulent
for a long time in the ice-box, but will not withstand heating above 60° for
any time.
Darling (1909) describes a case of human sarcosporidiosis occurring in
Panama, from which he studied the organism and came to the conclusion that
it was probably a different species from the one already described as occurring
in man. He gives a good historical review. Later he decides that morpho-
logically his human sarcosporidia are identical with Sarcocystis muris.
BiBUOGRAPHY.
Darling. The Archives of Internal Medicine, 1909, and The Journ. of Exp.
Med., 1910, XII, 19.
Laveran and Mesnil, 1899, Compt. rend. see. Biol.
Th. Smith. Journ. Exp. Med., 1901, VI, 1, and Journ. of Med. Res., 1905,
XIII, 429.
CHAPTER XLV.
THE MALARIAL ORGANISMS. BABESIA.
Introduction. — The malarial organisms are a group of protozoan
parasites found to be the cause of a definite group of specific infec-
tious fevers in man, called by the somewhat misleading term malaria,
a term which signifies "bad air."
They are classed as sporozoa, order hsemosporidia, and are consid-
ered by the majority of observers as forming one genus, plasmodium.
Hartmann thinks that this group should be placed in the new order, hinu-
cleata, which he has created under the flagellata. He considers that they
have lost by their endo-globular parasitism most of the characteristics of this
order, but that in a few stages, he points out, they still show the flagellar and
binucleate phases, two of the most important characteristics of this order.
So far as is known, the only means by which the malarial organisms
are transmitted to man is mosquitoes of the genus anophdes. A
part of the life cycle of the organisms is carried on in the body of these
mosquitoes. The parasites develop in man within the red blood
corpuscles which they finally destroy, thus producing the anaemia and
pigment granules peculiar to malarial fevers.
Historical Note. — The fevers caused by these organisms were recognised
and studied as early as 400 B. C, but it was not until 1880 that the true
nature of the dancing pigment which had been observed long before was
determined. At that time Laveran announced that he had discovered a
parasite in the blood which he claimed was the cause of the disease and he
p\iblished a good description of several of the stages in the Ufe of the organ-
ism. The public remained at first almost entirely uncon\'inced of the para-
sitic nature of these bodies. Many still believed that the bacillus described
shortly before by Klebs was the cause of the fevers. Among others, Marchia-
fava and Celli in Italy believed that Laveran's organisms represented areas of
degeneration within the red blood cells, though Laveran himself demonstrated
the organisms to them. When they began, however, to study the fresh tissue
themselves they changed their opinion and later they published a number
of valuable contributions on this subject. They gave the organism described
by them the inappropriate name, Plasmodium malarup. Laveran's researches
were later confirmed by many other observers, and, though not all of Koch's
laws have been verified in this case, the fact that a protozoan, the pla.*-
modium, causes malaria is accepted as proved.
In 1885 (tolgi showed that quartan fever depends upon a specific form of
the parasite, and that the malarial paroxysm always coincides with the
sporulation or segmentation of a group of parasites. Thus, in a single in-
fection with the quartan variety a paroxysm occurs every fourth day, with
a triple infection on successive days, segmentation with its accompan>nng
paroxysm occurs daily. Golgi and others soon showed that tertian fever and
aestivo-autumnal fevers were each due to a distinct variety of the Plas-
modium. These varieties are at present regarded by some as distinct genera,
596
THE MALARIAL ORGANISMS, BABESIA, ^ 597
by others as species, belonging to a single genus. Councilman first called
attention to the diagnostic value of the different forms which appear in the
blood.
Though it had been thought for nearly 2000 years that malaria is trans-
mitted by insects, the question was not definitely settled until Ross in 1896
clearly demonstrated that the haematozoa of birds were transmitted by a
certain species of mosquito. These investigations of Ross were soon con-
firmed by Grassi, Bignami, and others. MacCallum's observations on the
sexual forms of halteridium were a great advance, and Bignami, Grassi, and
others soon proved that all varieties of malarial fevers are transmitted from
man to man by mosquitoes of the genus AnopheUs. (Jrassi worked out the
complete hfe cycle of the pernicious type (sestivo-autumnal), while Schaudinn
(1901) did the same for the tertian form.
Materials and Methods for Study. — If a case of malaria is at hand the
organism may be examined aHve under the microscope by allowing a cover-
glass to drop gently upon a drop of fresh blood placed upon a clean glass
slide. For finer differential points, however, smears should be made. The
making of these smears is a simple matter. There are the cover-glass and
the slide methods, both of which have their peculiar advantages. To make
a cover-glass preparation, two square, very thin (hence flexible) cover-glasses
are cleaned. Holding one with tnumb and index fingers by opposite corners,
the tip of a drop of blood obtained by needle puncture of finger or lobe of
ear is made to touch the centre of the cover-glass, and the second clean
cover-glass held similarly is allowed to fall upon the first one in such a manner
that the corners do not coincide. The blood droplet spreads by capillarity
into a thin film, which is a sign to pull the two covers apart in the plane in
which they lie; good results depend upon cleanliness, rapidity, and success
in sliding the two covers apart.
A simpler way is to polish two slides. The tip of the exuded blood drop is
made to touch one slide near one end and the edge of the second slide, held at
an acute angle to the first one, is made to bisect the drop, which will spread
at the point of contact by capillarity across the slide. Upon puUing the
second or spreading slide over the first slide, never changing the angle and
applying gentle pressure, a thin layer of blood suitable for examination will
be formed. A slide made in this manner should be dried immediately by
agitation in the air. It may then be fixed and stained in various ways. The
following staining methods may be recommended:
Jenner's Stain. — Clear pictures of parasites, which, however, show no
chromatin ; hence unsuitable for study of finer differential points.
Nocht -Romano wsky Method. — Very suitable, but retjuires accurate mix-
ture of several fluids just before using, which afterward have to be thrown
awav.
Wright's Stain. — Practically identical with Goldhorn's one-solution stain
(vide infra) y but less rapid; powerful chromatin stain and general blood stain.
Polychrome Methylene Blue (Ooldhom).— To prepare the stain dissolve
1 gram lithium carbonate in 200 c.c. clean water and add 1 gram methylene
blue. Shake and dissolve. Pour into porcelain dish over water-bath, stirring
frequently until blue color changes to a rich purple. Run through cotton in
funnel; make up to 200 c.c. To 100 c.c. add 5 per cent, acetic acid until a
faint pink is just visible on litmus-paper above level of point discolored by
the dye. Now add the remaining 100 c.c. of dye and allow to stand in open
dish for forty-eight hours. Run once more through cotton into clean bottle.
It is not necessary to use distilled water, and satisfactory results are ob-
tained with all the different forms of methylene blue tried. B-X Gruebler
is preferable.
Fix the smear by immersion in commercial wood alcohol for fifteen to
thirty seconds; wash well and stain for about ten to fifteen seconds in poly
chrome; wash and stain for from fifteen seconds to sixty in *u per cent"
598 , PATHOGESIC MICRO-ORGASISMS.
aqueous eosin. Waah again in water and dry in air without beat. Body of
parasites blue; chromatin is red to purple.
Results may be varied by using polychrome or eosin for differeot lengths
of time. Admirable preparations may be obtuned, even when there is pre-
cipitation, by just rinsing the smear a little in 50 per cent, ethyl atrohol.
This will remove any precipitation.
The simplest method- of staining the parasite is probably the following,
recommended by Goldhorn for the staining of mast -cells: Saturate wood
alcohol with methylene blue. Pour on dry smear for live to ten seconds and
wash in water. Parasite blue.
Ooldhom's One-Bolntioii Stftin.— To Goldhom's polychrome methylene-
blue {vide supra) add weak, watery (i to A per cent.) eosin until the filtrate
is of a pale blue color; the exact amount of eosin will depend upon the degree
of alkalinity of the polychrome and upon the amount of unaltered methrlene
blue in the polychrome.
The precipitate is washed with water and dried without heat and pro-
tected from dust. When absolutely dry it is dissolved in commercial wood
alcohol, making a 1 to 2 per cent, solution.
The smear is dried without heat and held for a second or two io the
dye. It is then dipped slowly into a vessel with clean water, film sidr dtnr*:
it should not be plunged into the water. The staining depends upon the
interaction of the water with the film of dye adhering to the blood. Hold
preparation in the water for a few seconds, then move it about for a moment,
and rinse in clear water; clean lower side of the sUde, as precipitation will
have taken place here; hence, do not introduce into water with film side up.
Dipping the preparation for a moment into 30 per cent, ethyl alcohol re-
moves smudges and precipitate.
Oiemu's Method (see p. 624) gives excellent results.
Robs' Hsthod of ExaminiiiK a Larg« Qaantit; of BhUrUl Blood in Oafl
Film. — A large drop of blood (about 20 c.mm.) is placed on a glass slide
and is slightly spread over an area which can be covered by an ordinary'
cover-glass. This is allowed to dry in the air or it is warmed over a flame
without heating it more than enough to fix the hemoglobin. The drv film
is then covered with an aqueous solution of eosin (10 per cent.) and aflowed
to remain about fifteen minutes. This is then gently washed off and a weak
alkaline methylene-blue solution is run over the nim and left for a few seconds,
when the preparation is again gently washed. After drying it is ready
for examination.
The Parasite. —Three distinct species of amiarial organisttis in man
have been described: Plasmodium vivax (causing tertian fever),
Plasmodium malaricE (causing quartan fever), and Plasmodium
falciparum (also known as Laverania malaricE and causing lestivo-
autumnal fever). The last species has been divided by certain
authors into two varieties, a quotidian and a tertian. On the op-
posite page is a table of the chief diflerences between these forms.
Each of these species undergoes the two phases of development
already alluded to, one within the red blood cells of human beings
^iKo <.<;^v,,<.i r.i.,,spj; the other within the digestive tract of the mosquito
se). The form changes which the parasite undergoes
whole cycle in both hosts are shown on Plate III, for
isite, which may be considered a typw of all. Briefiy,
icribed as follows.
Cycle (Schizogony) Occturiiig in the Blood of Man.—
n is often difficult to Hnd in fresh blood. A pale urea
THE MALARIAL ORGANISMS. BABESIA.
599
<
Is
s
a
2
o
z
o
i-3
^
aiS
^»^
§8
Is!
o
us
s
-
«
<
a
J
o .
^
■"2"?
90
^•c
S5
i^
90
»«
<
a:
O
<
<
<
o
. u
u
s
z
u
u
u
OS
30
u
z
as
u
o
s
X
u
a
flS
3
I
S *
^i
2 fl g b
'•J 2 fl 2 ©•
3
S o « © ©
■c * .S_^0--
« g^S^.a-33
c
e
3
O
00
** P S i L
^ <> C c o
^ 08 - fl Z ti
©
OS
?o£ails
cS u >
CO
3
i
II? *5S|
II Nil
'c^ b
§
« -I obi?
U3 si C.S c ^x
©
©
>
©
a. « O
2< 4-> U ©
© 3
o
M
© e
aJ
o
3
i
1
9
3
O
<
©«■
^^ © oe a o
111
© g h g
;5 ©
a
3
o
■«r
a
O ©
• © I ^
E cE 1^
J= 3 $ 3 ;s
CI o
e
3
O
J3
00
^ C ©
ki ©
c B o © Sr
a>»g2«
o
Z
> JtN (tcS,
*j on .
3 « ©
«• '^ s ^
if
si
8^
"ax |''**i. ©
^S o > 2 5 ■©
*•*♦* S Sx
^
8! .
>
p.i: o a.2 " >^
^. c « > c © — w
c
a
•J
M
600 PA THOGENIC MICRO-ORGA NISMS.
is seen on an otherwise unaltered red corpuscle, situated usually
eccentrically, about one-tenth the size of the red corpuscle or about
one-fourth its diameter, when at rest presenting a rounded appearance,
but usually actively amoeboid, throwing out distinct pseudopodia,
never remaining long in the same focal plane, frequently dipping, so to
speak, into the substance of the corpuscle. It is often called the hyaline
form because it is free from pigment, but it is not hyaline in the proper
sense of the term. It is also called the ring form, because of its resem-
blance to a ring in stained preparations; but it is never a true ring.
The ring appearance is produced by the formation of a large food
vacuole. The young organism passes from the surface to the interior
of the red corpuscles and grows there at the latter's expense,
The forms intermediate between this and the segmentation stage ap-
pear in the fresh blood simply as larger parasites, which are readily
found on account of the reddish-brown pigment granules that they
contain. These granules begin to appear several* hours after the or*
ganism has infected the red blood cell. At this time the organism is
usually actively amoeboid and the granules have a lively dancing
motion, due to protoplasmic currents in the parasite. The infected
corpuscle is swollen and paler, in forms other than tertian the infected
red blood cells are smaller than normal
When the parasite has approached nearly to its full growth, it
occupies the greater portion of the corpuscle, which is now more
difficult to make out. The pigment is still more evident, so that this
form is therefore most readily found. At this stage amoeboid move-
ments are not so active. When full growth is reached, segmentation
occurs. The forms up to the period of segmentation are called
schizonts.
The morphologic changes which have been going on in the parasite
preparatory to segmentation are best studied in properly stained smear
preparations. In the living organism, they become presently suffi-
ciently distinct to be followed; the pigment gathers more or less centrally
into a compact mass, and a peripheral notching indicates that the
parasite is preparing to divide into a number of segments called
merozoites; the number of these segments varies in the different species.
(See table.) Suddenly the segments separate as small spheroidal
bodies, the young parasites. A corpuscular remnant and the pigment
float away and are ultimately ingested by phagocytic cells. The
young parasites attach themselves to red corpuscles as before and the
human cycle is repeated (see Plate V for unstained organisms).
In a suitably stained preparation (any of the modifications of
Romanowsky's stain, p. 597) the young parasite (see Plates \T and VII
for the different species) appears to be a disk consisting of a central
pale, unstained area, known as the achromatic zone, and of a basic
(blue) periphery, the body, including a metachromatically stained,
rounded, compact (red) chromatin mass, the nucleus, which tends to
give the parasite the form of a signet ring.
* See table for number of hours in each species.
THE MALARIAL ORGANISMS. BABESLA. GOl
Later stages up to a certain number of hours show simply changes
in size and outline of the body. The nucleus then divides by simple
mitosis. Later it breaks up by amitotic division into an increasing
number of small masses. By the time the chromatin division is
completed the chromatin masses will have assumed a rounded form,
and will be seen to exhibit ultimately the same strong affinity for certain
dyes which is seen in the compact chromatin body of the young ring-
like form. At this stage the heretofore scattered pigment appears
in one clump. Good technique will always show a corpuscular remnant
even at this time. The achromatic zone mentioned will be seen to
develop with the chromatin, and when the next step, namely, the
division of the body of the parasite, is seen to be completed, there will
be as many achromatic bodies as there are chromatin bodies, each
division having an equal share of the basic mother-body, each repre-
senting the young parasite (merozoite).
A certain number of the full-grown parasites do not segment and
these are the forms which commence the life cycle in the mosquito.
These forms grow to produce the sexual forms, the macrogametocyte,
or female organism, and the microgametocyte, or male organism.
When mature these forms are generally larger than the mature
schizont of the same species, the female organism being usually larger
than the male and containing more food granules and a smaller nucleus.
In the sestivo-autumnal forms they are crescentic in shape, while in
the other species they are spherical. In the circulating blood of human
beings they show no further changes except to become freed from the
corpuscle; but when the blood containing them is withdrawn and
exposed for a short time to the air, an interesting series of changes
in the microgametocyte is observed. The crescentic bodies are
transformed into spherical bodies; the pigment of the microgameto-
cytes becomes actively motile, due to internal agitation of the chro-
matin fibrils, which presently emerge as flagella-like appendages.
Their movements are very rapid, causing corpuscles to be knocked
about, and finally they become detached as the microgametes, or male
elements, and go in search of the female element. In birds, one may
actually observe the process of conjugation in slide preparations even
without the aid of a moist chamber and heat. This transformation of
male bodies never occurs in the human blood. It will be seen
that it belongs to the sexual cycle which occurs in the stomach of
the mosquito.
The Sexual Cycle (Sporogony) Developing in the Mosquito.—
The common mosquito, often day-flying, belongs to the genus Culex;
it cannot carry human malaria. It is easily distinguished from its
night-flying or dusk-flying relatives, Anopheles (the malarial carrying
mosquitoes comprise about eight genera of the sub-family anophelinee),
by its assuming a different posture on the perpendicular wall. ^Vhile
the Culex holds the body more or less parallel with the surface, the body
of the Anopheles stands off at a marked angle. Other differential
points are the following (see Fig. 190):
602 PATHOGENIC MICRO-ORGANISMS.
Wings of Culex are unspotted; those of Anopheles are spotted (except
in one rare species).
The proboscis of Anopheles points toward the resting surface,
while that of Ctdex does not do so.
Anopheles species bite usually in the early evening, while those
of Ctdex bite almost at any hour of the day.
The male mosquito is readily told from the female by its plumed
antennae, those of the female being inconspicuous.
The eggs and the larvce of the two genera are quite distinct as
may be readily seen by glancing at Fig. 191. The anopheles mos-
quitoes breed in practically any kind of a collection of water, though
some species prefer slow running water to quiet pools. The best known
domestic carriers are usually found in barrels and cisterns.
If an ordinary mosquito (Ctdex) is allowed to imbibe the blood of
a malarial patient whose blood shows gametocytes there will be simply
a digestion of such blood in the mosquito, and no development of the
malarial organisms results. If, however, certain species of Anophdes
ingest such blood, immediate changes follow. It should be remem-
bered that only female mosquitoes are blood sucking; hence, they
alone can be responsible for the spreading of the disease. It should
also be remembered that if the blood imbibed by the anopheles does
not contain gametocytes, though it may contain earlier stages of the
malarial organisms, no amount of such blood can cause general in-
fection of the mosquito. The sexual cycle is similar in all species of
the parasite.
The flagellation of the male parasite described above will promptly
take place in the stomach of the anopheles, 4 to 8 microgametes being
formed; these conjugate with the female element (Plate III) in a
manner comparable to the impregnation of the ovum of higher animals
by spermatozoids. The macrogametocyte becomes a macrogamete
by the formation of a reduction nucleus which is thrown out of the
organism (Plate III, Figs. 13a and 14a).
The product of conjugation, the ookinet (zygote), remains for a
number of hours in the juices of the chyme stomach, changing gradually
from a spherical, immobile body into an elongated wormlet endowed
with motility (Plate III, Fig. 17). This penetrates the epithelial
Hning of the stomach and rests in the tunica elastico-muscularis (Plate
III, Figs. 8-20); here it changes into an oval, then into a round
body, which grows in the course of the next few days enormously,
forming a cyst which projects into the body cavity. Meanwhile the
chromatin will have become very active. It will have divided into nu-
merous nuclei, which become arranged around inactive portions, and
filamentous sporozoites develop from this chromatin and surrounding
protoplasm (Plate III, Figs. 21-24). These sporozoites ultimately
fill the cysts, which rupture, setting them free into the cavity of the
mosquito's body (Plate III, Fig. 25); they then are carried by the
lymph to all parts of the body of the mosquito and thus reach a glan-
dular structure in the thoracic cavity of the insect, the so-called salivarv
/6 ^
Uife-cycJeof Plasmodium Vivax. (After Grassi and Schaudlnn.)
The humsn cycle ta above the trsnavcne line, somewhat rearraiie«l by Kinlialt uid HartmuiD.
letc; 13ft und 14b. givHth of tha microBsmete; 15b.
oflkinet: IS (o 20, entrance of the ofikiDst into the Btomurh wall
22 and 23. nucleu laultiplication in the sporont; 24 and 2S. fori
of (he aporoioitea to the salivary gland: 27. salivary (land of th>
Bcation 1 to 17c,. 1200 to I; 18 (o 2Tc., SOO to 1.)
ont; 7, fori
nation ot the
of tbe micro-
il 14a. matu
mUoD of the
ifieation: 17
iiito; 20 to 2
e.BporoKony:
i; 2e,pa»ace
^th iporoio
it««. {Ma«ni.
THE MALARIAL ORGANISMS. BABESIA.
I 4t ♦
Chief comoBTBtivE rhancif riHiica of Cuia: md At
C-uin (II laid together in "soiaU boat." those of Ai
C. CM hanga nearly bI njiht snclee to wstcr surfHer. tl
C. (SI wben reeUn* i» held parallel to wall in a curvcc
about M" and i»8i™Bht; worn of C. (7) are generall] _...,.
C. the psipie (9) of Ihr female an very Aon. of the male aie
palpie (10) of both sexes are about equal in lenath with the proboena.
■phtlH. (From Kolle and Heoch.) Egg of
•phclri 12) separate aad rounded. Laria of
weof v1. (4>areparalleltOBuifaee. Boilyof
position, that of A. (S) stands at an angle of
~ ipolted. those of .4. (8> are spoiled: in
than (he probo
604 PATHOGENIC MICRO-ORGANISMS.
gland (poison gland), in which they accumulate in large numbers
(Plate III, Figs. 26-27). This gland is in immediate connection with
the biting and sucking apparatus. If, now, such an infected mosquito
** bites'' a human being, the lubricating fluid of the puncturing appa-
ratus will carry sporozoites into the latter's blood and the human cycle
begins. The stages of development in the mosquito require from
seven to ten days, but only when the temperature is favorable.
Effect on Man (Pathogenesis). — As the organism grows at the expen^^e
of the red blood cells the principal change is in the blood. Melaiu&niia, or
the formation of pigment granules from the destroyed red blood cells, is one
of the most characteristic features of malaria. As the disease progresses the
red corpuscles show varying changes in form arid haemoglobin content, not
only the infected corpuscles, but others as well, thus showing that the organism
produces either primarily or secondarily some toxic substances. The pig-
ment occurs in two forms, melanin and hflemosiderin. The second only gives*
the reaction for iron and is found in the internal organs, while the first is found
everywhere in the circulating blood. The pigment is taken up by the leuko-
cytes. There is usually a definite reduction of both red and white blood cor-
puscles, which is more marked in tertian and quartan malaria than in aestivo-
autumnal. There is a relative increase in the number of mononuclear
leukocytes. The spleen shows marked hyperplastic inflammation and
pigmentation.
After death, which sometimes takes place in cases of pernicious aestivo-
autumnal fever, there are scattered areas of intense congestion and of paren-
chymatous inflammation in the various internal organs, together with the
presence of large numbers of the parasite.
Toxin Production. — ^The relationship between segmentation an<l
paroxysm is always noted in tertian cases, and it is reasonable to sup-
pose that the occurrence of the paroxysm is referable entirely to the
liberation of toxic substances resulting from metabolic activity of the
parasite within the corpuscle. That there should be a toxic product
seems highly probable, and its amount must be considered in heavy
infections. Cases showing an infection of 1 to 5 per cent, of all cor-
puscles are not infrequent; the destruction of from 50,000 to 200,000
or more corpuscles per cubic millimetre of blood leads to the rapid
deglobularization of the blood; hence the deficiency in numbers; add
to this the effects of the metaboUc products, and little is left to the
imagination to explain the pronounced anaemia.
Immunity from malaria appears to exist as natural and acquired
immunity.
Prophylaxis. — The fact that, with the extermination of the malarial
carrying mosquitoes, malarial fevers in man would be made impossible,
remains established; the parasite must have its chance of rejuven-
escence in the mosquito's stomach.
The various methods of extermination are fully described in l)ooks
which go minutely into the subject. The method of giving small
doses of quinine to human beings exposed to Anopheles, and of thus
getting rid of the organism itself within man, should be considered.
In hot climates especially, where it is practically impossible totallv to
Description of Plate IV.
1. Typical young tertian form; the corpuscle shows incipient degeneration; cor-
puscle to left above shows a blood platelet.
2. Abnormal young form, showing small accessory chromatin body.
3. Two parasites; one a normal young form; the second a large form in crenated
corpuscle is an unusual abnormal form with very large achromatic area.
4. 5, 6. E^tivo-autumnal parasites; single, double, and triple infection; central
elongated chromatin bodies. These forms are about the largest usually seen in the
peripheral blood; no degeneration of corpuscle.
7. Tertian parasite, about ten hours old; marked degeneration of corpuscle.
8. Double infection of a corpuscle in tertian fever; marked degeneration of corpuscle.
9. 10. 11. Large tertian parasites showing division of chromatin previous to seg-
mentation.
12 and 14. Complete s^mentation of tertian parasite.
13. Double infection of corpuscle, one parasite reaching maturity, but showing
unusually small segments; the second one atrophied.
15. Tertian parasite, old case; while the parasite is only half-grown, the chroma-
tin has split into several compact masses. Degeneration of infected corpuscle.
16. Dwarfed tertian parasite, smaller than a red corpuscle, but showing five compact
chromatin bodies; resemblance to quartan rosette.
17. Microgametocyte of tertian malaria; prominence of blackish pigment surrounding
a large achromatic zone in which the microgametes lie coiled up.
18. Tertian macrogametocyte.
19 to 23. Crescentic bodies of estivo-autumnal malaria.
19. Typical gametocyte; pigment surrounding achromatic area; no chromatin shown ;
the "bib" is present. (Male?)
20. Semiovoid gametocyte. (Female?)
21. Pigment removed. ElUptical achromatic area in which the microgametes are
seen.
22 and 23. Pigment removed; chromatin more compact; possibly female elements.
24. From a case of pernicious malaria with rich infection; only hyaline forms in
peripheral blood. Below, a large blood-platelet.
Note. — As the amplification is not uniform, a comparison of the parasites with the
blood corpuscles shown should be made in order to have a correct conception of
their size.
PLATE IV
'^"
.-3',
7
5
• ;•
^
s ' /i>
-^c- .;
i ■
■IT 1
* !
<
U -'■
i
Photographs or Tertian and Estivo-autumnal Malarial
Parasites in Dirferent Stages of Development.
(Goldhopn.)
THE MALARIAL ORGANISMS. BABESIA. 605
destroy the breeding places of the mosquitoes, this method is especially
serviceable.
Points in Diagnosis. — By a study of the circulating parasite the
examiner should be able to tell not only the species present, but also
the progress the disease is making. Malarial parasites can always
readily be found in recent primary infections, and it is usually only in
old cases that the search becomes diflScult; one is, however, generally
rewarded by finding them if one looks long enough for them.
A helpful sign is the finding of pigment in mononuclear leuko-
cytes, which are seen about the time of a chill or of the period symp-
tomatically corresponding to it. Free pigment cannot be used as a
means of diagnosis, as it may be impossible to tell it from dirt or dust.
In a primary infection of long standing, the gametocytes may be found,
and in relapses and in those cases treated by quinine, many atypical
forms appear. A small dose of quinine may drive all parasites except
the sexual forms out of the peripheral circulation; at all events, the
finding of them becomes, in the absence of gametocytes, a matter of
time and experience, especially also as they may be much altered in
appearance. The part most and first affected is the blue staining
body; later follow eccentricities of the chromatin, such as multiple
bodies, and dwarfing, just such changes as might have occurred in
time, if the body had been allowed to combat the parasite without the
aid of drugs. In both cases the fever curve becomes atypical. It
should be remembered that there is no quotidian form originating in
this country. Quotidian paroxysms occurring here are either a double
tertian or a triple quartan infection. The notion that the parasites
can be found only at the time of the paroxysm is still in the minds of
many; it is erroneous. The gametocytes are quite resistant to qui-
nine and other drugs, and it appears as if cases in which these forms
are seen are much more prone to relapse than promptly treated recent
primary infections. The macrogametocytes may remain quiescent
for years in the blood, and then under certain conditions, probably
through parthenogenesis, may again begin to develop and multiply,
thus bringing about relapses.
In thesestivo-autumnal forms the crescentic gametocytes are gener-
ally few, but at times large numbers of them develop. Of course,
they are absolutely characteristic. The young parasites are more or
less characteristic in stained preparations (Plate VII). There may
be as many as seven parasites in one corpuscle. Later the few heavy
pigment granules are characteristic.
In fatal cases the formation of crescents may not take place; the
blood infection with young parasites is then enormous, every field of
the microscope showing numbers of them.
In the study of aestivo-autumnal fever as well as in that of the other
forms, it is to be remembered that crescents when found indicate that
the disease is of some standing, for such sexual forms are not formed
until the asexual propagation is waning. The recognition of these
ovoidal and crescentic bodies is easy. But as there are no readily
606 PATHOGENIC MICRO-ORGANISMS,
discoverable pigmented forms in the peripheral blood in the early
stages, it is necessary to be thoroughly familiar with the young aestivo-
autumnal forms. Polychrome staining for them cannot be too much
recommended, as there is little that is characteristic about them when
they have been stained with methylene blue alone. Many a serious
error has been made by adhering to the antiquated idea that parasites
should be looked for in the fresh blood, as these young, non-pigmented,
so-called hyaline forms cannot be readily recognized by the inex-
perienced, while it is an easy matter to know and classify them when
properly stained.
The recognition of the quartan parasite in its early stages in the
fresh blood is not as diflScult as that of the tertian form, because the
outline is more distinct; but in stained preparations it is often indis-
tinguishable from the latter. The living amoeboid young form or
schizont is more refractive than the young living tertian schizont,
more like the sestivo-autumnal form, and it is just as sluggish in its
movements. Here, too, the corpuscle is often shrunken and looks
as if it contained more hsemoglobin than in the case of infection with
the tertian parasite.
The growing parasite rapidly becomes pigmented, but it shows
fewer, larger, less motile pigment granules than the corresponding
tertian one; moreover, the pigment is arranged around the periphery
of the organism, while in the tertian form it is distributed through-
out the protoplasm. (Plate II, Fig. 13 a and b.) The quartan
parasite is apt to form a band across the infected corpuscle. Seg-
ments are few in number, as a rule, and the parasite remains dwarfed,
while the infected red blood cells are normal in size. The segments
are generally arranged symmetrically around the central pigment,
giving the so-called daisy or marguerite appearance to the parasite at
this stage.
In tertian fever, the granular degeneration which the infected cor-
puscles early undergo is diagnostic. In the first few hours it resembles
the ordinary granular stroma degeneration with basic aflSnity, while
it is later seen that the aflSnity of the then more numerous granules
is more acid, or at least the staining is no longer orthochromatic, the
blue being superimposed by a red; in other words, these granules
stain later metachromatically. The greater the loss or transformation
of the heemoglobin, the greater the number of granules. This holds
good only for tertian parasites, the aestivo-autumnal variety causing
practically no appreciable change though the same technique be used.
Malariid-like Parasites in Other Animals. — ^Two genera of protozoa
closely related to the malarial organisms have been found in birds:
(1) the proteosoma or haemoproteus; (2) the halteridium; both found
in owls (HcPTnop-oteus noctuw Celli and Sanfelice). Points in their
life history have been brought out by various observers, especially
by Ross and by MacCallum. The complete life cycle of both forms,
as worked out by Schaudinn, is considered by him and his followers
to be of fundamental importance to the understanding of the re-
THE MALARIAL ORGANISMS. BABESIA. 607
lationship of bloocl parasites. Schaudinn states that these organ-
isms pass through a flagellate stage in the intestinal tract of the common
mosquito (CtUex pipiens) which had previously fed on owls infected
with the intracellular organisms (halteridium and haemoproteus).
Novy considers that this mosquito flagellate stage of Schaudinn is
simply a growth of trypanosomes in the mosquito's intestinal tract
which are normally found there, and that Schaudinn did not suflSciently
control his work to warrant his conclusions.
Malarial-like organisms have been found also in monkeys, cattle,
dogs, and frogs, but they have been little studied.
An interesting article by Bemberg-Gossler on the malarial organisms
in monkeys has just appeared. In it the author describes a binucleate
phase of these plasmodia and agrees with Hartmann in his recent
classification of these organisms (see p. 596).
GENUS BABESIA (PIBOPLASBIA).
It was not until 1888 that there was a hint as to the real nature of
the actual cause of ** Texas fever" (bovine malaria, tick fever, hcemo-
globinuria) and allied diseases which attack field cattle in many parts
of the world. Then Babes described inclusions in red blood cells in
Roumanian cattle sick with the disease, though he did not decide upon
the nature of the organism. No new studies were reported until 1893,
when Theobald Smith and Kilborne gave such a complete description
of this disease and its cause as occurring in Texas cattle that little
that is new has since been discovered.
These authors describe as the cause of Texas fever, pigment-free
ameboid parasites appearing in various forms within the red blood cells
of infected animals. The organisms may be irregularly round and lie
singly or they may be in pear-shaped twos, united by a fine line of
protoplasm.
Because of these double pear-shaped forms Smith and Kilborne
named the organism Pyrosoma bigeminum^ and they placed it pro-
visionally among the haemosporidia. These authors also showed
that the contagion was carried by a tick (see below). Their work
has been corroborated by many investigators in different parts of the
world. Hartmann places this genus in his new order Binucleata,
and he considers it an important form for showing the relationship of
the endocellular blood parasites to the flagellates. Schaudinn, in 1904,
was the first to call attention to the occurrence of nuclear dimorphism
in J5. cants and hovisy and Luhe, Nuttall and Graham-Smith, Breinl and
Hindle and others have confirmed this observation. The second
* The generic name Pyrosoma^ already in use for a well-known Ascidian genus,
was altered to PiropUuma by Patton in 1895. In the meantime Starcovici (1893)
had given the name Babesia bovia to the form described bv Babes; and as this
form seems to be identical with that described by Smith anci Kilborne the correct
name of the genus should be Babesia, while the species parasitic in cattle should
be called Babesia bigemina.
608 PATHOGENIC MICRO-ORGANISMS.
nuclear mass is generally in the form of a small granule similar to the
blepharoplast of undoubted flagellates.
Morphology of the Parasite (Plate II, Fig. 14).— In the exami-
nation under 1000 diameters of fresh blood of sick cattle, according
to Smith and Kilborne, are seen, in the red blood cells, double pear-
shaped forms and single rounded or more or less irregular forms.
The size varies, though generally it is the same among the l>odies
in the same red blood cell. The average size is 2/t to 4fi long and
l^ju to 2fi wide. The pointed ends of the double form are in appo-
sition and generally touch, though in unstained specimens a connec-
tion between them cannot be seen. The axis forms either a straight
line or an angle. The protoplasm has a pale, non-granular appearance,
and is sharply separated from the protoplasm of the including red
blood cell. The small forms are generally fully homogeneous, whereas
the larger ones often contain in the rounded ends a large rounded
body, 0 . 1/i to 0 . 2/£ in size, which is very glistening and takes a darker
stain. Within the largest forms in the centre of the thick end is a
large round or oval body, 0.5/t to l/£, which sometimes shows ameboid
motions. Piana and Galli-Valerio (1895 and 1896) and other observers
have since described definite ameboid motion of the whole parasite.
The motion of the whole parasite on the warm stage is not produced
by the formation of distinct pseudopodia, but by a constant change
of the boundary. The changes can succeed each other so quickly
that it is scarcely possible to follow them with the eye. The motion
may persist for hours. The single ones show motion, while the double
ones remain unchanged. The parasites take most basic aniline stains
well. The Romanowsky method or its modifications gives the best
results.
Stained by this method, the smallest forms appear as tiny rings,
about one-sixth the diameter of the red blood cell. A part of the
rim takes the red nuclear stain, the rest is blue. In the large mature
pear-shaped organisms a loose mass of chromatin is at the rounded end
and a dense, compact mass is situated nearer the pointed end. These
mature, pear-shaped forms, Nuttall states, are the mark of distinction
between Piroplasma (Babesia) and other intracorpuscular blood para-
sites. These pyriform bodies are generally present in pairs, and
occasionally, in the acute form of the disease, sixteen pairs may be
seen in a single blood cell.
The number of red cells infected is about 1 per cent, of the whole.
If the number increases to 5 per cent, or 10 per cent, it generally
means the death of the animal. The parasites quickly disappear
from the blood after the disappearance of the fever. In fatal cases
many parasites are found in the red blood cells of the internal organs.
They vary in number according to the stage at which death occurs,
are most abundant in the kidneys (50 to 80 per cent, of all red corpuscles
infected), and are found in fewer numbers in the liver, spleen, and other
internal organs.
R. Koch has described a bacillar form which he found in large
PLATE V
Fig. 1.— Tertian Malarial Plasmodium.
1. Hyaline form. 7. Segmenting forms. 9. Non-flagellate form. (Macro-
2. Pigmented ring form. 8. Flagellate form. (Mioroga- gamete.)
3 to 6. Pigmented forms. met^cyte.) 10. Segmenting form aft«r de-
struction of red corpuscle.
Fig. 2.— Quartan Malarial Plasmodium.
1. Hyaline forms. 8. Segmentinjj forms after the 9. Flagellate form. (.Microga-
1' to 5. Pigmented forms. destruction of red corpus- metocyte.)
6 and 7. Segmenting forms. 10. Non-flagellate form. (Macro-
gamete.)
Fig. 3.— Tertian ^^Istivo-autumnal Malarial Plasmodium.
1 and 4. Hyaline ring form. 8 Young intracorpuscular ores- 10. Flagellate form. (Microga-
2. 3 and 7. Pigmented ring form. cent. metocyte.)
5 and 6. Pigmented forms. ^- Segmenting forms. U to 14. Crescentic forms.
Fig. 4. -Quotidian -«Cstivo-autumnal Malarial Plasmodium.
lto4. H valine rin^ forms. Some 8. Segmenting forms. Segmen- 10. 11. 13 and 15. Crescentic
ceils show infection with tation complete within in- forms
more than one organism. fected red blood con)U8cle. 12. Ovoid form
5 to 7. Pigmente<l forms. In 6 9. Flagellate form. (Microga- id \i«„ n ^ii„* * /»#
one hyaline form. metocyte.) ^^- Non-flagellate forms. (Ma-
•^ ' crogamete.)
Note.— Mark the larger siae and greater amount of pigment in the tertian tpstivo-autumnal Plasmodium.
PLATE VI
Fig. 1. — Tertian Malarial Plasmodium. Stained by Oliver's
Modification of \A^ right's Stain.
1 to 4. Ring forms of tertian 11 to 14. Nearly full-grown forms. 18. Segmenting forms after cle-
parasite. showing diffusion of the struct ion of red corpuscle.
5. Uing form. (Conjugation chromatin. jg I'laRellum. (Microgamete.j
form of Kwing.) 15 to 17. Segmenting forms 20 Sporozoite
6 to 10. Pigmented organisms. within red corpuscle.
Fig. 2.— Quartan Malarial Plasmodium. Stained by Oliver's
Modification of Wright's Stain.
1 to 4. Ring forms of quartan 10 to 12. Segmenting forms of 13. Segmenting Htagc after <ie-
parasite. quartan parasite. structi<ni of retl corpuscle.
5, 6, 7, 8. 9. Pigmented! parasites.
Note. — Chromatin of nucleus .stained red ; protoplasm ntained blue ; vesicular portion of
nucleus un'^tained.
PLATE VII
.^stivo-autumnal Malarial Plasmodia. (Tertian.) Oliver's
Modification of Wright's Stain.
1, 3, 4, 5, 6, 7, 8, 9. 10 and 15. 12. Red corrjuscle showing in- 2. > to 36. Crescentic forms of
King forms of tertian fection with two *' ring :psti\«)-;iutumnal plasmo-
a-Htivo-autumnal plasmo- forms." tiium (tertian).
^*"™* 18 and 19. l'igmente<l forms, just 29. Ovoid form.
2. Intracellular form. prior to segmentation. 37. Segmenting form.
11. 13. 14, 16 and 17. Pigmented 20. 21, 23 and 24. Round and 3g Sporozoites
ring forms. ovoid forms developed a. Segmenting form of quotid-
from crescents. i^n .T^tivo-autumnal phis-
22. Macrogamete. mo»lium.
Note. In this plate the tertian a-stivo-autumnal Plasmodium is shown. The staining reactions of
the quotidian Plasmodium are exactly similar.
Fio. 1
PLATE V
10
6
8 !
9
K,
-' "N.
10
Fig. 2
'V*..
^
3 s
9
8
Fio. 3
Fio. 4
<'
V ^. ^
>'9
♦f
13
II
(»}
Tr
12
PLATE VI
•'.^
0
r
Af"
11
3
12
Fio» 1
C
,.«'-
o
' .1 ^*' *
10
*i
6
8
IV
19
^'
*■"
^^ 13
18
14
9
17
20
Fio. 2
1
10
5
K^
13
•i8
2
11
i:3
9
-*;?
^"
6
r*
12
/ir5
7
<2>
8
k
PLATE VII
+
9
(>
r-
9
o
1()
■^
17
o
it
20
25
30
..^,
vr
35
14
U
♦ r*
18
21
t^
26
^
^^7^
15
12
22
23
.<-..'
.'f*
.'*«.'
>»?
27
32
13
a
16
19
24
31
VJ; 33
28
*^..
->
^
29
34
36 d^^^^^^e
37
y
r
THE MALARIAL ORGAXISMS. BABESIA.
609
numl)ers in red blood cells of acute fatal cases in East Africa. Be-
tween these and the pear-shaped forms he found all grades. This
variety is probably a distinct species.
Flagella-like appendages in Babesia have been discribed by several
observers as occurring in the blood in mammals. More frequently
ihey have been seen in the tick and in attempted cultures. Some of
them have been interpreted as possible microgametes (Hartmann,
Calkins), others as true flagella (BreinI and Hindle), still others as
fine pseudopodia (most observers).
Smith and Kilborne showed that the infection is caused by a species
of tick, yiargaropus annvlalus. Say (Bodphilvs bovis) (Fig. 192), and
(Boophilua bovia), X 16.
Kossel gives Ixodes redivius as the tick causing transmission of the
germ in the hsemoglobinuria of Finland cattle.
The ticks feeding upon the blood of cattle and other mammals
become sexually mature at their last moult. They then pair, and
the fertilized females, after gorging themselves with the blood of
their host, drop to the ground. Each female then lays about 2000
eggs, and within the shell of each egg a large quantity of blood is
deposited to serve as food for the developing embryo. The female
then shrivels up, becoming a lifeless .skin. The newly hatched larvK
containing in their abdomens some of the mother-blood, crawl about
until they either die from starvation or have the opportunity of
passing to the skin of a fresh host. If the mother-tick has drawn
its supply of blood from cattle infected with piroplasma, her larvie
arc born infected with the parasite and become the means of dis-
seminating the di.sease further. This mode of dissemination ex-
plains the long incubation period of the disease (forty-five to sixty
days — thirty days for the development of the larvjc and the remainder
for the development of the parasite within the host). It is po.ssible
that the tick embryo acquires ihe infection secondarily from the
610 PATHOGENIC MICRO-ORGANISMS.
blood it absorbs in the egg, and that the parasites do not pass through
the ovum itself as in Nosema bombycis. This species of tick J/, afmu"
lotus has been found also on sheep and ponies.
So far, it has not been possible experimentally to inoculate animals
other than cattle with these parasites. Calves withstand the infec-
tion better than older animals and a certain degree of immunity is
reached in some of the older cattle in infected districts. The piro-
plasmata taken in by such animals may remain as harmless parasites
for some time. If, however, such cattle are weakened from any
cause, their resistance to the organism may be lowered and they
may therefore pass through a more or less severe attack of the disease.
Symptoms of the Disease. — ^Fever (40° to 42° C), anorexia, weakness,
increased pulse and respiration, decreased secretion of milk, hsemoglobinuna ,
at the height of the fever, causing the urine to appear dark red like port wine
or darker. The urine may contain albumin even if the hemoglobinuria is
absent, but there are no red blood cells present, the color being due to the
coloring matter of the blood only. There is icterus of the mucous membrane
if much blood is destroyed.
The prognosis varies in different epidemics from 20 to 60 per cent. Death
may occur in three to five days after first symptoms appear. Recoveiy is
indicated by a gradual fall of the fever.
Treatment. — Quinine in large doses seems to have helped in some epi-
demics. Nuttall, Graham-Smith, and Hadwen have reported curative effect*
from trypanblau in both canine and bovine babesiosis (Piroplasmosis).
Prophylaxis. — Stalled cattle are not infected, but it is impractic-
able to keep large herds of cattle stalled. If the cattle are kept from
infected fields for one or two years and other animals (horses and
mules) are allowed to feed there the ticks may disappear. The
burning of the field for one season may have a good effect. If ani-
mals cannot be taken from infected fields such fields should be
enclosed.
Ticks on animals may be killed by allowing the cattle to pass through
an oil bath (paraffin, cottonseed oil, etc.), whereupon the ticks die
from suffocation. The bath should be repeated after a week in order
to kill any larvae which may have developed. All animals sent from
infected regions should receive this treatment. Animals apparendy
healthy before the treatment, after the disturbing influence of the
bath often develop the disease in an acute form and die.
Certain birds in Australia seem to feed on the ticks, therefore such
birds might be propagated.
Various attempts have been made to give protection by the inocu-
lation of fresh (not older than two or three days) blood from slighdy
infected animals. Some partial results have been reported, especially
when the inoculations were made during the cold months. In Aus-
tralia, the inoculation of defibrinated blood from animals which have
just recovered from the infection, but whose blood still contains some
parasites, has been tried. So far no absolute protection has been pro-
duced, neither does the parasite-free serum of animals which have
entirely recovered from the disease seem to contain protective qualities.
THE MALARIAL ORGANISMS, BABESIA, 611
Blood Organisms. — Blood organisms similar to those described in
the hsemoglobinuria of cattle have been found in cases of red water
fever of cattle in England. They also occur in monkeys, dogs, sheep,
horses, and pigeons. Nocard and Motas, who have made an extensive
study of these parasites in the malignant jaundice (heemoglobinuria,
malaria, or biliary fever) of dogs, state that though the parasites are
morphologically similar to those infecting cattle, yet it is impossible
to infect cattle or any other animal tried with them. They must
therefore be considered a physiologic variety.
Nuttall and Graham-Smith have recently completed a series of articles in
which they have reported a minute study of canine piroplasmosis, and have
drawn a cycle showing the usual mode of multiplication in the circulating
blood. They consider B. canis a species distinct from B. bovis and B. pitheci
(found by Ross, in 1905, in blood of a species of cercopi-thecus) though no
morphologic differences are given.
Christophers has described probable sexual stages of development in the
tick R. sanguineus, so that he has drawn a complete life cycle of the organism.
Various attempts at artificial cultivation have not met with much success.
Bibliography.
Berenberg-Gossler . Beitr&ge zur Naturgeschichte der Malariaplasmodien.
ArchivfttrProtistenkunde, 1909, XVI, 245.
Christophers, Journ. of Trop. Med., 1907, X, 323.
Craig. "The Malarial Fevers," in Osier's Modem Medicine. Philadelphia,
Vol. 1, 1907, also "The Malarial Fevers" etc., 1909, Wm. Wood & Co., New York,
first edition.
Howard, "Mosquitoes," in Osler^s Modern Medicine. Philadelphia, Vol. I,
1907.
Kinoshita. Arch. fQr Protistenk., 1907, VIII, 294.
Koch. Zeitschr. f. Hygiene, 1901, XLV, 1.
Marchiafava and Bignami. "Malaria," in Twentieth Century Practice, New
York, 1900.
Miyajami. Philip. Journ. of Science, 1907, II, 83.
Nuttall and Graham-Smith. Journ. of Hygiene, 1905-1906-1907. Also in
Parasitology, 1909, II, 215, 229, 236.
Schilling, in Kolle and Wassermann's Handbuch der Pathogenen Mikroorgan-
ismen, Ergknzungsband, 1st Hft., 1906.
T. H. Smith and Kilborne. U. S. Depart, of Agriculture, 1893, Bull. No. 1.
Thayer and Hewetson. "The Malarial Fevers of Baltimore," Johns Hop.
Hosp. Rep., Vol. V, 1895.
CHAPTER XLM.
SMALLPOX AND ALLIED DISEASES. SCARLET FEVER. MEASLES-
SMALLPOX (VARIOLA) AND ALLIED DISEASES.
IntrodactioiL — ^The diseases smallpox, cowpox, vaccinia, horse-
pox, sheeppox, if not identical, are closely allied. Indeed, the follow-
ing facts seem to prove that at least cowpox and variola are very closelv
related, if not essentially the same disease: First, smallpox virus
inoculated into calves produces, after passage through several animals,
an aflFection exactly similar to cowpox. The successfid inoculation
of the first series of cattle from smallpox is a matter of great diffi-
culty, but so many experimenters have asserted that this has been
done that there seems to be no doubt as to its truth. In our labo-
ratory not one of many attempts to accomplish it has been successful.
Second, both when occurring in nature and when produced by experi-
ment the lesions of the two diseases are similar. Third, monkeys have
been successfully protected against either disease by previous inocula-
tion of the other; also, observations go to show that human beings
inoculated with cowpox vaccine are not susceptible to inoculation with
smallpox virus, and that those who have within a varied time passed
through an attack of smallpox cannot be inoculated successfully with
cowpox vaccine. These facts seem positively to prove that the two
diseases are produced by organisms originally identical, one being
modified by its transmission through cattle, the other through human
beings.
Variola is perhaps the most regularly characteristic of the diseases
of man. It is highly infectious and is controlled only by vaccina-
tion. Notwithstanding the fact that we know definitely the exact site
of the infective agent in this disease and that certain experimental
animals are susceptible to inoculation of the material containing the
infective agent, most investigators are still undecided in regard to the
nature of the chief exciting factor. A few, however, claim diat certain
bodies found chiefly in the epithelial cells of the skin and mucous mem-
branes in the specific lesions are protozoa causing the disease.
Definition. — Smallpox (Synonyms: Variola, la variola, Blattem,
Pocken, Vajuola) is an acute infectious disease characterized by an
epidermic eruption of vesicles and pustules which, upon healing,
produce cicatrices of varying extent and depth.
Historical Note. — The first undoubted description of the disease was
given by Rhazcs in the tenth century, but it is evident that he did not con-
sider it a new disea.*«e. To trace its original home seems to be impossible.
It may have developed first in certain regions in Asia and Central Africa
612
SMALLPOX AND ALLIED DISEASES. 613
where it is at present endemic and is said to be uncontrolled by vaccination.
Many outbreaks of the disease in the United States can be traced directly
to the importation of African negroes.
The disease, carried by the intercommunication, principally of war and
commerce, was widespread when Edward Jenner showed conclusively in
1798 that vaccination with cowpox afforded protection. Now the few cases
of variola that occur are seen in those who, through neglect or ignorance
(sometimes willful), have not been vaccinated.
Etiology of Variola and Oowpox. — It has been repeatedly shown
that no bacteria similar to any of the known forms have a causal
relation to these diseases. In our own laboratory we. are able, by the
inoculating of rabbits' skins, to produce extremely active vaccine
virus in large quantities, absolutely free from microdrganisms which
grow under the conditions of our present methods of bacterial culti-
vation. Such pure active vaccine, when emulsified in equal parts of
glycerin and water and filtered through two or three thicknesses of
the finest filter-paper, gives a slightly opalescent filtrate, which in
the hanging drop under high magnification shows many very tiny
granules with an occasional larger one, and in smears shows no formed
elements giving characteristic stains. This filtrate, from which no
growth can be obtained on artificial culture media, when rubbed over
a freshly shaved rabbit's skin after the method of Calmette and Gu^rin,
or when used to vaccinate human beings, gives an abundant typical
reaction.
These facts show that some, at least, of the infective forms cannot
as yet be made to grow outside of the body, that such forms are very
tiny, and that they do not stain characteristically with our usual methods
of staining. In a few experiments we were unable to filter the virus
through a Berkefeld filter under forty pounds' pressure, but this may
have been due to the fact that we did not dilute our virus sufficiently.
Since then Bertarilli has reported moderately successful results from
his filtration experiments.
Since Guarni^ri in 1892 claimed that certain inclusions present
in the epithelial cells of the lesions of smallpox in a rabbits' cornea
(Fig. 192) were parasites, much attention has been given to the study
of these bodies, commonly known as "vaccine bodies," yet opinions
still differ as to their nature. The most recent important studies of
these bodies have been made, on the one hand, by Councilman and
his associates, who believe them to be protozoa, and, on the other, by
Ewing, who believes that all of the forms so far described are degenera-
tion products, some specific, others not.
Councilman believes that there are two cycles of development of
the "parasite," one intracellular and the other intranuclear, and that
the intranuclear infection occurs only in smallpox. The intracellu-
lar cycle is simple, showing only "multiplicative reproduction," while
the intranuclear cycle is more complicated, probably sexual in character.
Calkins, working with Councilman, described a cycle of development
in which we believe are included many forms due to degeneration of the
614 PATHOGENIC MICRO-ORGAMSMS.
host cells alone. Calkins now thinks that his original tentative cvde
was too elaborate. He still firmly believes that the bodies are protozoa,
but that they belong among the rhizopoda and not among the micro-
sporidia where he first placed them.
Prowazek and others believe that the organisms of this group of diaeafles. »s
well as of rabies, scarlet fever, trachoma, and a few others, are all tiny coccus-
like forms which have the power of producing an envelope from (he host c*ll
substance, such envelope with its contained organism constituting the specific
body which others have called a protozoon. Prowatek calls the group
Chlamydozoa and says they probably stand between the bacteria and the
protozoa in systematic classification. From our studies on this whole group
of diseases we have come to the conclusion that there is no close relationship
between the trachoma bodies and the intracellular bodies of rabies, small-
pox and scarlet fever.
Fia, 193
le bodia." Tissue
In our own work on sections, which has extended irregularly over a
period of several years, we have gotten results which are somewhat
confusing, principally so because of the non-uniformity of the ap-
pearances of these bodies, both by different methods and by the same
methods at different times. There is no doubt that, whatever the
nature of the bodies, they are easily affected by methods used for
fixing, hardening, and staining them. This accounts in part for the
varied results reported. However, in the most perfectly prepared
specimens, judged according to the appearance of the red blood celk
leukocytes, .and tissue cells at a distance from the lesions, we have
found that the vaccine bodies, especially in corneal infection, show a
more or less constant series of changes, somewhat similar to those
described by Calkins in his "gemmule formation" and by Tyzzer
in his development of the vaccine bodies. This series of changes
might lie represented somewhat schematically as in Fig. 194.
SMALLPOX AND ALLIED DISEASES, 615
One can easily see that such tiny bodies as these possible spores,
with no definite characteristic staining qualities, would be with diffi-
culty, if at all, differentiated from the mass of cell granules in the
degenerated areas of the lesion; and, as the outline and structure of
most of the other forms seem to be easily disturbed, the whole question
as to their nature is, from a morphologic standpoint alone, a very
difficult one to settle.
Fio. 194
o
/
© ^
Schematic representation of vaccine bodies seen within the epithelial cells in the lesions of
smallpox and vaccinia: 1, spore (merosoite, sporoEoite ?) ; 2, small form which stains solidly
with basic stains: 3, larger form which contains central, more darkly staining granule; 4, larger
form, with more lightly staining reticular cytoplasm. This form and the next may have amcDboid
outline, and there ms^ be larger amoeboid forms whfch might be interpreted either as the grown
single form or as the fusion of two or more forms; 5, form containing two central, darkly staining
bodies; 6, form containinij many bodies taking basic stains more or less intensely; 7, form con-
taining a central body stamini^ faintly with basic dyes, and small rounded bodies about it, some
taking basic and some acid stains- 8, same as 7, except that many of the bodies surrounding the
centra] body are definitely ring-shaped, and all take the acid stain. Thrae forms vary in sixe;
some are larger than the host nucleus; 9, form breaking up (spores set free?).
Our best results on corneas have been obtained with the following tech-
nique: Fix in Zenker's fluid for from four to eight hours; wash in running
water overnight; place in 95 per cent, alcohol (changing in two hours to
fresh) for twenty-four hours, then in absolute alcohol for twenty-four hours.
Imbed in paraffin. The cuts should be from 3At to 5/* thick. Stain with (1)
eosin and methylene blue (Mallory) — eosin half an hour, methylene blue two
minutes; (2) Heidenhain's iron haematoxylin; (3) Borrel modified by Calkins.
The vaccine bodies may be studied for a short si me in the living
cornea by rapidly excising an inoculated cornea, spreading it on a
shallow agar plate and dropping a thin cover-glass over it. The
structured bodies are very clearly differentiated from the rest of the
cell contents, and interesting changes have been observed in them.
Too little work has been done, however, by this method, to draw
any further conclusions in regard to their nature. Councilman and Tyz-
zer have photographed these living cornea bodies with the ultra-
violet light, and the structure has come out as the chromatin structures
of known living cells.
Pathogenesis. — For Lower Animals. — Various animals seem to contract the
disease, or a modification of it, in nature. Horsepox, sheeppox, and cowpox,
all show similar pathological changes. Experimentally, probably all mam-
mals are susceptible though in varying degrees. Most of them are more
sensitive to vaccinia than to variola. The epidermis of rabbits, for instance,
shows a beautifully typical eruption after inoculation with vaccine virus,
w^hile material from smallpox eruptions produces only diffused redness.
The corneal "take," however, in both instances, is similar in intensity.
Monkeys are ecjually susceptible to both forms of the disease.
For Man. — Without vaccination human beings seem to be equally suscep-
tible to infection with variola, whatever their race or their condition in
life or in whatever part of the world they live.
616 PATHOGENIC MICRO-ORGANISMS,
The immunity caused by successful vaccination is not permanent,
and varies in its duration in different individuals. Although it usu-
ally gives protection for several years and may give it for ten or fifteen
years, it is not well to count on immunity for more than one year,
and whenever one is liable to exposure it is well to be vaccinated.
If this vaccination were unnecessarv it will not be successful, while
if it is successful we have reason to believe the individual was open
at least to a mild smallpox infection.
Protective Substances Present in the Serum of Animals after Suc-
cessful Vaccination. — It has been frequently shown that the blood
serum of a calf some days after an extensive vaccination possesses
feeble protective properties, so that the injection of one or two
litres of it into a susceptible calf would prevent a successful vaccina-
tion. A further and more convincing fact has been demonstrated
by Huddleston and others, namely, that when active vaccine is mixed
in certain proportions with serum from an animal which had just
recovered from a successful vaccination, and the mixture is inocu-
lated into a susceptible animal, there is no reaction.
The Preparation of Vaccine.— For most of the following sugges-
tions we are indebted to Dr. J. H. Huddleston, who has had the im-
mediate charge of the production of vaccine for the New York Health
Department for some years:
Seed Vims. — A sufficient amount of vaccine virus should be on hand
to vaccinate forty to fifty persons. Five children in good health, and
not previously vaccinated, should then be vaccinated, each in a spot
the size of a ten-cent piece. On the fifth day after vaccination the
top of the resulting vesicle should be removed and sterilized bone slips
be rubbed on the base thus exposed. From one to two hundred slips
on each side of the slip may be charged from each child. The slips
should be allowed a moment in which to dry and then be placed in a
sterilized box, in which, if kept in cold storage, they will probably re-
main efficient for at least two or three weeks. Rabbits are now used
by us alternately with children to obtain seed virus.
Animals. — The preferable animals are female calves, from two to
four months of age, in good condition and free from any skin disease.
These can easily be vaccinated on the posterior abdomen and inside
of the thighs by placing them on an appropriate table. It is possible
that, on account of the character of the available supply, older animals
may be desirable, but the calves take more typically and are more
easily handled. When an animal is too old to be thrown and held with-
out difficulty it may be vaccinated on the rump, each side of the spine;
but the skin there is tougher than on the posterior abdomen and
inside of the thighs, and the resulting virus, though efficient, is not
so easily emulsified.
Vaccination. — The hair should be clipped from the entire l>odv
when the animal is first brought into the stable and the calf should
be cleaned thoroughly, including the feet and the tail. Just before
vaccination the posterior abdomen and insides of the thighs are
SMALLPOX AND ALLIED DISEASES. 617
shaved and the skin beneath washed in succession with soap and
water, sterilized water and alcohol, and then dried with a sterile towel.
On this area there are now made about one hundred scarifications,
each from one-quarter to one-half of an inch square. The scarification
is made most easily by cross-hatching with a six-bladed instrument
the blades being about one-thirtieth of an inch apart. The scarifica-
tion is superficial, but brings a small amount of blood. An area
as small as specified is less likely to become infected than a larger
one. The scarifications should be separated from each other by
an interval of at least one-half to three-quarters of an inch. After
they have been made they should be dried with a sterile towel or with
sterile cotton and rubbed with the charged slips. One to two slips,
depending on the amount of virus each slip holds, should be suflScient
for vaccinating each vesicle.
Collection. — On the fifth or sixth day, depending upon the rate of
development of the vaccine vesicles, they should be ready for col-
lection. The entire shaved area is washed with sterile water and sterile
cotton, and the crusts are picked off. The soft, pulpy mass remaining
is then curetted off with an ordinary steel curette and the pulp placed
in a sterilized vessel. After the curettage, serum exudes from the
torn base of the vesicle, and ivory slips may be charged in this. The
pulp should be mixed with four times its weight of glycerin and water
(50 per cent, glycerin, 49 per cent, water, 1 per cent, carbolic acid),
and this is done most effectively by passing the mixture between the
rollers of a Doring mill. The more watery the pulp, especially if it
is not to be used immediately, the smaller should be the proportion of
glycerin. The emulsion so produced can then be put up for issue in
vials. The slips charged with the serum from the calf may also be used
for vaccinating. Capillary tubes require especial means of filling,
and small vials filled and corked answer the purpose admirably.
Propagation. — Subsequent animals may be vaccinated in any one of
the three ways: (a) slips may be charged from typical vesicles on
primary vaccinations, just as with the first calf, and used for seed
virus; (6) slips charged with the serum from the calf may be used to
vaccinate a second calf; (c) the glycerinated emulsion may be used
to vaccinate succeeding calves, but in the last case it is necessary to
keep the emulsion a varying length of time — often two or three months —
before it is fit for use in vaccination of the calf, since the employment
of fresh glycerinated pulp on a succession of calves leads to prompt
degeneration of the vaccine and to the production of infected vesicles.
Oare of the Calves. — All bedding is avoided and an exclusively
milk diet given; thus much of the otherwise unavoidable dust is done
away with.
Laboratory. — The laboratory should consist of at least three rooms :
(a) stable; (jb) operating-room; (c) laboratory-room. It should be
possible to make and keep all the rooms clean. The stable and oper-
ating-room should be flushed with a hose and hot water daily. Ex-
creta should be removed immediately. The calves can be kept clean
618 PATHOGENIC MICRO-ORGANISMS.
if they stand on a raised and perforated platform which i§ so short
that the defecations cannot fall on it and if they have no bedding.
They must be fastened to keep them from kicking the scarifications.
In the health department, when a calf is removed, its stall and plat-
form are scoured with a brush and sodium carbonate solution. The
stable should be provided with a shovel, broom, hose, horse clipper,
cord, and with halters, buckets, scrubbing brushes, and sponges. The
operating-room should be well-lighted and provided with a table
and with stools.
The only requisites for the table are that it should be heai'y and
firm; that it should have holes through the top so arranged that straps
can be pasesd through them to hold the calf down, and a vertical
strip on one side of the table to which the upper hind leg of the calf
can be fastened. The calf can be thrown upon the table easily by two
attendants.
The laboratory should also be well-lighted and furnished with
tables, chairs, desk, case for instruments, and refrigerator. It should
also have both a steam and a dry-air sterilizer, a set of scales weighing
to grams or centigrams, and a blast lamp and bellows. In stock there
should be one to two thousand bone slips for seed virus and ten to
fifteen thousand smaller slips for issue; two or more scarifiers; a curette;
four to six razors for shaving the animals; a razor strop; a pair of large
scissors, curved on the flat, for clipping the animals; a burette, from
which glycerin flows while the vaccine pulp is being ground; a burette
holder; a Doring vaccine grinder; clinical thermometers to take the
temperature of the animals; six to twelve small glass dishes with covers;
a hard-rubber syringe, of four-ounce capacity, to make suction; absorb-
ent cottpn; glass vials and corks; and several pounds of soft glass
tubing, three-eighths of an inch in calibre, to store virus emulsion.
There should also be gowns and caps for the attendants. Sodium
carbonate, bichloride of mercury, bromine for a deodorizer, alcohol,
carbolic acid, and glycerin are the chemicals needed.
For issue for public vaccinations there are also needed packing-
boxes, rubber bands, sheet wadding, needles, and wooden tooth-
picks for removing the virus from the vials and rubbing it on the
sacrifications.
Yield. — The material obtained from the five children should vac-
cinate at least five calves; it may easily vaccinate fifteen calves. Ten
grams of pulp and two hundred charged slips would be an average
yield from a calf, and that, when made up, should suffice to vacdnate
at least fifteen hundred persons. Calves vary immensely in the yield.
Of two calves vaccinated in precisely the same way one may furnish
material for five hundred vaccinations and the other for ten thousand
vaccinations.
The Durability of Olycerinated Vims in Sealed Tubes. — As a re-
sult of testing from time to time an immense number of specimens
of vaccine, the conclusion has been reached that vaccine properly
put up should keep at least three months. From time to time a single
SMALLPOX AND ALLIED DISEASES. 619
lot of virus will fail by the end of one month. Sometimes this is due
to the glycerin, as when it has some chemical impurity or it is not
diluted sufficiently. When kept below the freezing point it holds its
activity for a longer time.
Bacteria in Vaccine. — It is impossible to prepare vaccine on a
large scale so that it is at the time of its removal free from bacteria.
In fact, there are usually very large numbers of one or more varieties
of bacteria present. When the stable and animals have been kept
clean the bacteria comprise usually very few varieties; when dirty
conditions prevail the bacterial varieties are more numerous. The
number of bacteria found varies enormously. The largest number
found by us in vaccine pulp from the calf was 126,360 in one loop-
ful, and the smallest number 523. Discrete vesicles at the borders
contain many less bacteria than the confluent ones caused by the in-
oculation at the scarification. The pulp has many more bacteria
than the serum of the vesicles. The period which elapses before
glycerinated virus becomes sterile is also quite variable, but does not
depend in any direct way upon the number of bacteria originally pres-
ent. A very large number may disappear rapidly, and a few persist.
After two or three weeks the number of living bacteria is usually
greatly diminished, especially after addition of glycerin-carbolic mix-
ture, when they entirely disapppear. Pathogenic bacteria other than
the practically non-virulent skin staphylococci are not found when
animals are properly kept and vaccinated.
Rabbit Vaccine. — Upon rabbits a practically bacteria-free vaccine
can be obtained, and many laboratories now use rabbits not only to
intensify the virus, but to free it from bacteria. (See p. 613 for
method of obtaining vaccine from rabbits.)
Inoculation of Human Beings. — Efficient vaccine should be inoculated
• in a portion of skin no more than one-sixteenth inch in diameter.
8GARLET FEVER.
Scarlet fever is an acute febrile, highly infectious disease, char-
acterized by a diffuse punctate erythematous skin eruption, accom-
panied by catarrhal, croupous, or gangrenous inflammation of the
upper respiratory tract and by manifestations of general systemic
.infection.
. Historic Note. — The disease was probably known long before the
Christian era, but the present name does not appear until the time
of Sydenham (1685), who differentiated the disease from measles.
The cause is still undetermined.
Occurrence. — It is very generally disseminated, but is much more
common in temperate climates tban in the tropics.
Etiology. — ^The specific exciting factor is thought by many to be
a streptococcus, of the Streptococcus pyogenes type, but the evidence in
favor of this view is very slight.
Recently Mallory reported the presence in scarlet fever of certain
bodies which he considered protozoa and the probable cause of the
620 PATHOGENIC MICRO-ORGANISMS.
disease. He summarized his observations as follows: '*In 4 cases
of scarlet fever certain bodies were found which in their morphology
strongly suggest that they may be various stages in the developmental
cycle of a protozoon. They occur in and between the epithelial celb
of the epidermis and free in the superficial lymph vessels and spaces
of the corium. The great majority of the bodies vary from 2/£ to 7/£ in
diameter, and stain delicately but sharply with methylene blue. They
form a series of bodies, including the formation of definite rosettes with
numerous segments, which are closely analogous to the series seen \n
the asexual development (schizogony) of the malarial parasites, but
in addition there are certain coarsely reticulated forms which may
represent stages in sporogony or be due to degeneration of the other
forms." He has given the name Cyclasterion scarlatinale to these
bodies in consequence of the frequent wheel and star sha|>es of the
rosettes. In our laboratory Field in 1905 examined 10 scarlet fever
autopsies and 20 specimens of living skin taken from patients at diflfer-
ent stages of the disease, together with a number of control specimens
taken from measles, antitoxin rashes, and diptheria; but he was only
able to find a few of Mallory's less characteristic forms, and these only
in the scarlet fever autopsy cases.
Duval (1904) made the announcement that in fluid obtained through
blistering the skin of scarlet fever patients by a very quick method
he has obtained bodies which he interprets as forms of Mallory's
protozoon.
Field obtained similar bodies by the same method in both scarlet
fever and measles cases, and in four cases of scarlatiniform anti-
toxin rashes, more in the first two groups than in the last. He ob-
tained them in no other cases so far examined. Field came to the
conclusion that the majority of them are from degenerated leukocytes.
Since 1905 we have continued the studies on the etiology of scarlet
fever, both from the protozoan and the bacteriologic standpoints. We
have examined the skin of 46 new cases (17 living) and other organs
from 5 autopsies, and though we have found interesting bodies in the
tissue taken from the livivg, as well as from the dead, some corre-
sponding to ^lallory's less definite forms, we have been unable to
demonstrate morphologic characteristics distinct enough to place
these bodies among the microorganisms. (Plate VHI, Fig. 1.)
MEA8LE8.
Field states that he found a moderate number of delicately staining
nucleate bodies in the skin and blister fluid of measles as well as in
scarlet fever, but does not suggest their nature.
A tiny influenza-like bacillus has been found by several observers
in the blood and nasal discharges of measles cases, but nothing has
been proved in regard to its causal relationship to the disease.
Hektoen (1905) produced measles in two human cases by the in-
oculation of the blood drawn from an infected case at an early stage
of the disease.
PLATE VIII
'l I
12) '-?^
-^, . >
'^^^
^:^'i^-
n Gienun's fuchain aiid mrtliyJeiie-l
a (eoaln und methylene bJuo). 2. Yuri' lu d f f
dewTiplian see Uxt. 3. Smear of Amm h f d ^
1 the lufRT blue-stnineil nerve oelb; A', n J us f pell
SMALLPOX AND ALLIED DISEASES. 621
Ewing reports the finding of peculiar granules or ring-shaped
structures in apparent vacuoles about epithelial nuclei and in capil-
laries and lymph spaces of the skin. These were in large numbers in
a case of hemorrhagic measles and in smaller numbers in other cases.
Ewing thinks that the most probable hypothesis in regard to their
nature is that they represent a coagulated albuminous material derived
from the blood and from degenerating epithelium. We have also
found large numbers of similar bodies in a fatal case of acute measles,
and fewer forms in less severe cases.
TRACHOMA.
A good deal of work has been done recently on the etiology of
this eye disease, which is a progressive follicular inflammation of the
conjunctivfle followed by cicatrization. Prowazek in 1907 announced
that the cause of the disease is a tiny organism which grows in a
characteristic manner in the conjunctival epithelial cells. The or-
ganism itself he says is very small, so small that at first it cannot be
seen, only the mantle which it produces is demonstrable; this stains
blue with Giemsa, and as the organisms grow in bunches, one sees at
first in the neighborhood of the nucleus only a bunch of tiny blue
coccus-like bodies. The organism finally appears as a tiny red
granule within the blue body. As it continues to increase in numbers
and size the blue mantles finally disappear, leaving a mass of small
rounded or slightly elongate red bodies. The bodies are only found
in the early acute cases. Prowazek named them Chlamydozoa on
account of their mantle, and thinks they should occupy a place between
bacteria and protozoa (see also pp. 488 and 623).
We have examined about 260 cases, chiefly school children, diag-
nosed clinically trachoma; and, while we have found ** trachoma
bodies" in many (14) of the early acute cases (23), the others have
shown nothing, thus indicating (if these bodies are diagnostic) either
that the great majority of our school children have not true trachoma
or that the "bodies" are too few in these chronic cases to be of
practical aid in diagnosis.
Bibliography.
CouncUmann and his co-workers. Journ. of Med. Research, 1904, Xll, 1.
Osier's Modem Medicine. Philadelphia, Vol. II, 1907.
Ewing. Epithelial Cell Changes in Measles. The Journ. of Inf. Dig., 1909,
VI, 1.
Ewing. Journ. of Med. Research, 1904, XII, 509.
Field. Journ. of Exper. Med., 1905, VII, 343.
Prowazek and HaWerstaedter, Zur Aetiologie des Trachoms. Deutsche med .
Woch., 1907, XXXIII.
Mallory. Journ. of Med. Research, 1904, X, 483.
McCoUomy in Osier's Modern Medicine. Philadelphia, Vol. II, 1907.
Williams and Flournoy, Studies from the Rockefeller Institute for Medical
Research, 1905, Vol. III.
CHAPTER XLVII.
RABIES. YELLOW FEVER.
RABIES.
Introduction. — Rabies (synonyms: Hydrophobia, Lyssa, Hunds-
wuth, Rage) is an acute infectious disease of mammals, dependent upon
a specific virus, and communicated to susceptible animals by the
saliva of an infected animal coming in contact with a broken surface,
usually through a bite. The name rabies is given to the disease
because of its most frequent and characteristic symptom — ^furor or
madness. Hydrophobia (Greek, fear of water) is another name com-
monly used, which is also given because of a frequent symptom of the
disease, the apparent fear of water. Lyssa is a Greek word indicating
still another symptom, i.e., swollen follicles on the under surface of
the tongue. Within the gray nerve tissue of rabid animals are peculiar
protozoon-like structures known as **Negri bodies" which are diagnostic
of rabies. The nature of these bodies is still a question of dispute.
Historical Notes. — Rabies is probably one of the oldest diseases in exist-
ence, but because of the occurrence of so few human cases, and because
the disease develops so long after the bite, its source was for a long time not
known nor was it recognized as a separate disease. Hippocrates does not
mention it in his writing, but Aristotle about 50 years later (about 300 B. C.)
speaks of its being purely an animal disease and being carried by the bite
of one animal to another. Celsus in the first century was the firet to give
in writing a detailed description of human rabies. He s{>eaks of it being
produced by the bite of rabid animals and states that the wound must be
thoroughly bathed and then burned with a hot iron in order to prevent
the development of the disease, for after symptoms appear death always
follows. As Celsus was not a physician he must have gotten his knowledge
from writings which have since been lost. Other writers soon after gave
very true descriptions of the symptoms and handling of the disease.
Many hundred years passed after this without adding anything to our
knowledge of the disease, though authors on the subject were numerous.
Van Sweiten in 1770 observed the paralytic form of rabies in human beings.
At this time several authors, among them Morgagni and Zwinger, believed that
the bite of a dog which was not suffering from rabies might produce the dis-
ease in man. In 1802 Bosquillon brought forth the original idea that belief in
the existence of infectious material in rabies was a chimera and that hydro-
phobia was simply due to fright. This false idea had adherants for a long
time ; even now, by a few people, it is throught to be a true one.
Among the host of good observers who studied the disease during the
latter part of the nineteenth century, Pasteur stands out as the discoverer, in
1880, of the fact that the disease may be prevented by inoculating gradually*
increasing doses of the virus into the person or animal bitten. This treat-
ment with some modifications, the details of which will be given later, is
still used, though many efforts have been made to develop an efficient senmi
treatment. Pasteur, as well as numerous other investigators, tried to dif^-
G22
RABIES. 623
cover the specific cause of rabies, but all of their results were negative. The
importance of making a quick diagnosis had become so evident that the efforts
of many workers were directed toward this end alone.
Pasteur and his immediate followers relied for their diagnosis entirely upon
rabbit inoculations, and this meant a fifteen to twenty days' wait before the
patient knew whether or not the treatment he was receiving was necessary.
In 1898 this time was shortened to about nine days in our laboratory by
Wilson, who found that guinea-pigs came down with the disease much more
quickly than rabbits. From time to time it has been thought that certain
histologic findings were diagnostic; for instance, the "rabic tubercles" of
Babes, and the areas of "round and oval-celled accumulation in the cerebro-
spinal and sympathetic ganglia'' of Van Gehuchten and Nelis, were said to
be specific, but further study has shown that they are not absolutely specific
for rabies. In many cases the whole picture of the grosser histologic changes
is sufficiently characteristic to warrant the diagnosis of rabies, but often it is
not so.
It is not until Negri, in 1903, described certain bodies (Negri bodies) seen by
him in large nerve cells in sections of the central nervous system, that anything
was found which seemed absolutely specific for hydrophobia. Negri claims
that these bodies are not only specific for rabies, but tnat they are probably
animal parasites and the cause of the disease.
Negri's later studies confirm his previous work and add some new facts
in regard to the structure of the larger bodies.
His work, especially as far as the diagnostic value of these bodies is con-
cerned, has been corroborated by investigators in almost all parts of the
scientific world, among them workers in our own laboratory who not only
determined their worth in diagnosis, but investigated their nature.
In our work emphasis was placed upon the fact that the demonstration of
the ** Negri bodies" by our *' smear method" (see p. 624) wonderfully sim-
plified the process of diagnosis. As a result of our studies we concluded that
the Negri bodies are not only specific for rabies, but that they are living organ-
isms, belonging to the protozoa, and are the cause of the disease; giving as our
reasons the following facts: (a) They have a definite, characteristic mor-
phology; (6) This morphology is constantly cyclic, that is, a definite series of
forms indicating growth and multiplication can be demonstrated; (c) The
structure and staining qualities, as shown especially by the smear method of
examination, resemble those of certain known protozoa, notably of members
of the rhizopoda.
Since this report was published many more cases of rabies have been
more or less studied by us and our former conclusions have been more
firmly established. Indeed, the evidence as to animal nature of these
cell inclusions seemed so convincing that Williams in 1906 gave them
the name Neuroryctes hydrophobicB,^ Calkins has since studied these
bodies and agrees with Williams as to their nature. He called attention
to the similarity between their structure and that of the rhizopoda.
A number of observers, however, still believe that the Negri body
as a whole is principally the result of cell degeneration and that the
specific organism may be contained within it. Prowazek includes
rabies with his " chlamydozoan diseases*' (see p. 621). To anyone
who has studied the two diseases, however, there can be no question
in regard to the essential diflference between the " trachoma bodies "
and the ** Negri bodies."
> Proceedinga of the N. Y. Pathological Society, 1906, VI, 77.
624 PATHOGENIC MICRO-ORGANISMS.
Material and Methods for Study. — In New York one may almost con-
stantly obtain fresh brains of rabid animals^ from veterinary hospitab or
from the laboratories handling this material. Two methods have been used
in helping to study the principal site of infection.
(1) Animal inoculations; (2) Sections and smears.
The first method is used as a decisive test in diagnosis when results from
the second method are doubtful.
The technic of the smear method used at present in the Research Labora-
tory of the New York City Health Department is as follows;
1. Glass slides and cover-glasses are washed thoroughly with soap and
water, then heated in the flame to get rid of oily substances.
2. A small bit of the gray substance of brain chosen for examination is
cut out with a small sharp pair of scissors and placed about one inch from
the end of the slide, so as to leave enough room for a label. The cut in the
brain should be made at right angles to its surface and a thin slice taken,
avoiding the white matter as much as possible.
3. A cover-slip placed over the piece of tissue is pressed upon it until
it is spread out in a moderately thin layer; then the cover-slip is moved
slowly and evenly over the slide to the end opposite the label. Only slight
pressure should be used in making the smear, but slightly more should be
exerted on the cover-glass toward the label side of the slide, thus allowing
more of the nerve tissue to be carried farther down the smear and producing
more well-spread nerve cells. If any thick places are left at the edge of the
smear, one or two of them may be spread out toward the side of the slide
with the edge of the cover-glass.
4. For diagnosis work such a smear should be made from at least three
different parts of gray matter of the central nervous system : first, from the
cortex in the region of the fissure of Rolando or in the region corresponding
to it (in the dog, the convolution around the crucial sulcus); second, from
Ammon's horn, and, third, from the gray matter of the cerebellum.
5. The smears are partially dried in air and fixed for about ten seconds in
neutralized methyl, alcohol to which one- tenth per cent, picric acid is added
6. The excess of alcohol is removed by pressing fine filter-paper gently
over the smear.
7. The methylene-blue-fuchsin staining mixture recommended by Van
Gieson is poured over the slide, warmed until it steams, poured off", and the
smear is washed in running tap water, and allowed to dry, the excess of water
being removed with fine filter-paper.
The staining mixture recommended by Van Gieson is made by us at pres-
ent in the following proportions :
5 c.c. distilled HjO; 10 drops sat. ale. sol. meth. blue; 2 xirops sat. ale.
sol. basic fuschsin.
This mixture in room temperature in diffuse daylight will keep for a day.
and possibly two. In the dark at room temperature it retains its staining
powers a little longer. At ice-box temperature it lasts a much longer time,
probably indefinitely.
With this method the Negri bodies stain magenta, their contained granules
blue, the nerve cells blue, and the red blood cells yellow.
Other methods we have found useful for staining smears are the following:
Oiemsa's Solution. — Smears are fixed in neutralized methyl alcohol for
one minute. The staining solution recommended last by Giemsa* (1 drop of
* Azur II — Eosin 3 . 0 g.
Azurll 0.8
Glycerin (Merk. chem. pure) 250.0 c.c.
Methyl alcohol (chem. pure) 250.0
Both glycerin and alcohol are heated to 60° C. The dyes are put into the al-
cohol and the glycerin is added slowly, stirring. The mixture is allowed to stand
at room temperature overnight, and after filtration is ready for use.
The solution is prepared ready for use by Griibler, Leipzig.
RABIES. 625
the stain to every c.c. of distilled water made alkaline by the previous addition
of one drop of a 1 per cent, solution of potassium carbonate to 10 c.c. of the
water) is poured over the slide and allowed to stand for one-half to three
hours. The longer time brings out the structure better, and in twenty-four
hours well-made smears are not overstained. After the stain is poured off,
the smear is washed in running tap water for one to three minutes, and
dried with filter-paper. If the smear is thick, the '* bodies" may come out a
little more clearly by dipping in 50 per cent, methyl alcohol before washi^ig
in water; then the washing need not be as thorough. By this method of
.staining, the cytoplasm of the "bodies'* stains blue and the central bodies
and chromatoid granules stain a blue-red or azur. Generally the larger
** bodies" are a darker blue than the smaller, the smallest of all may be very
light. The cytoplasm of the nerve cells stains blue also, but with a success-
fully made smear the cytoplasm is so spread out that the outline and struc-
ture of most of the "bodies" are seen distinctly within it. The nuclei of
the nerve cells are stained red with the azur, the nucleoli a dull blue, the
red blood cells a pink-yellow, more pink if the decolorization is used. The
** bodies" have an appearance of depth, due to their slightly refractive
qualities.
The Eosin -methylene -blue Method Recommended by Mallory. — The
smears are fixed in Zenker *s solution* for one-half hour; after being rinsed in tap
water they are placed successively in 95 per cent, alcohol -f iodine one-quarter
hour, 95 per cent, alcohol one-half hour, absolute alcohol one-half hour,
eosin solution twenty minutes, rinsed in tap water, methylene-blue solu-
tion fifteen minutes, differentiated in 95 per cent, alcohol from one to five
minutes, and dried with filter-paper. With this method of staining, the
cytoplasm of the "bodies" is a magenta, light in the small bodies, darker
in the larger; the central bodies and chromatoid granules are a very dark
blue, the nerve-cell cytoplasm a Hght blue, the nucleus a darker blue, and
the blood cells a brilliant eosin-pink. With more decolorization in the alco-
hol the "bodies" are not such a deep magenta, and the difference in color
between them and the red blood cells is not so marked.
The technique of the section work is as follows: (1) The small pieces are left
in Zenker's fluid for three to four hours; (2) washed in tap water for five
minutes; (3) placed in 80 per cent, alcohol -f- iodine* (enough tincture of
iodine added to give port-wine color) for about twenty-four hours; (4) 95
per cent, alcohol -f iodine twenty-four hours ; (5) 95 per cent, alcohol twenty-
four hours; (6) absolute alcohol from four to six hours; (7) cedar oil until
cleared; (8) cedar oil-f paraffin 52° two hours; (9) paraffin 52° two hours
in each of two baths; (10) boxing; (11) sections are cut at 3 to 6/*, dried
in thermostat at 36° C. for about twenty-four hours protected from the
dust, and stained according to the eosin and methylene-blue method recom-
mended by Mallory. The most important point in the technique is the time
the material is allowed to remain in Zenker. According to our experience,
two hours* fixation is not enough, three to eight hours is very good, and
wit h every hour after eight hours the results become less satisfactory. Left
in Zenker overnight the tissue is granular and takes the eosin stain more or
less deeply, both of which results interfere with the appearance of the tiniest
"bodies," especially of the very delicate, tiny forms found by us in sections
from fixed virus.
In regard to the rest of the technique, it is sufficient to say that the changes
to the different fluids are made with great regularity, and the final differen-
tiation in alcohol of the stained sections is done most carefully.
In the sections made in this way we have been able to demonstrate clearly
very tiny forms, as well as good structures in the larger forms.
> See p. 522.
^ Better results are obtained by treating the tissue with iodine after the sections
are cut, just before staining, as they then do not need to be so long a time in the
iodine solution — ten minutes to half an hour being sufficient.
40
626 PATHOGENIC MICRO-ORGANISMS.
Harris has recently published a new staining method for both sections sod
smears (see bibliography), which brings the larger bodies out clearly, but
which doea not seem to give eoough differentiatioD between the smallet
. bodies and the nucleoli of the nerve cells.
MorpholoB7 of the Negri Bodies (Plate VIII, Figs. 2 and 3).-
The largest forms measured are about 18/( and the smallest about
0.5/1. They are round, oval, oblong, triangular, or amceboid. The
latter are more numerous in the fixed virus of rabbits and guioea-pijts.
Their structure is shown especially well in smears. WTiatever the
variety or species of animal infected, the bodies present the satae
general characteristic structure; i. e., a hyaline-like cytoplasm with
an entire margin, containing one or more chromatin bodies haring
a more or less complicated and regular arrangement.
Their structure varies to a certain extent with their size. In fi-^ted
virus, with an occasional exception, only tiny forms are found (Plate
VIII, Fig. 2 a-d). These are rounded or sometimes wavy in outline.
Negri body showing central chromatin with ring of anull granules. X 2000.
as if posses.sing slight amceboid motion, sometimes elongated, extend-
ing along the rim of the host-cell nucleus, or along one of the ncne
fibrils, as if moving there; with eosin and methylene blue they take a
delicate light magenta stain, very similar to that taken by the small
serum globules in the blood vessels. Many of the organisms, however,
show a small chromatin granule, situated more or less eccentrically.
sometimes on the very rim of the body. In the larger forms the
granule is large, in the smaller it cannot always be seen; some of the
larger forms show from two to several granules and occasionally there
is a body with the definite central body and the small granules about il.
Detailed Charad eristics of Structure in ihelAirge Forms (Fig. 195 and
Plate VIII, Fig. 2 e-f). — In smears, as well as in sections, the cytoplasm
appears quite homogeneous; there is no evidence of a reticulum or of
a granular structure out.side of the definite chromatoid granules.
RABIES. 627
The smears, however, have brought out one important point in regard
to the cytoplasm more clearly than the sections, and that is that it is
more basophilic than acidophilic in staining qualities. With the
Giemsa stain, as we have already seen, it takes the methylene-blue
stain more than the eosin-red, and even with the simple eosin methy-
lene-blue stain the protoplasm appears as a deep magenta unless
much decolorized.
In studying the central bodies of these organisms, as they appear
in the smears, one of the first things noticeable is that they are not
surrounded by a clear space — that there is no sign of a vacuolar ap-
pearance in the body. This is a very different appearance from that
given in the sections, and it shows that the vacuoles seen in the sec-
tions are artefacts due to the technique. We notice next that in the
great majority of the organisms the central body stands out clearly,
as decidedly different in structure, and slightly so in staining quali-
ties, from the chromatoid granules which surround it. The general
tj'pe of the structure of the central body is that of many well-known
protozoan nuclei.
The chromatin is arranged in a more or less granular ring around
the periphery of the central body or nucleus, leaving an achromatic
or more acid-staining centre in which is situated, generally eccentrically
a varying-sized karyosome (Plate VIII, Fig. 2p). There are a
number of variations from this principal tj^e, according to stage
of development. Often the whole nucleus answers to the description
of the compound karyosome given by Calkins in his description
of the protozoan nucleus. In the tiny "bodies," as we have said,
the chromatin can only be seen as a dot; in those a little larger it
may be a large solidly staining granule, or a ring or rod, the latter
often hour-glass-shaped. In forms large enough for the character-
istic structure to be developed and to be clearly seen, the central
body may show evidence of fragmentation (Plate VIII, Fig. Iq, etc.).
Just such evidence of fragmentation is shown in many protozoan nuclei
preparatory to division, notably among the rhizopoda. It is interesting that
forms showing this phase, and, moreover, very similar in general appearance
to some of the forms seen here, have been depicted by Doflein in the early
stages of the life-cycle of Nosema lophii, a myxosporidium, parasitic in the
ganglion cells of a fish Lophius piscatorius.
•
The fragmented particles seem to be leaving the nucleus in cer-
tain forms, and in this way presumably the chromatoid granules are
produced, thus forming chromidia.
The chromatoid granules are most frequently arranged in a more
or less complete circle about the nucleus. They are somewhat ir-
regular in outline and size, being occasionally ring-shaped, some-
times elongated, often in two's, due probably to active changes of
growth and division. They take generally a more mixed chromatin
stain than the chromatin of the nucleus.
Evidences of Division. — All stages in transverse division are seen.
Many evidences of budding are also seen. The chromatoid granules
628 PATHOGENIC MICRO-ORGANISMS,
divide and pass out with part of the cytoplasm as a bud. This bud-
ding or unequal division appears to take place very early in the growth
of the organism and to continue throughout growth until the parent
body forms a mass of small organisms which may then break apart at
the same time. The budding accounts for the number of small and
large forms in a single cell.
Number. — ^They vary in number according to the stage of the
disease and to the infectivity of the part.
Site. — They are situated chiefly in the cytoplasm and along the
fibres in the branches of the large nerve cells of the central nerv-
ous system. In parts of smears which are more broken up the bodies
may appear as if lying free, and it is these bodies, if the pressure
be not too great in smearing, that show the structure best. In some
cases the bodies are distinctly localized in small scattered areas of the
central nervous system. We have always found bodies in the spinal
cord in abundance, but here they are especially prone to be locaIize<l
in discrete groups of cells.
That the organisms are present in various glands of the body (salivarj*
thyroid, suprarenal capsule, etc.) is shown by the virulence of emul-
sions from these organs. Cows' milk (Westbrook, McDaniel) and
blood (Marie) have also been shown to be slightly virulent.
Diagnosis of Babies. — In our laboratory, for the past five years,
or since we have used the smear method in routine diagnosis, there
have been about fifteen hundred cases in all examined, including
suspected rabies and controls.
These are divided into two groups, the first comprising the cases
sent in from outside, for diagnosis only, and the second, the experi-
mental cases.
Since the publication of our work in May, 1906, in our routine
work we have considered the presence of the Negri bodies in smears
as diagnostic of rabies and have made no further tests except in those
cases which we have used in our experimental work. Through
this experimental work,' however, we have added three hundred cases
to the list of those which had the comparative tests, and our former
conclusions have been more firmly established.
In all of our work controlled by careful animal inoculations we have
never yet failed to have typical rabies develop in animals inoculated
with material showing definitely structured Negri bodies. Negative
results after inoculation with such material must be interpreted at
present as due to some error in technique, such as regurgitation, or
hemorrhage at the time of inoculation, emulsion improperly made,
not enough of the virulent material taken because of localization of
the organisms, etc.
Possibly individual resistance of the animal inoculated might play a part.
We have used principally guinea-pigs, and some of them have shown enough
irregularity in regard to the time in which they have come down with the
disease to suggest a varied individual susceptibility, if other factors can be
ruled out.
RABIES. 629
On the other hand, material in which we have failed to demon-
strate typically structured bodies has produced rabies. All of this
material, however, since we have improved our technique, has shown
suspicious small forms similar to those found in rabbit-fixed virus.
But any decomposing brain may also show in smears bodies very
similar to these tiny forms, therefore it is difficult to rule out rabies
in such cases. Of course the animal test will probably always have
to be used with brains that are too decomposed to show any formed
elements except bacteria, unless a reliable chemical test can be
discovered.
So far we have not had rabies produced by fresh brains showing no
Negri bodies and no suspicious forms, but a few observers have claimed
that such material has produced the disease. Therefore, until we can
standardize our technique, we must in all such cases use animal inocu-
lations. We may, however, be reasonably certain that a case showing
such negative material was not a case of rabies. We may summarize
our knowledge in regard to the worth of the smear method in diagnosis
as follows:
1. Negri bodies demonstrated, diagnosis rabies.
2. Negri bodies not demonstrated in fresh brains, very probably not rabies.
3. Negri bodies not demonstrated in decomposing brains, uncertain.
4. Suspicious bodies in fresh brains, probably rabies
The localization of the Negri bodies is an important point in mak-
ing diagnoses. We have found well-developed bodies distinctly local-
ized in different parts of the brain in several instances. In one horse
there were small widely scattered areas of well-structured forms through-
out the cerebellum, while tiny indefinite forms were scattered through
the rest of the brain examined. In two human brains well-developed
forms were found in the corpus striatum and not in the rest of the brain.
In several dogs the localization has also been marked.
The Complement Binding Test in Babies.— This test has been
tried by Heller (1907), Friedberger (1907), and Baroni (1908), with
negative results. Berry (1910), in our research laboratory went over
this work thoroughly and obtained similar negative results.
Effect of Chemic and Physic Agents on Babic Virus.— Rabic virus
appears to become attenuated under certain conditions of tempera-
ture; indeed, if it be subjected for about an hour to 50° C. or for
half an hour to 60° C, its activity is completely destroyed. A 5
per cent, solution of carbolic acid, acting for the same period, exerts
a similar effect, as do likewise 1 : 10(K) solutions of bichloride of
mercury, acetic acid, or potassium permanganate. The virus also
rapidly loses its strength by exposure to air, especially in sunlight;
when, however, protected from heat, light, and air it retains its viru-
lence for a long period.
Pathogenesis. — Natural Infection. — The disease occurs in nature
among the following animals given in order of their frequency: dogs,
cats, wolves, horses, cows, pigs, skunks, deer, and man; in fact, as all
630 PATHOGENIC MICRO-ORGANISMS,
warm-blooded animals are more or less susceptible to inoculations, all
may presumbly contract the disease when an open wound is brought in
contact with infectious material of a rabid animal.
Rabies occurs in almost all parts of the world. It is most common in
Russia, France, Belgium, and Italy; it is not infrequent in Austria and
in those parts of Germany bordering on Russia. In this hemisphere
it is infrequent in Canada, but in the United States the cases are in-
creasing in numbers, especially during the last year when there have
been several epidemics in some of the northwestern States. In Cali-
fornia several cases have recently been reported. In England, Mexico,
and South America it occurs occasionally; while in England, North
Germany, Switzerland, Holland, and Denmark, because of the en-
forced quarantine laws, and to the wise provision that all dogs shall
be muzzled, it is extremely rare. In Australia it is unknown, probably
because the law that every dog imported into the island must first
undergo a six months' quarantine has always been enforced. In the
vicinity of New York the disease seems to be on the increase.
The contagion is supposed always to be carried through the bite of
a rabid animal or through the sputum of such an animal coming in
contact with an open wound.
In this connection the question as to how long the sputum of a rabid dog
may remain virulent after it drops from the animal is an interesting one.
A case came under our observation in 1906 which illustrates this point. A
child of six years came down with typical rabies in a neighborhood where
there had recently been several cases of canine rabies, but no history of a
bite could be obtained. The parents were sure she had not been bitten.
Six weeks before, however, the child had fallen in the street and cut her
cheek severely on a jagged stone. The wound was cauterized and healed
without further trouble. A mad dog had been on that street just before this
occurred. It is reasonable to suppose that the stone had on it some of the
sputum from that dog, and so the child was infected. Such a case would not
occur very often, but the possibility should be considered.
In regard to the question as to whether the bite of apparently healthy
animals may give the disease, it may be said that, judging from labora-
tory experiments, some animals may have a light attack of the disease
and recover spontaneously, though such cases, if they occur, are prob-
ably extremely rare. That the bite of an infecfed animal may give the
disease before that animal shows symptoms has been proved. Nine
days is the longest time reported between a bite and the appearance of
symptoms. Therefore, if an animal is kept under observation two
weeks after biting another, without developing symptoms, he may be
pronounced free from suspicion.
Occupation seems to have an effect upon the number of cases among
humans in one way. Those people who are much in the country or on
the streets — in other words, those who might come most frequendy
in contact with rabid animals — most frequently contract the disease;
otherwise neither age, sex, nor occupation has any effect.
The time of the year seems to have little effect, though most cases
RABIES. 631
are said to occur during the summer months. The numbers vary
with different years. In 1907 for instance we had as many cases in
January as in August and in September and more in June than in any
other month.
The certainty with which the disease may be produced after a bite
and the rapidity of its development have been found to be governed
by three factors: (1) the quantity of the rabic virus introduced;
(2) the point of inoculation; (3) the strength of the virus as deter-
mined by the kind of animal which affords the cultivation ground
for the growth of the organism. It is a matter of common observation
in man that slight wounds of the skin of the limbs and of the back or
wherever the skin is thick and the nerves few either produce no results,
especially when bites are made through clothes, or are followed by the
disease after an extremely long period of incubation; while in lacerated
wounds of the tip of the fingers where small nerves are numerous or
where the muscles and nerve trunks are reached, or in lacerated
wounds of the face where there is also an abundance of nerves the
period of incubation is usually much shorter and the disease generally
more rapid.
These facts explain why only about 16 per cent, of human beings
bitten by rabid animals and untreated appear to contract hydrophobia.
Since the establishment of the Pasteur treatment for the disease,
the percentage of developed cases after bites is very much less, a
fraction of 1 per cent.
Symptoms. — There is always a decided incubation period after the bite
which varies within quite wide limits, but in the great majority of cases it
is from twenty to sixty days. Any period after six months is an exception;
the shortest we have on record is fourteen days and the longest authentic
period is seven months. A very few apparently authentic cases have been
reported as developing in about one year, but reports of any time beyond
this must be received with doubt. After treatment, however, a few cases
have been reported as occurring later than this, but even here the question
of reinfection is not absolutely ruled out. We had a case illustrating this:
One of our patients, a man who helped a dog veterinarian, was treated after
a severe wound from a rabid animal, and fourteen months later came down
with typical hydrophobia; but we found that since his treatment he had be-
come very careless wi^h cases of rabies because he considered himself immune.
He was warned that there might be danger, but six weeks before his death
he put a wounded hand into the mouth of a rabid animal. There seems to
be no doubt but that it was a case of reinfection after loss of protection from
the treatment rather than one of delayed rabies.
The wound heals as other wounds and sometimes shows no further symp-
toms. Occasionally, however, redness and swelling of the scar have been
reported; oftener there are pains extending from the scar along the nerve
paths to the brain.
The symptom-, may be divided into three stages. First, the prodromal or
melancholic stage; second, the excited or convulsive stage; and, third the
paralytic stixge.
When the second stage is the most pronounced the disease is called furious
or convulsive rabies; when this stage is very short or practically lacking and
paralysis begins early, the disease is called dumb or paralytic rabies.
In the dog rabies appears in the two typical forms, the furious and the
632 PATHOGENIC MICRO-ORGANISMS.
paralytic. The principal symptoms of each form may be summarized as fol-
lows : (a) Furious rabies; change of behavior, biting (especially at those to whom
the animal has been affectionate before), increased aggressiveness, charac-
teristic restlessness, loss of appetite for ordinary food, with desire to eat
unusual things, intermittent disturbance of consciousnQgs, paroxysms of
fury, peculiar howling bark, rapid emaciation, paralysis, beginning in the
hind limbs, death in great majority of cases in three to six days (exceptionally
slightly longer) after the beginning of symptoms. (6) Paralytic rabi^it:
short period of excitation, paralysis of the lower jaw, hoarse bark, appetite
and consciousness disturbed, weakness, with paralysis spreading in great
majority of cases, and death four to five days after first symptoms. There
may be a number of cases showing transition types between these two form.-*.
La Human Beings. — Furious Rabies. — The first definite symptoms are
difficult and gasping breath with a feeling of oppression and difficulty in
swallowing, the latter, the most characteristic symptom. It is caused by
convulsive contraction of the throat muscles. The attacks are brought out
when attempting to drink or swallow. The very thought of drinking may
bring one on ; and though there is no fear of water itself, there is fear of taking
it because of the effect it produces. The convulsive attacks finally become
more or less general over the whole body; in certain cases some parts are more
aflfected by reflex excitation than others; for instance, there may be slight
or no photophobia, while in exceptional cases, more frequently in dogs, the
hydrophobia is also absent.
Most of the special reflexes are increased. Pupils become irregularly con-
tracted and widened until they finally remain fixed.
Human beings are seldom dangerous to the people about them: they do
not make aggressive bites. In their convulsions they may bite things placed
between their teeth, but not otherwise. At this time there is an increased
flow of saliva, and one should avoid the contact of this with opened wounds'.
It may be so increased that the patient may try to get rid of it by taking
it from the mouth with the hand and throwing it about. As a general thing,
however, the patient has full possession of his senses between the con\iilsive
attacks until very late in the disease.
Few changes have been noticed in the urine. The bowels are generally
constipated, the temperature is increased from 38° C. to 40*^ C, at first ^nth
morning remissions. Just before death it may rise as high as 42.8*^ C (In
lower animals the temperature sinks below normal just before death.) The
pulse is generally over 100 and is irregular. This stage lasts from one to
four days. Death may occur during a convulsion, but more ofter there is a
paralytic stage which lasts from two to eighteen hours. The convulsions
become less frequent and the patient becomes weaker until finally there is a
complete paralysis. At the beginning of this stage the patient may be able
to drink water better than formerly. Death may occur at any time through
paralysis of the heart or respiratory center.
Paralytic Rabies. — This form occurs quite seldom in human beings, more fre-
quently in dogs, but not so often as a convulsive form. It is supposed to occur
in humans and dogs after a more severe infection. Instead of periods of
convulsions, the various muscles simply tremble and become gradually weaker
until complete general paralysis supervenes. Sometimes paral3rsis develoj)??
very quickly and may be general before death from syncope or asph\^ia
occurs. This form generally lasts longer than ordinary rabies. Between
these two typical forms of rabies there are many different types, gi\ing
quite different pictures of -the disease.
In certain cases which have been badly bitten, treatment with protective
inoculations may not save the patient, but may cause the disease to mani-
fest itself quite late and then the symptoms may be milder than in untreated
cases, though death finally results.
Length of the Disease. — The majority of the cases of furious rabies die
RABIES, 633
on the third or fourth day after the symptoms show themselves. The limits
of the reported cases are one to fifteen days, though there are reports of only
one or two cases dying on any day over the ninth to the fifteenth. As the
time when the symptoms really begin is difficult to notice, these statistics are
probably only approximately correct. In paralytic rabies the average time
of death is five days.
Treatment. — The old treatment of rabies consisted simply in
encouraging bleeding from the wound, or in first excising the wound
and then encouraging bleeding by means of ligatures, warm bathing,
cupping-glasses, etc. ; the raw surface was then freely cauterized with
caustic potash, nitric acid, or the actual cautery. It is doubtful whether
the disease ever manifested itself after such heroic treatment if the wound
were small and the treatment was begun soon after the bite; but when
the wounds were numerous or extensive, the mortality was still high.
As it was often impossible to apply cauterization to the wound rapidly
or deeply enough to ensure complete destruction of the virus, Pasteur
and others were led to study the disease experimentally in animals
with the hope of finding some means of immunization or even cure;
these investigations finally resulted in the discovery of methods of
preventive inoculation applicable to man.
Pasteur's Method of Preventive Inocolation.— Pasteur's treat-
ment is based upon the fact that rabic virus may be attenuated or
intensified under certain conditions. He first observed that the tissues
and fluids taken from rabid animals varied considerably in their viru-
lence. Then he showed that the virus may be intensified by success-
ive passage through certain animals (rabbits, guinea-pigs, cats) and
weakened in passing through others (monkeys). If successive inocu-
lations be made into rabbits with virus, either from the dog or the
monkey, the virulence may be so exalted beyond that of the virus
taken from a street dog, in which the incubation period is from twelve
to fourteen days, that at the end of the fiftieth passage the incubation
period may be reduced to about six or seven days. This, the strongest
virus obtainable, was called by Pasteur the ''fixed virus," This fixed
virus was used by Pasteur in his preventive treatment and has been
since used as follows.
A series of spinal cords taken from rabbits dead from ** fixed
virus" infection are cut into short segments and suspended in sterile
glass flasks plugged with cotton stoppers and containing a quantity
of some hygroscopic material, such as caustic potash; these are kept
at a temperature of about 22° C. The cord when taken out at the
end of the first twenty-four hours is found to be almost as active
as the fresh untreated cord; that removed at the end of forty-eight
hours is slightly less active than that removed twenty-four hours pre-
viously; and the diminution in virulence, though gradual, progresses
regularly and surely until, at the end of the eighth day the virus is in-
active. An emulsion of the cord kept until the fourteenth day is made,
and a certain quantity injected into the animal that has been bitten;
this is followed by an injection of an emulsion of a thirteenth-day
634 PATHOGENIC MICRO-ORGANISMS.
cord; and so on until the animal has been injected with a perfectly
fresh and, therefore, extremely active cord, corresponding to the fixed
virus. Animals treated in this way were found by Pasteur to be
absolutely protected, even against subdural inoculation with con-
siderable quantities of the most virulent virus; and thus Pasteur's
protective inoculation against rabies became an accomplished fact
As it would be undesirable to inject any but persons who had actually
been bitten by a rabid, or presumably rabid, animal Pasteur con-
tinued his experiments in order to see whether it would not be possible
to cure a patient already bitten. He carried on, therefore, a series
of experiments which led to the discovery that if the process of inocu-
lation be begun within five days of the bite in animals in which the
incubation period was at least fourteen days, almost every animal
bitten can be saved; and that even if the treatment be commenced
at a longer interval after the bite a certain proportion of recoveries
can be obtained. Thus the application of this method of treatment
to the human subject was not tried until it had been proved in animals
that such protection could be obtained and that such protection would
last for at least one year and probably longer.
The chance of success in the human subject appears to be even
greater than in the dog or' rabbit, seeing that, on account of the resist-
ance offered by the human tissues to the virus, the period of incuba-
tion is comparatively prolonged. Thus there is an opportunity of
obtaining immunity by beginning the process of vaccination soon after
the bite has been inflicted, the protection being complete before the
incubation period has passed. In his earlier experiments Pasteur in-
jected on each succeeding day emulsions from a cord dried for one
day less until cords dried five days were reached; but later he used
those dried for only three days. This was the "simple" ten-day
method. It was soon evident that although this method was effica-
cious where the wounds were not severe and were confined to parts
in which the nerve supply was not extensively interfered with, it was
often quite inadequate in serious cases, as of wounds about the face
or of wounds inflicted by a mad wolf, the virus of which is more ac-
tive and the lesions made more severe than that of the rabid dog of
the streets. In these latter cases the injections which, in the simple
treatment, were spread over five days were made in three days; then,
on the fourteenth day, a fresh series of injections or, rather, repe-
titions, was begun, which lasted until the twenty-first day. This was
called the ** intensive method."
Present Administration of Pasteur's Treatment in Hmnan Beings^
— A small portion (about 1 cm.) of the cord is rubbed up thoroughly
with three cubic centemeters of bouillon until a complete emulsion is
made; this is then injected by means of a hypodermic syringe, first on
one side of the hypochondriac region and then on the other. With
the observance of thorough asepsis no local reaction to speak of take-s
place nor are abscesses ever formed.
Inoculations. — The series of inoculations given in the Research
RABIES.
635
Laboratory in treating human cases after an average bite are as
follows :
Mild Treatment
Intensive Treatment
FOR Severe Cases.
1st day,
2d day,
3d day,
4th day,
5th day,
6th day,
7th day,
8th day,
9th day,
10th day,
nth day,
12th day,
13th day,
14th day,
15th day,
16th day,
17th day,
18th day,
4, 3, 2,
(duration,
14 and 13-day cord 12 and 11-day cord
(Repeated in afternoon)
12 and 11-day cord 10 and 9-day cord a.m.,
8 and 7-day cord p. m.,
10 and 9-day
8 and 7-day
6-day
5-day
4-day
3-day
2-day
4-day
3-day
2-day
4-day
3-day
2-day
4-day
3-day
2-day
until the end
16 to 21 days).
cord
6-day cord
cord
5-day cord
cord
4-day cord
cord
3-day cord
cord
2-day cord
cord
4-day cord
cord
4-day cord
cord
1-day cord
cord
4-day cord
cord
3-day cord
cord
2-day cord
cord
4-day cord
cord
1-day cord
cord
4-day cord
cord
3-day cord
cord
2-day cord
4, 3j 2, until the end
(duration, 21 to 26 days).
Some Pasteur Institutes (Berlin; Washington, D. C.) begin treatment
with the eighth-day cord.
Results. — The results of Pasteur's method of protective inoculation,
as recorded in the reports issued in the Annales de VInstiiut Pasteur and
those of other antirabic institutes in Italy, Russia, Roumania, etc., are
very favorable. Since 1886, when the treatment was first commenced at
the Pasteur Institute in Paris, over 30,000 persons bitten by rabid, or pre-
sumably rabid, animals have received preventive inoculations, with a mor-
tality of only 0 . 5 of 1 per cent. The mortality of those bitten on the face
or head was 1 .25 per cent.; of those bitten on the hand, it was 0.75 of
1 per cent.; of those bitten on other parts of the body, a little over
0 . 25 of 1 per cent. As a rule, only those persons are treated who have
been bitten on the face or hand or whose clothes have been lacerated
so that the virus has passed into the wounds. Taking only the cases in
which rabies has been confirmed in the animal by a competent examiner,
the mortality of the cases treated at the Pasteur Institute in Paris is
only 0.6 per cent. — a proof, it would seem, of the trustworthiness of
the statistics. In view of this fact there can no longer be any doubt
of the value of Pasteur's antirabic treatment. It has been stated by
some that the percentage of persons killed by the bites of rabid animals
is inconsiderable; but, according to the reliable statics of I^blanc,
from 1878 to 1883, out of 515 persons bitten in Paris, 83 died of hydro-
phobia, a mortality of 16 per cent.; some authorities place the mortality
at a much higher figure. According to recent statistics of Kerr and
Stimson, during 1908, 111 persons died of rabies in the United States.
Extensive bites on the face and head are considered to be particularly
dangerous; the mortality of these is said to have been 80 per cent.
636 PA THOGENIC MICRO-ORGA XISMS.
The bites of wolves seem to be more fatal than the bites of dogs or
other animals; the mortality of these, in spite of the most intensive
treatment, is stated to be still 10 per cent., as against a previous mor-
tality, without specific treatment, of 40 to 60 per cent- But even
Pasteur's antirabic treatment is unavailing when symptoms of the
disease have manifested themselves.
On the whole, the results we have obtained in the New York De-
partment of Health from cases treated by this method have been very
encouraging.
Other Methods of Immunization. — Others methods of immunization
against rabies have been proposed by different investigators. But
almost all of these methods have proved on trial to be unsatisfactonr
and unreliable, besides being not devoid of danger. As early as 1889
Babes and Lepp conceived the idea that it might be possible by means
of the blood to transmit conferred immunity against rabies from one
animal to another; but although the success of these investigators was
not great, Tizzoni and Schwartz, and later Tizzoni and Centanni,
worked out a method of serum inoculation and protection in rabies
which is worthy of attention. In this method not the rabic poison
itself, but the protective material formed is injected into the tissues.
These observers showed that the serum of inoculated animals is capable
of destroying the pathogenic power of the rabic virus — not only when
mixed with it before injection, but when injected simultaneously or
within twenty-four hours after the introduction of the virus into the
body.
Marie, Poor, and others have corroborated these results. The
latter in our laboratory has gotten strong virus-destroying serums
from hyperimmunized sheep and horses. Babes, Marie, and others
now recommend treatment by sensitized virus. Poor has tried this
on some of the lower animals and, though his results have been
encouraging, they have not been satisfactory enough to warrant the
treatment of human beings by this method.
The Cauterization of Infected Wounds. — We believe that in cases
in which the Pasteur treatment cannot be applied great benefit may be
derived from the correct use of cauterization with fuming nitric acid,
even twenty-four hours after infection, and that even in cases in which
the Pasteur treatment can be given, an early cauterization will be of
great assistance as a routine practice and should be very valuable, as
the Pasteur treatment is frequently delayed several days for ob^nous
reasons, and then does not always protect. In the case of small
wounds all the treatment probably indicated will be thorough cauter-
ization with nitric acid within twelve hours from the time of infection.
Our experience in dealing with those bitten by rabid animals goes to
show that physicians do not appreciate the value of thorough cauter-
ization of the infected wounds.
Pasteur Treatment by Mail. — ^For several years we have made a
practice of sending the treatment by mail when the patients could
not go for treatment. The results have been good.
YELLOW FEVER. 637
Preventive Measures in Animals. — ^Faif more important than any
treatment, curative or preventive, for hydrophobia in man is the preven-
tion of rabies in dogs, through which this disease is usually conveyed.
Were all dogs under legislative control and the compulsory wearing
of muzzles rigidly enforced for two years where rabies prevails, hy-
drophobia would practically be stamped out. This fact has been
amply demonstrated by the statistics of rabies in countries (e.gr., Eng-
land) where such laws are now in force.
Literature.
Berry. The Complement Binding Test in Rabies. Journ. Exp., MM., 1910,
XII.
Harris. A Method for the Staining of Negri Bodies. Journ. of Infect. Dis-
eases, 1908, V, 566.
HogyeSj Lyssa^ in Nothnagel's Specielle Pathologic u. Therapie, Wien, 1897.
Kerr and Stimson. The Prevalence of Rabies in the United States. The Journ.
of the Am. Med. Assoc., 1909, LIII, 989.
Marie. L^Etude exp^rimentale de la Rage^ Paris, 1909.
Williams and Lowden. Journ. of Infect. Diseases, III, 1906, 460, with full list
of literature to date on Negri bodies.
YELLOW FEVER.
Yellow fever is an acute infectious disease of tropical countries with
no characteristic lesions except jaundice and hemorrhage. Other
lesions that exist are those common to toxaemia.
Historical Note. — There have been many extensive studies on the etiology
of this disease with numerous announcements of the discovery of its specific
cause. Not one of the latter, however, has been corroborated. The Bacillus
icteroides of Sanarelli (1897), found in the circulating blood and in the tissues
of most yellow fever patients, was thought by many to be the real organism,
and for some time it was the subject of most minute studies with the result
that it, too, has been placed with the rejected organisms.
The epoch-making investigations of the United States Army Commission
composed of Walter Reed, James Carroll, Aristides Agramonte, and Jesse
W. Lazear (1901), established the truth, that this disease, like malaria, is
carried from one infected person to another through the agency of a mosquito.
Finley in 1881 was the first positively to assert that the mosquito was the
transmitter of the disease. He was, however, unable to prove his theory, and
it remained for the commission conclusively to show that a distinct species
of mosquito carried the infection.
The work of the American commission was fully corroborated by the French
commission and by other workers.
The principal facts established by the commission have been
summed up by Goldberger as follows:
1. Yellow fever is transmitted, under natural conditions, only by
the bite of a mosquito {Stegomyia calopus) that at least twelve days
before had fed on the blood of a person sick with this disease during
the first three days of his illness.
2. Yellow fever can be produced under artificial conditions by the
subcutaneous injection of blood taken from the general circulation
of a person sick with this disease during the first three days of his
illness.
3. Yellow fever is not conveved by fomites.
638 PATHOGENIC MICRO-ORGANISMS.
4. Bacilliis icteroides (Sanarelli) stands in no causative relation
to yellow fever.
Though the specific parasite remains yet undiscovered, facts have
been brought out by these studies which give some idea of its character.
1. It seems to require two hosts (a mammal and an arthropod)
for the completion of its life cycle (analogies, Plasmodium mnlari^y
Piroplasm^ bigeminum). (The recent discovery by Stimson of a
spirochete-like organism in the tubules of a yellow fever kidney is
suggestive in this connection.)
2. There is a definite time between the bite of the mosquito and
the infectivity of the blood (average, five days), and a definite time
that the blood remains infective (three days).
3. The blood during these three days is still infective after passing
through the finest-grained porcelain filters (Chamberlain B and F).
4. The blood loses its virulence quickly (forty-eight hours) when
exposed to the air at temperature of 24° to 30° C. When protected
from the air by oil and kept at the same temperature it remained
virulent longer (five to eight days). Heated for five minutes at
55° C. it becomes non-virulent.
5. The bite of an infected mosquito does not become infectious
until twelve days (at a temperature of 31° C.) after it has bitten the
first patient.
The cause of the disease still remains undiscovered, notwithstanding
much study of human blood and other tissues and of infected mos-
quitoes. The infective blood filtrates show nothing with the dark-
field illumination except small motile granules similar to those found
in healthy persons.
Certain facts relating to the disease seem to point to protozoa as
the cause; for instance, the necessity for a second host and the long
incubation time required before that host becomes infective after bit-
ing a yellow fever patient.
The higher monkeys seem to be susceptible, though no complete
experiments have been made with them.
The Yellow Fever Mosquito (Fig. 196).— The name Stegomyin was
suggested by the English entomologist Theobald, who separated this
genus from the genus Culex, with which it was formerly classed.
It was first given the specific name fdsciata, but Blanchard proved
that this had already been used and the name calojms (Meigen, 1818)
was found to be the proper one. The salient characteristics of Steg-
omyia are: (1) The palpi in the male are as long or nearly as long,
as the proboscis; in the female the palpi are uniformly less than one-
half as long; (2) the legs are destitute of erect scales; (3) the thorax
is marked with lines of silvery scales. Stegomyia, or at least Steg-
omyia calopus, is spread over a wide range of territory, embracing
manv varieties of climate and natural conditions. It has been found
as far north as Charleston, S. C, and as far south as Rio de la Plata.
There is no reason to believe that it may not be present at some time
or other in anv of the intermediate countries. In the United States
YELLOW FEVER.
639
specimens of Stegomyia calopus have been captured in Georgia, Louisi-
ana, South Carolina, and eastern Texas. The island of Cuba is
overrun with this insect. The fact that Stegomyia calopus has been
known to exist at various times in Spain and other European countries
may account for the spread of yellow fever which has occurred there
once or twice in former times; the same may be said of the country
farther north in the United States, where Stegomyia calojms has not yet
been reported, but which have suffered from invasions of yellow fever.
Fig. 196
"4
*'^' .
\
^tegomyia calopus. 1. Full-grown female. X 8. 2. Eggs, natural sixe. 3. Larvie and pupae,
natural size. 4. Larva. X 25 (Koile and Wassermann).
Brackish water is unsuited for the development of Stegomyia larvae.
The species Stegomyia calopus seems to select any deposit of water
which is comparatively clean. The defective drains along the eaves
of tile roofs are a favorite breeding place in Havana and its suburbs;
indoors they find an excellent medium in the water of cups of tin or
china into which the legs of tables are usually thrust to protect the
contents from the invasion of ants, a veritable pest in tropical countries.
The same may be said of shallow traps, where the water is not fre-
quently disturbed.
Like other Culicidce, it prefers to lay at night. It is eminently a
town insect, seldom breeding far outside of the city limits. Agra-
monte never found Stegomyia calopus resting under bushes, in open
fields, or in the woods; this fact explains the well-founded opinion
that yellow fever is a domiciliary infection.
640 PATHOGENIC MICRO-ORGAXISMS,
The question of hibernation in the larval stage is important. Agra-
monte failed to get larvee that could resist freezing temperature,
and found that in the case of Stegomyia calopus this degree of c*old
was invariably fatal.
The possibility of their being capable of life outside their natural
element must also be considered from an epidemiological point of
view. The dry season in the countries where this species seems to
abound is never so prolonged as completely to dry up the usual breed-
ing places. Experimentally, adult larvse removed from the water
and placed overnight upon moist filter-paper could not be revived
the following morning.
The question of the life period of the female insect is of the greatest
importance when we come to consider the apparently long interval
which at times has occurred between the stamping out of an epi-
demic of yellow fever and its new outbreak without introduction of
new cases. The fact is that Stegomyia calopus is a long-live<l in-
sect; one individual was kept by Agramonte in a jar through ^larch
and April into May, in all for seventy-six days after hatching in the
laboratory.
These mosquitoes bite principally in the late afternoon, though
they mav be incited to take blood at anv hour of the dav. Thev are
abundant from March to September, and even in November Agra-
monte was able to capture them at will in his office and laboratory.
The mosquito is generally believed to be incapable of long flights
unless very materially assisted by the wind. At any rate, the close
study of the spread of infection of yellow fever shows that the ten-
dency is for it to remain restricted within very limited areas, and that
whenever it has travelled far beyond this, the means afforded (railway
cars, vessels, etc.) have been other than the natural flight of the insect.
Experiments have demonstrated that not all mosquitoes which
bite a yellow-fever patient become infected, but that of several which
bite at the same time some may fail either to get the parasite or to
allow its later development in their body. This condition is similar
to that seen in Anopheles, with regard to malaria.
How long do infected mosquitoes remain dangerous to the non-
immune community? This question cannot be definitely answered
at present; there is good presumptive evidence that the mosquito
may harbor the parasite through the winter and be enabled to trans-
mit in the spring an infection acquired in the fall. There is reason to
believe that the mosquito, once infected, can transmit the disease at
any time during the balance of its life. Freezing temperature, how-
ever, quickly kills the insect.
OUo. Qelbfieber. In Kolle and Wassermann's " Handbuch d. path. Mikrooitt."
Zweites Erg&nzungsband, Erstes Heft, 1907.
Reed and Carroll. Journ. Exp. Med., 1900, V, 215.
Reed and Carroll and Agramonte. Journ. Am. Med. Assoc., 1901, XXX VI, 413.
The Yellow Fever Institute Bulletin, No. 16, Yellow Fever, Etiol., Symp. ami
Diagnosis, by Goldberger, gives a good r<5sum^ with full literature to 19()7.
GLOSSARY.
(L. aggressus, attacked), name given by Bail (1905) to a
hypothetic substance in exudates which are produced by living organisms
inoculated into animals. The substance is supposed to be an endotoxin,
liberated from the bacteria through bacteriolysis. It is supposed to act by
paralyzing the polynuclear leukocytes, thus preventing phagocytosis. It
thereby allows the bacteria to become more aggressive, hence the name.
Alexin (oAc^civ, keep off, defend), name given by Buchner (1889) to
what he believed to be the single protective substance in normal blood.
The term was retained by Bordet to designate that constituent of normal
and immune serums which does not withstand heating to 55° C. and which
is one of the factors in lytic processes. Synonym, complement.
Amboceptor (L. ambo from dfjL<l>a, both, -|- capare, take), name applied
by Ehrlich to that substance of the blood which withstands heating to
55° C. and which attaches itself both to the foreign cell and to the comple-
ment in order to produce lysis. It is increased during immunization.
Synonyms, immune body, sensitizing substance (substance sensibiliza-
trice of Bordet), copula, desmon, preparator, interbody.
Amitosis (a, negative prefix, -|- furo^, a thread, -h osis), direct nuclear
division without the formation of the thread-like chromosomes, asters, and
spindle.
Anaphylactin {dva, upon, back again, -I- <^vXdcr<rciv, watch, guard),
term used by Gay and Southard (1907) to designate a hypothetic sub-
stance contained in horse serum and certain other organic substances,
which is an irritant to animal cells, causing them to become sensitive
to the poisonous element in the organic substance.
Anaphylaxis, the term introduced by Richet (HX)")) for the phenom-
enon of .sensitization to a foreign proteid, e. g., guinea-pigs inoculatedwith
horse serum become, after a period, poisoned by a second inoculation
which would otherwise produce no injury.
Anisogamy (dvio-o-s, unequal, -h yoftos, marriage), fertilization by the
union of two unequal cells.
Antagonism (dyraywviarfm, struggling against), the opposition one or-
ganism exerts upon another either within or without the body.
Antigen (avr*', against, -|- ycVos, race, stock), name given to those sub-
stances which are capable of producing antibodies. Synonym: haptin.
Autogamy (avros, self, -l- yofto?, marriage), self-fertilization. Fertili-
zation bv the union of nuclei within the parent cell.
Bactenol3rtlC (paKrypiov, a little stick, -h Xwris, a loosening), term de-
scribing the solvent power of blood serum for bacteria.
Blepnaroplast (pk€<lHipov, eyelid, -l- irXoo-o-civ, mold, form), a secondary
nucleus in certain protozoa, forming motor apparatus, or acting as a
kinetic center.
Centrosome (Kcvrpov, centre, -h <r<o/ia, body), a small cell-organ which
is regarded as the active centre of cell-division.
41 641
G42 GLOSSARY.
Gomplement (L. complementum, that which fills up completelj),
that constituent of normal and immune serums which is destroyed bv
heating to 55° C. and which unites with the immune body (amboceptor)
to produce lysis.
Ghitin (x^^wv, a tunic), the name given by Odier to the horny organic
substance which forms the integuments of insects and some other animals.
Composition, CijHjgNjOio.
Gmorophyl (x^<«>pos, yellowish-green, -|- <^vXXov, a leaf), the yellow-
green pigments common to most plants; also found in a few protozoa.
Chromatin {xP^y^t color), the deeply staining substances of the
nucleus, consisting of nuclein or nucleic acid.
Ghromatophores {xp^ij^, color, -f 4>6poi, bearing), a general term
applied to the colored bodies (plastids) found in plant and animal cells.
Ghromidium (xpw/ia, color -h iSiov, dim.), a name given by Hertmg
(1902) to the chromatin particles which pass from the nucleus to the cyto-
plasm and there perform nuclear functions.
Ghromosomes (x/ow/uui, color + <ru)/Aa, a body), deeply staining bodies
which are formed from the chromatic nuclear network during cell
division.
Gonunensal (L. com, together + mensa, a table), living in harmless
union. One organism living on or in another without harming either.
Gopula (L. copula, a bond, link), a fertilized protozoan cell.
Gytol3rtic {kvtos, a hollow (a cell), H- Xwris, a loosening), term de-
scribing the solvent action of the blood serum on any cell.
Gyt(^lasm (kvto^, a hollow (a cell), -h irXacTfm, anything formed), that
part of the cell protoplasm which is outside of the nucleus.
Ectoplasm (cktos, without, -h -rrXaa/mj anything formed), the exterior
denser cytoplasm of a cell.
Entoplasm (cvrds, within, -|- plasm), the inner, more fluid portion of
the cytoplasm.
Gamete (ya/xcny, a wife; ya/xcriys, a husband), one of two conjugating
cells, destined to die unless it unites in fertilization with another cell.
OametOCyte, the sexual cell which resolves itself into the individual
gamete.
Oenus (L. genus, birth, origin, race), in biology, a classificatory group
ranking next above species.
Haptin {airroi, to bind, + in), synonym of antigen.
Haptophore (aTrTw, to bind, -l- 4>opo^, bearing), term applied to the
group of atoms which effects the specific binding to a corresponding
group of atoms in certain foreign cells.
Holozoic Nutrition (o\o<:, whole, -h fcoiKos, animal), entirely animal-
like nutrition.
Hypnocyst (wvos, sleep, -h Kvans, cyst), a sleeping or quiescent cyst.
ImiQuhe body (L. immunis, exempt from public service, free),
synonym of amboceptor.
Isogamy (lo-os, equal, -f ya/A09, marriage), the conjugation of two
gametes of similar form .
Karyokinesis (Kapvov, a nut (nucleus) -l- KtViyo-is, movement, change),
the series of active changes which takes place in the nucleus of a living
cell in the process of division. Synonym: mitosis.
Lysis (Xixrts, a loosening), the general solvent power of the blood for
foreign substances.
Macrogamete (/xaKpd? large, -l- yufiervj, a wife), female mating cell.
GLOSSARY. 643
Maturation (L. maturare, to ripen), term used to designate the series
of complicated processes which occur during the ripening of a germ cell.
Merzoites (fJ^po^, a part, + ^wov, animal, H- ittjs, like), a reproductive
germ produced by a protozoon without fertilization.
Metazoa (/^cTa, after, H- iioov, an animal), animals ranked above the
protozoa, each consisting of many cells.
Microgamete (fiUpo^, small, + yafitrrjs, husband), male mating cell.
Mitosis (fJ^To^, thread, + osis), synonym of karyo kinesis, so called
because of the thread-like changes in the nuclear chromatin during
division.
Oocyst (wpv, an egg, H- kixttis, a cyst), fertilized cyst containing spores.
Opsonins (©xov, anything giving a zest to food; a relish), substances
in blood serum which combine with the bacteria and thus prepare them
for being taken up more easily by the phagocytic cells.
Parasite (irapdj beside, + ariro^, food, live at another's table), an organ-
ism which lives on or in, and at the expense of another organism called
technically the host.
Perq)lastic (^<pt, around, H- irAooTos, mold, form, H- ic), applied to
flagella or cilia formed from the cell substance about the nucleus.
Feritrichal (Tcpe', around, H- Opti, a hair), applied to flagella or cilia
springing from the cell membrane.
Precipitin (L. prae, before, + caput, the head, literally, falling head-
long), any substance developing in the serum as the result of the inocula-
tion of the animal with a foreign substance and which precipitates that
foreign substance.
Protista (TrpwTMrra, the very first, superlative of ir/owros, first), name
{)roposed by Haeckel (1868) for a third kingdom, including the lowest
orms of both animal and plants.
Protozoa (irpwro?, first, -h ^wov, animal), first-formed animals; the
name given to the simplest animal forms, those consisting of a single cell.
Receptors (L. re, back, -h capare, take), atom groups in cells which
Ehrlich conceives to have affinities for toxins and similar substances.
Reduction Division, a complicated process in maturation whereby
the nuclear chromatin is reduced in amount preparatory to the formation
of the gametes.
Saprophyte (o-aTrpos, rotten, + <I>vt6v, a plant), an organism that grows
on decaying vegetable matter.
Schizogony (o-xticiv, cleave, split, + yovia, generation), the multiple
asexual reproduction of protozoa.
Schizont ((rxi^civ, cleave, split, -h ont), the mother cell which gives rise
to the merozoites.
Somatic (o-co/AaTtxos, pertaining to the body), pertaining to vegetative
growth.
Species (L. species, kind, a particular sort, etc.), a group of similar in-
dividuals which differ from other members of a genus.
Sporoblast (arwopd, seed, -|-)8AaoT09, a germ), the mother cell which
gives rise to sporozoites.
SporOC3rst {<rtropd, seed, -f Kwrrt?, cyst), the resistant outer covering
of the spore.
Sporogony {airopd, seed, + yovuL, generation), multiple sexual repro-
duction with the formation of spores.
Sporozoites (awopd, seed, -f ^wof, animal, -I- Irrj^, like), a young repro-
ductive germ, formed in a sporoblast after fertilization.
644 GLOSSARY,
Symbiosis ((rvfifiuoa-i^, a living together), the living together of certain
orffanisms, each of which is necessary to the other.
S3nigailiy (<rw, together, + yofto?, marriage), sexual reproduction.
Toxoid (roimov, poison, -h oid), toxin which while still combining i^ith
antitoxin has become so altered that it no longer causes poisonous effects.
Ehrlich supposes the haptophore group remains intact after the destruc-
tion of the toxophore group.
Toxon (roiiKov, poison, -h on), name given to a secondary toxin pro-
duced by diphthena or other true toxin-producing bacteria when this
differs in its characteristics from the toxin of primary importance.
Toxophore (ro^txdv, poison, H- 4>opo^, bearing), term applied to the
group of atoms which is the carrier of poisonous action to the cell.
Trophozoite (tt/ckm^i/, nourishment, ^wov, animal, H- irrf^, like), the
young vegetative cell.
Virulence (L. virulentus, full of poison), the power possessed by or-
ganisms to produce injury by growth in a living host with the formation of
poisonous substances. The variations in virulence of an organism in
different species of host's are due more to the ability of that organism to
grow than to its ability to produce poisonous substances.
Zygote (fvyoiTos, yoked), a fertilized cell, produced by the union of
gametes in lower plant or animal.
Zymophore {ivfirj, leaven, ferment, -h <^po?, bearing), term applied to
the group of atoms which exerts a ferment action on the cell.
INDEX OF AUTHOES.
Abbott, 490
Abel, 210, 450
Axramonte, 637, 639
Akermann, 262
Albarran, 262
Albrecht, 394
Alessi, 57
Altmann, 580
Ambrjz, 14
Ammon, 624
Anderson, 162, 426
Ant bony, 371
Appel, 56
Anstotle, 622
Arloing, 338
Aming, 351
Aronson, 110
Arrhenius, 216
Ashbum, 487
Atkinson, 212, 215
Avery, 251
B
Babes, 607, 623, 636
Baelz, 547
Baeslack, 562
Bail, 160
Bailey, 95, 99
Baker, 559
Balbiani, 592
Baldwin, 251, 330
Ball, 98
Banzhaf. 212
Baraneck, 330
Barbagallo, 535, 544
Bard, 195
Baron, 629
Bassett, 277
Bassi, 5
Baumgarten, 310
Beattie, 593
Beebe, A. L., 204
Beebe, S. P., 580
von Behring, 6, 112, 147, 149, 151, 211,
237, 327, 330
Beijerinck, 481, 487
Bergey, 208, 503
Bemberg-Gossler, 607, 61 1
Berry, 629, 637
Bertarelli, 484, 576, 583, 613
Bertram, 594
Besredka, 182
Beyerinck, 98
Biedert, 343
Bienstock, 246, 254
Biggs, 217
Bignami, 597, 611
Billroth, 5
Blanchard, 638
Blandford, 567
Blochmann, 586
Boeckman, 127
Bohne, 304
Boinet, 351
Bolduan, 384
Bollinger, 460
Bolton, 237
Bonhoff, 395
Boni, 13
Bonome, 419
Booker, 416
Bordet, 89, 150, 155, 157, 161, 163, 216,
484, 577, 580
Bordoni-Uffreduzzi, 387
Borget-Gengou, 577
Borrel. 351, 425, 485, 488, 580, 615
Bosquillon, 622
Bossowski, 367
Bostroem, 460
Bouchard, 265, 413, 417
Bradford, 558
Braun, 531
Breinl, 559, 566, 568, 607, 609
Bretonneau, 195
Brieger, 86, 236, 446
BrUl, 270
Broeden, 559
Brooks, 588
Brown, 332
Bruce, 408, 558, 563, 568
Bruck, 577
Brunner, 416
Buchanan, 98
Buchner, 55, 79, 83, 150, 159, 331
Bumm, 402, 408
Bunge, 35
Burn, 47
Buschke, 482, 576
645
646
INDEX OF AUTHORS.
Busse. 481
Batschli, 13, 621
Buxton, 271
Cabot, 270
Cahn, 252
Calkins, 519, 526, 529, 531, 533, 549,
562, 580, 583, 609, 613, 623, 627
Calmette, 327, 425, 613
Cameron, 99
Canon. 356
Capaldi, 298, 300
Capitan, 417
Cardiac, 112
Carey, 279
Carle, 232
Carlisle, 583
Carroll, 637, 640
Carter, 583
Casagrandi, 535, 544
Castellani, 580, 583
Celli, 535, 596, 606
Celsus, 622
Centanni, 636
Cemovoaeanu, 240
Chagas, 558
Chamberland, 435
Charlton, 279
Charrin, 56, 413, 416
Chassin, 262
Chester, 76
Chevreul, 4
Chowning, 426
Christophers, 556, 611
Clairmont, 390
Claussen 4^0
Clegg, 350, 465, 471, 535, 543, 549,
564, 568
Cobbett, 201, 208
Cohendy, 249
Cohn, 5, 236
Coley, 374
Colles, 576
Collins. 170, 279, 390
Conradi, 252, 287, 298
Councilman, 399, 533, 542, 549, 597,
613, 615, 619
Courmont, 338
Craig, 487, 535, 544, 549, 611
Cramer, 21
Cunningham, 455, 535
Curtis, 481
Cushing, 287
Cygnaeus, 285
Dale, 528
Dansner, 580
Darling, 127, 554, 556, 583, 595
D'Arsonval, 56
Davaine, 5, 429, 552
Davis, 206
Dean, 175
Deneke, 443, 455
Denys, 172, 329. 330
Dieudonn^, 55
Dock, 546, 549
Doflein, 531, 558, 588, 592, 593
Donitz, 241
Donn6, 586
Donovan, 553
Dorset, 316
Doariae, o59
Doutrelepont, 351
Dreyer, 164
Drigabki, 287, 298
Ducrey, 410
Dujardin, 521
Dunham, 277, 441, 453
Durham, 42, 155, 163, 270, 292, 567
von Dusch, 4
Dutton, 558, 565, 582
Duval, 276, 350, 620
Eberth, 282
Ehlers. 414
Ehrenberg, 569, 571
Ehrlich, 6, 31, 89, 151, 163,"'214, 237,
342, 566, 568
Elser, 393, 399
Emmerich, 50, 255, 413, 449
Endo, 298
Engelmann, 521
Eppinger, 459, 465, 470
Epstein, 109
Ernst, 414
Escherich, 246, 254, 264
von Esmarch, 54, 485
Evans, 559
Ewing, 575, 580, 583, 613, 619, 621
F
Faguet, 471
Falcioni, 269
Fantham, 592
Fehleisen, 5, 369, 374
Feinberg, 13
Fenger, 419
Ferre, 471
Field, 239, 620, 621
Finger, 576
Finkler, 291, 443, 455
Finley, 637
Fiocca, 535
Fisch, 237
Fischer, 13, 108, 272, 576
Flexner, 167, 170, 274, 394, 466, 486.
535, 570, 583,
Floumoy, 583, 619
I'lagge, 232, 220
Fod, 384, 387
Fontaine, 482
Foote, 288
Fomet, 580
Foulerton, 458, 471
Foumier, 575
Fox, 289, 305
Fraenke 97
Frankel,'49, 343, 364, 381
Franklands, 490
ISDEX OF AUTHORS.
G47
Freeman, 130
Freytag, 439
Friedberger, 629
Friedlander, 267, 381
Frosch, 287, 487, 527, 538
FuUerton, 19
FOrbinger, 1 28, 408, 473
Gabbett, 343
Gaffky, 282, 367, 439
Gage, 27
Galli-Valerio, 608
Galziekte, 559
Gamalela, 456
Garr4, 365
Gartner, 268, 271, 325
Gasperini, 463
Gasten, 459
Gay, 162
Gaylord, 580
Gengou, 161, 484
Gerlief, 435
Gessard, 412
Ghon, 394
Gibson, 212
Giemsa, 427, 570, 577, 624
Gilbert, 269
Gilchrist, 481
GillUand, 327
Goldberger, 427, 637, 640
Goldhorn, 570, 598
Golgi, 596
GoU, 408
Gomez, 427
Goodwin, 171, 274, 395
Gottschlich, 14, 454
Grassi, 533, 597
Greig, 559
Greig-Smith, 98
Gross, 536
Gniber, 42, 155, 163, 292
Griibler, 29, 522, 624
Gruby. 474, 557
Guaml^ri, 384, 613
Guerard, 217
Gu^rin, 613
Guignard, 413
Guilliermond, 13, 481
Gwyn, 269
H
Haas, 151
Hadwen, 610
Haeckel, 14, 519
Haffkine, 426, 452
Halberstaedter, 621
Halle, 262
Hamilton, 206
Hansen, 101, 249, 349, 481
Harris, D. L., 626, 637
Harris, H. F., 535, 537, 543, 549
Hartmann, 529, 531, 551, 580, 584, 596,
607, 608
Harz, 460
Hasse, 482
Hastings, 410
Hauser, 414, 455
Hebra, 474
Heidenhain, 522
Heiman, 407
Hektben, 180, 621
Helbrigel, 97
Heller, 629
Henle, 3
Henni, 240
Henrijean, 234
Herman, 343
Herter, 245, 254, 441
Hertog, 531
Hertwig, 13, 521, 524
Hess, 143, 321
Hetsch, 603
Heuss, 323
Hewetson, 611
Hewlett, 207, 269
Heymann, 621
Higley, 229
HiU, 17
Hindle, 559, 607, 609
Hiss, 34, 66, 183, 277, 298, 384
Hodenpyl, 312, 318
Hoffmann, 4, 571, 575, 583
von Hoffmann, 109, 207
Hogyes, 637
Holt, 508
Home, 195
Houston, 494, 499
Howard, 611
Huddleston, 616
Hueppe, 82, 349, 370, 423
Hundswuth, 622
Hilnermann, 269
Hunter, 330
Huntoon, 393, 399
I
Israel, 460
Jaeger, 392
Jansen, 435
Jehle, 279
Jenner, 597, 613
Jennings, 525
Jobling, 397
Joblot, 520
Jochmann, 395, 484
Johnson, 292
Johnston, 269
Jordan, 442, 490
Jullien, 575
Jiirgens, 542, 547, 559
Justinian, 423
Kanthack, 567
Kaposi, 477
Kartulis, 483, 533, 543, 547, 549
Kayser, 288
Kempner, 321, 559, 567
Kent, 170, 558
648
INDEX OF AUTHORS.
Kerandel, 568
Kern, 251
Kerr, 254, 635, 637
Kessler, 483
Kilborne, 607, 609, 611
Kinoshita, 611
Kisskalt, 531, 584
Kitasato, 6, 31, 57, 147, 232, 237, 284,
424, 431
Klatsch, 538
Klebs, 195, 575, 596
Klein, 175, 254, 442
Klocker, 483
Knapp, 581, 583
Knight, 207
Knoepfelmacher, 486
Knorr, 240, 376
Koch, 5, 6, 29, 32, 54, 60, 78, 107, 160,
282, 310, 327, 343, 357, 359, 369,
376, 429, 439, 443, 447, 608, 611
Kohler, 47
Kolle, 291, 395, 455, 531, 603
Kolliker, 521
Koplik, 129
Korte, 165
Kossel. 323, 609
Krambals, 414
Kraus, 171
Krause, 369, 484
Kromer, 396
Kruse, 142, 274, 414, 535, 543
Kuhne, 5, 33
Kuline, 5
Kummel, 495
Kurpjuweit, 252
Kurth, 17, 269
Kutchler, 456
Kutzing, 101
Ladenburg, 86
Ladowski, 419
Lafleur, 533, 542, 549
Lamb, 582
Lambert, 242, 384, 387
Lamdl, 533
Landemann, 330
Landsteiner, 486, 576
Lang^ 531, 591
von Langen beck, 460
Lankester, 7, 531, 594
Lartiga'j, 414
T Q t,zpi* 2 ^4
Laveran, 6, 554, 559, 563, 567, 568,
595, 596
Lazarus, 452
Lazear, 637
Leber, 363
Leblanc, 635
Leclef, 173
Lee, 29
Leeuwenhoeck, 2, 520
Lehmann, 74, 75, 91
Leiner, 486
Leishman, 173, 553, 554
Lemke, 247
Lentz, 277, 287
Lepp, 636
Lepriere, 395
Lesage, 535, 546
Levaditi, 486, 571, 574, 576
Levin, 245
Levy, 288, 416
Lewis, 443, 559
Leyden, 547
Libman, 373
Liborius, 78
LieberkUhn, 450
Liebermann, 474
Lindner, 481
Lingard, 559
von Lingebheim, 394, 401
Linnsus, 520
Lipschiitz, 488
Lister, 5
Loeffler, 6, 25, 32, 35, 36, 65, 195, 373,
417, 487
Lon^cope, 270
Losch, 533, 535
Loew, 413
Lowden, 637
Lowenstein, 332
Loe wen thai, 580
LGwit, 13
Lubenau, 316
Luhe, 607
Lundsgaard, 482
Lustgarten, 348, 573
M
Maassen, 90
MacCallum, 410, 597, 606
McCoUom, 621
MacConkey, 258
McDaniel, 208, 628
McDonald, 394
MacFadycan, 487
Mc Farland, 79, 241
MacFayden, 246, 327
McKenzie, 181
MacNeal, 254, 552, 559, 563, 568
Madsen, 90, 216
Mahnsten, 588 *
Maklezow, 246
Mallory, 484, 537, 620, 621, 625
Malmst, 588
Manson, 546, 559
Manz, 594
Maragliano, 336
Marchiafava, 596, 611
Marie, 239, 628, 636, 637
Marmorek, 65, 338, 376, 384
Marshall, 170
Martin, 180
Martini, 277
Massart, 13
Massol, 249
Maupas, 521
Mayer, 568
Meier, 580
Meigen, 638
INDEX OF AUTHORS.
649
Meissner, 421
Meltzer, 56
Mesnil, 554, 559, 563, 567, 568, 595
Metchnikoff, 150, 158, 172, 245, 254,
451, 455, 575, 582
Meumir, 112
Meyer, 239
Meyerstein, 304
Michaeb, 30
Miescher, 594
Migula, 13, 23
Mifler, 443, 456
Minchin, 563, 592, 594
Mitchell, 482
Mitrophanow, 14
Miyajami, 611
Moeller, '64
Mohler, 321
Moore, 531, 559
Mora, 253
Morax, 239
Morgagni, 622
Morgenroth, 155
Moro, 337
Moser, 379, 484
Motas, 611
Muhlens, 571, 574, 580, 583
Muir, 158, 180
M Jller, 522
Muntz, 97
Musgrave, 465, 471, 535, 543, 549, 564,
568
N
N\BARRO, 559
Nais, 593
Nakanischi, 13
Negri, 623
Neisser, 159, 197, 237, 349, 402, 406,
455, 575,
Nelis 623
Nencici, 86
Nepvieu, 559
Netter, 268, 386
Neufeld, 173, 180, 390
Neumann, 74
Nicolaier, 232
Nicolle, 427, 556
Nierenstein, 566, 568
Nocard, 269,465,488, 611
Nocht, 568
Nocht-Romanowsky, 556
Noguchi, 579, 583
Norris, 580, 583
North, 307
Novy, 80, 527, 552, 559, 562, 563, 568,
571. 580, 582, 583, 607
Nuttall, 150, 245, 607, 610, 611
O
Oeldecker, 323
Oergel, 449
Ogston, 361, 369
Ophuls, 481
Opie, 180, 588
Orth, 364
Osier, 330
Otto, 640
Paltauf, 473
Pansini. 268
Pappenheimer, 583
Park, 167, 203, 215, 274, 277, 306
Pasquale, 414, 455, 535, 543
Passet 369
Pasteur, 4, 6, 50, 68, 78, 94, 102, 138,
361, 381, 429, 432, 439, 497, 622, 631,
634. 636
Patterson, 544, 549
Patton, 554, 556, 607
Pearce, 386
Pearson, 101, 327
Perrin, 571, 583
Petri, 70, 90, 447
Petruschky, 63, 287, 376, 380
Pettenkofer, 289, 449
Pfaundler, 264
Pfeiffer, 150, 290, 295, 353, 400, 451,
456
Pfuhl, 456
Piana, 608
Pick 212
von i^irquet, 218, 219, 336
Pitfield, 35
Plenciz, 2
Plimmer, 558
Pollender, 5, 429
Poor, 636
Popper, 486
Porges, 580
Posner, 547
Pottevin, 110
Prescott, 492
Prior, 291, 443, 455
Proskauer, 108
Prowazek, 488, 531, 533, 536, 549, 559,
571, 584, 614, 621, 623
Prudden, 306, 312, 318, 364, 373
Quincke, 535
R
Rabinowitsch, 321, 559, 567
Rainey, 594
Ransom, 238
Rattone, 232
Raynaud, 147
von Recklinghausen, 5
Reed, 637, 640
Reichert, 15, 47
Remak, 476
Remlinger, 484
Rhazes, 612
Ribbert, 364
Richardson, 56, 304
Richet, 162
Richmond, 208
Ricketts, 423, 426, 483
Rimpau, 173
Rinafleisch, 5
650
INDEX OF AUTHORS.
Riviere, 471
Rixford, 481
Roentgen, 313
Roger, 262
Rogers, John, 242
Rogers, Leonard, 554, 556
Romano wsky, 231
Roos, 535
Rosenau, 162, 312
Rosenbach, 361, 366, 369
Rosenow, 385
Rosen-Runge, 304
Ross, 554, 597, 598, 606, 611
Rouget, 559
Roux, 6, 203, 435, 451, 488, 575
von Ruck, 330
Ruediger, 375
Ryssell, 277
Ruzicka, 14
S
Sabouraud, 474
Sa braces, 471
Sachs, 580
Salkowski, 446
Salmon, 465, 576
Salter, 208
Sanarelli, 637, 638
Sanfelice, 483, 606
Sanger, 364
Sauvage, 576
Schaudinn, 13, 18, 20, 524, 533, 539,
544, 549, 561, 569, 572, 576, 580, 583,
587, 589, 597, 606, 607
Schereschewsky, 570, 574, 583
Schering, 121
Schewiakoff, 586
Schilling, 611
Schimmelbusch, 414
Schleiden, 520
Schlosing, 97
Schmiedelberg, 86
Schneider, 85
Schoenlein, 476
Schottelius, 13, 245
Schottmuller, 269, 304, 390
Schroeder, 4, 321
Schroeder, M. C, 125
Schuerer, 392
Schultz, 298
Schultze, 577
Schulze, 3
Schiirmayer, 414
Schiitz, 417, 421
Schwann, 3
Schwartz, 636
de Schweinitz, 327
Sedgwick, 306
Seitz, 285
Senn, 559
Shick, 218
Shiga, 167, 274, 535
von Sholly, 205
Siedentopf, 46
Simon, 175, 571
Sirena, 57
Smith, Graham, 607, 6»0, 611
Smith, Greic, 98
Smith, Letcnworth, 503
Smith, Theobald, 92, 93, 162, 203,-272,
312, 340, 377, 594, 595, 607, 609,
611
Southard, 162
Spallanzani, 3
Spencer, 526
Spengler, 330
Spronck, 203
Stahl, 111
Stanesco, 574
Starcovici, 607
Steel, 558 •
Stem, 570
Sternberg, 107, 381, 492, 536
Stiles, 533, 549, 592
Stimson, 635, 637, 638
Straus, 320, 419
Strong, 270, 276, 535, 543, 549, 588
Studdiford, 379
Surra, 559
Swellengrebel, 13
Swift, 351, 578, 583
Sydenham, 619
Syngamy, 526
T
Tabardillo, 425
Talamon, 381, 389
Thayer, 611
Theiler, 559
Theobald, 638
ThierceUn, 253, 254
Thierfelder, 245
Thomas, 567
Thomassen, 465
Thue, 387
Tissier, 251, 254, 264
Tizzoni, 636
Todd, 558, 565
Tokishige, 482
Torrev. 406
Trembley, 521
Trevisan, 465
TriUat, 110
Trudeau, 327, 331
Trump, 207
Tsujitani, 536
Tuttle, 270, 467
Tyndall, 4
Tyzzer, 580, 614
U
Uhlenhuth, 344
Unna, 30, 351, 477
Uschinsky, 49
Van Ermenqen, 35
Van Erminghem, 271
Van Gehuchten, 623
Van Gieson, 537, 624
Van Leeuwenhoeck, 2, 520
Van Sweiten, 622
INDEX OF AUTHORS.
651
Vaughan, 86, 331
Viereck, 533, 542
Villemin, 310
Vincent, 231, 572
Virchow, 246
Voges, 49, 558
Volpino, 488
Von Dusch, 4
Von Hoffmann, 109
Von Recklinghausen, 5
W
Wads WORTH, 12 S
Waldeyer, 5
Walker, 427, 536
Walters, 351
Warrington^ 97
Wasielewski, 559, 594
Wasserman, 162, 395, 404, 414, 531, 577
Weber 323
Wechsberg, 159
Weeks, 357, 359,
Weichselbaum, 268, 364, 381, 392
Weigert, 5, 6, 14, 153, 154
Weigert-Ehrlich, 153
Weisner, 486
Welch, 33, 265, 366, 382, 440
Wenyon, 535
Wesbrook, 208, 628
Wherry, 485
White, 251
Widal, 163
Williams, 65. 199, 203, 209, 346, 356,
619, 623, 637
Wilson, L. B., 208, 426
Wilson, R. J., 43, 80, 121, 294, 623
Winogradsky, 92, 97, 98
Winslow, 306, 492
Wolf, 246
Wolffhiigel, 54, 72
WoUstein, 279, 357, 484
Woodcock, 568
Wright, 79, 390, 462, 471, 537, 554,
556, 566
Wright, S. E., 172, 180, 290
Wyssokowitsch, 364 *
Y
Ybrsin, 202, 424
Zenker, 625
Zettnow, 13
Ziehl, 31, 33
Zinsser, 184, 247
Zsigmondy, 46
Zwmger, 622
GENEEAL INDEX/
Abbe condenser, 37, 39
Abrin, 88
Abscess, chief producers of, 361
Absorption methods, 167
Achonon schoenleinii (favus fungus),
476
Acid-fast bacilli, 348
Acids, as disinfectants, 107
from carbohydrates, 92
effect of, on bacteria, 107
formation of, from alcohol, etc., 94
oxyfatty, 92
Actinomyces, 458, 460-465
isolation of, 463
occurrence, 464
Actinomycosis, 451 , 460
Aerobic bacteria, 50, 51
facultative, 51
obligatory, 82
Aerogenes capsulatus, bacillus, 254, 440
yEstivo-autumnal parasite of malaria,
598, 590, 605
Agar, nutrient, 62
Agglutinating strength of a serum, 171
Agglutination of bacteria, in hanging
drops, 42
group, 165
nature of substances concerned in,
163
relation between agglutinating and
bactericidal powers, 171
testing of, 42. See also Individual
bacteria.
Agglutinins, 163
absorption methods for differ-
entiation of, 167, 168
characteristics of, 164
development of, 165
group, 166, 167
loss of power of bacteria to absorb,
170
relative accumulation of specific
and group, 167
specific anci group, 166, 167
Agglutinoids, 164
Ag^essins, 160
Agitation, influence of, on bacteria, 56
Air, bacteriological examination of, 498
Alcohol as disinfectant, 109
Alcoholic fermentation, 102
Alexines, 151, 157, 158
Alkaline blood serum, 65
products and fermentation of urea,
85
Allogromia, 529
Alvei bacillts, 15
Amboceptor, 157
Ammonia in bacterial growths, demon-
stration of, 91
Ameba binucleata, 532
coli (see Entameba coli), 533
Amebse, 532
characteristics of, 532-538
cultures, 538-543
in different diseases, 547
material for class study, 536
morphology, 538
pathogenesis, 543
reproduction, 539
sexual phenomena, 540
sites of, in the human body, 536
source of, 545
stains for, 537
viability, 542
Amebic dysentery,
diagnosis of, 546
immunity from, 545
incidence of, 544
prognosis of, 545
symptoms, 544
tissue changes, 545
treatment oif, 546
Amebida, 529
Amido substances, 92
Amphitricha, 15
Anaerobic bacteria, 50, 78
associated with aerobic, 51
culture methods for, 78
pathogenic forms, 439
Anaphylaxis, 162
Aniline dyes, 29
basic and acid, 29
oil as mordant, 32
Animals, inoculation of, 185, 186
use of, for diagnostic and test pur-
poses, 185
♦For definition of bacteriological terms not found in Index, see Glossary, p. 641.
653
654
GENERAL INDEX.
Anopheles, 601
Antagonism, between living body and
microdrganisms, 144
Antagonists, 49, 50
Anterior poliomyelitis, 486
Anthrax bacillus, 429-436
biological characters of, 431
^owth in media, 431
identification of, 436
infection, how caused, 434
prophylaxis against, 435
malignant anthrax oedema, 434
morphology of, 429
non-spore-bearing varieties,
432
occurrence in cattle and sheep,
433
in man, 434
pathogenesis^ 433
spore formation, 432
staining, 431
bacteriological diagnosis of, 436
internal, 435
intestinal, 435
symptomatic, 436
Antibacterial sera, testing power of, 148
155
Antibodies in general, 146, 147
Antigens^ 157
Antiseptics, table of values, 112
action, 113
Antisera, 157, 158
Antistreptococcus serum, 376, 377, 378
serums in scarlet fever, 379
Antitoxic sera- testing power of, 148
Antitoxins, absorption of, 149, 240
Ehrlich's theory for production of,
152-155
in general (see also under Diph-
theria and Tetanus), 238
methods of administration of, 149
nature of, 152, 211
other theories as to production of,
154
as preventive, 149
production of, for therapeutic pur-
poses, 148, 212
relative development of and bac-
tericidal substances, 149
stability of, in the serum, 149
Antituberculous serum, 338
Apparatus, cleansing and sterilization
of, 59
Arnold's steam sterilizer, 61
Aromatic products of decomposition, 91
Arthrospores, 19
development of, 19
Articular rheumatism, 485
Ascitic fluid, 64
Aspergillus, 473
fumi^atus, 474, 475
Attenuation, 103
of virulence, 25
Autogenous vaccines, 183
Autoinfection, 142
Autopsy of test animals, 187
Azotobacter, 99
B
Babesia, 531 (see Biroplasma bigemi-
num), 596, 607-611
Bacillary dysentery, 274-276
Bacilli, acicl-fast, 348, 352
general characters of, 10
us (see also under individual
names), 10
acidophilus, 253
aerogenes capsulatus, 254, 440
sporogenes, 254
alcaligenes, 256, 257, 272
anthracis, 429-436
symptomatici, 436
of blue pus, 412
bifidus, 251
botulinus, 271
of bubonic plague, 423-426
bulgaricus, 249
butter, 352
butyricus, 20
capsulatus, 256, 257, 267
cholerse suis, 269, 273
Clostridium jjasteurianum, 98
coli communis, 255-267
definition of, 10
diphtheria, 195-231
-like, 205
of Ducrey, 410
of dysentery, 274-281
enteritidis, 268, 442
fsecalis alcaligenes, 272
of Friedlander, 267
of glanders, 417-422
of green pus, 412
grass, 348, 353
hofmanni, 207
icteroides (in yellow fever), 637,
638
influenza-like in whooping-cough,
360, 484
of influenza, 353-359
of Koch- Weeks, 359
lactis aerogenes, 267
lepne, 349-352
ot leprosy, 349
of Lustgarten, 348
of malignant oedema, 439
mallei, 417
mucosus, 12
capsulatus, 267
paracolon, 269
paradysentery, 274
parat3rphoid, 269
perfringens, 254
pestis, 423
pneumo-, of Friedlander, 267
prodigiosus, 21
proteus vulgaris, 96, 414
pseudodiphtheria, 206
pseudoinfluenza, 357
psittacosis, 269
putrificua, 254
pyocyaneus, 412
GENERAL INDEX.
655
Bacillus radicicola, 98
of rhinoscleroma, 268
of smegma, 348
of soft chancre, 410
subtilis, 96
of swine plague, 273
of tetanus, 232-245
of timothy grass, 352
of tuberculosis, 310-348
of typhoid, 282-310
Welchii, 254, 440
Bacteria, adaptation to environment of,
25, 80. 133, 134
aerooie, 50
anabolic power of, 82
in air, 95, 100
anaerobic 50, 78,
basic forms of, 9
behavior toward oxygen, 50
botanical relationship of, 7, 22
carriers, 141, 287
characteristics of, 7
chemical effects of, 81
composition of, 21
classification of, 7, 22
cultivation of, 69
definition of, 7
degeneration of, 17
destruction of, by chemicals, 103
dissemination of, 140
duration of life in pure water, 58
effect of temperature on, 51
effects of chemicals on, 58
elimination of, from the body, 143
through milk, 143
examination of, in hanging drops, 4 1
in tissues, 36
facultative, 57
general characteristics of, 7
growth and reproduction, 16
higher forms of. 11, 21
identification ot, in milk, 501
in industries, 101
influence of one species upon
another, 49
of quantity in infection, 135
of reaction of media on, 49
intestinal, 245
involution forms, 17
katabolic power of, 82
local effects of, 133, 134
loss of capacity to be agglutinated
or absorbed, 170
manner in which they excite
disease, 135-137
mesophilic, 52
morphology and structure of, 7
motility of. 16
natural haoitat of, 7
nitrification by, 72
nuclear substances in, 13
numerical estimation of, in milk,
500
nutrition of, 48
parasitic, 48
products of the growth of, 81
Bacteria, psychrophilic, 52
physiologic characteristics of, 15
relation of, to disease, 131
to next higher plants, 22
to other microorganisms, 7
to oxygen, 80
to protozoa, 7
to temperature, 70
reproduction of, 21
saprophytic, 48
in sewage, 99, 494
shape, 9
size, 8
in soil, 95
spore formation of, 19
staining of, 28, 30-34
structure of, 12
symptoms and lesions due to,
products of, 135
thermic effects of, 81
thermophilic^ 52
varieties of, in milk, 501
in vaccine vims, 619
in water, 489
Bacterial autoinfection, 142
cells, 12
structure of, 12
ferments, 102
invasion, 145
proteins, 84
species, 23
permanence of, 24
toxins, 86
vaccines, 172
Bactericidal sera, 144
power, 171
properties of blood, 150
substances, origin of, 158
Bacteriology, historical sketch, 1
Bacteriolysis, 156
Bacteriolytic sera, nature of. 155
Bacterium, characteristics of, 94
Bacteroids, 92
Balantidium coli, 530, 588
minutum, 588
Nyctootherus faba, o8S
Bailey-Denton filter, 497
Basic fuchsin, 29
Bedding, disinfection of, 115, 124
Beggiotoa, 21
Beriberi, 485
Berkefeld filter, 497
Bichloride of mercury, 106, 114
Biedert's method, 343
Bile as culture medium, 64, 304
Biniodide of mercury, 106
Binucleata, 551
• Bismarck brown, 29
Black-leg, 436
Blastomycetes, 472-479, 480
Blepharoplast, 524
Blister fluid, 190
Blood, bactericidal properties of, 150
flagellates, 552
as medium, 64
-serum coagulator, 65
656
GENERAL INDEX,
Blue, methylin, 29
thionin, 29
pus, bacillus of, 412
Bodo, 530, 551
lacertae, 584
urinarius, 584
Books, disinfection of, 124
Bordet-Gengou phenomenon, 161
in syphilis, 577
in yaws, 580
Botulinus bacillus, 271
Bouillon media, 60
diphtheria toxin, 62
glycerine-peptone nutrient, 62
mannite-peptone, 62
nutrient, 61
sugar nutrient, 61
sugar-free nutrient, 61
Bovine tuberculosis, 339
Bromine, 108
solution, 118
Broth (see Bouillon), 60
calcium, 384
Brownian movement, 16
Bubonic plague, 423
bacillus of, 423
biology of, 424
morpnology of, 424
pathogenesis, 425
staining, 424
diagnosis of, 426
immunity against, 425
Bunge's method, 35
Burn's method, 47
Butter, bacillus of, 352
Butyric acid fermentation, 94
Cadaverin, 86
Calcium broth. 384
compounds as disinfectants, 107
Calmette's ophthalmo-tuberculin test,
337
Camphor as disinfectant, 112
Capaldi plate medium, 300
Capsules of bacteria, 12
staining of, 33
Carbohydrates, action of bacteria on,
83, 92. 93
Carbol-fuchsin, 33
Carbolic acid as disinfectant, 105, 114
methylene blue, 33
Carbon dioxides, production of, by colon
bacillus, 259
Carbonic acid, 92
under pressure, 56
Carpets, disinfection of, 124
Carriages, disinfection of, 124
Catgut, disinfection of, 127
Cell membrane, 13
substance, 13
Cellulitis, streptococci in, 373
Cellulose, fermentation of, 95
Centrosome, 524
Cercomonas hominis, 552
Cerebrospinal meningitis, 392, 394-400
Cesspools, 118
Chancre, soft, bacillus of, 410
Charbon sjrmptomatique, 436
Chemotaxis, 58
Chlamydozoa, 614
Choanoflagellida, 530
Chloride of lime, as disinfectant, 10^,
114
of zinc, as disinfectant. 107
Chlorine, as disinfectant, 108
Chloroform, as disinfectant, 111
Cholera, Asiatic. 443
diagnosis of, 453
inoculation against, 452
lesions in man, 449
-red reaction, 446
serum therapy, 453
spirillum, 15, 443
agglutination, 453
allied organisms of, 454
biology of, 444
development outside of body,
447
distribution in body, 450
identification of, 453
immunity against, 452
morphology of, 443
occurrence outside of body,
447
pathogenesis of, 448
resistance and vitality of, 447
staining, 444
toxin of, 451
variations of, 453
spread of, 450
Chromatin, generative, 524
somatic, 524
Chromidia, 534
Cilia ta, 530, 587
Citric acid, 94
CladothriXj 21, 458, 459
asteroides, 460
liquefaciens, 459
Classification of bacteria, 7, 22
of protozoa, 529
Claviceps purpurea, 472
Cleansing solutions, 106
Closets, disinfection of, 116
Clostridium pasteurianum, 98
Cocci, characters of, 9
spherical form of, 9
staphylococcus pyogenes, 361
streptococcus pyogenes, 369, 620
Coccidia, 531
Coccidium bigeminum, 592
cuniculi, 589
Coccus, 9
Cold, intense, effect of, on bacteria, 52
Coley's streptococcus toxins, 374
Collodion sacs, 80
Colon bacillus, action on nitrogenous
compounds, 259, 260
association with other bac-
teria, 262
behavior toward carbohy-
drates, 259.
GENERAL INDEX.
657
Colon bacillus, biology of, 258
cultivation of, 258
curative vaccine treatment,
267
in cystitis, 265
in diarrhoea, 264
differential diagnosis from ty-
phoid bacillus, 257
as disease producer, 263
flagella, 258
gas production, 259
group of, 255
growth of, on common media,
258
immunization against, 266
indol, production by. 260
in inflammations of bile tract,
265
methods of isolation, 267
morphology of, 255
occurrence in man and animals
261
in other inflammations, 266
outside of intestines, 261
of pancreas, 265
passage through walls of inte-!-
• tines during life, 263
pathogenesis, 262
m peritonitis, 264
as pus formers, 266
reduction processes, 261
in sepsis, 263
staimng, 258
toxins, 261
treatment, 266
of urinary tract, 265
-typhoid intermediates, 268
Colonies, characteristics of, 73, 74
counting of, 70 - < 2
study of, in plate cultures, 73
various forms of, 74
Complement, 159
Bordet-Gengou phenomenon, 161
deflection of, 159
fixation of, 161
origin of, 158
Complete study of a bacterium, 94
Conidia, 473
Conju ictivitis, Koch- Weeks bacillus of,
359
Conradi and Drigalski medium, 301
(^onrad.'s bile enriching method, 304
Copper sulphate, as disinfectant, 106
capsule stain, 34
Corrosive sublimate, 105
Counting of colonies, 70-72
Cover-glass, preparations of, how made,
27
how stained, 28
how to render slips free from
grease, 27
thickness of, 40
Cowpox, etiology of, 613
relation to smallpox, 612
Creolin as disinfectant, 112, 114
Creosote as disinfectant, 112
42
Crescent bodies in malaria, 605
Cresol as disinfectant, 112
Oithidia, 553
Crystal violet, 29
Culex in malaria, 601
fa titans, 487
Cultivation of bacteria, 69
of protozoa, 527
Culture, ana robic, 78
artificial protozoan, o.iS, 552
block and hanging mass, 42
media, preparation of, 60
reaction of, 66
plate, making of, 70
pure, 75
storage of, 68
titration of, 67
Cyclasterion scarlatinale, 620
Cyst formation, 526
(Cytolytic serum, 155
C3^oplasm, 522
Dark ground examination, 46
Decolorizing of stained smears, 32
Decrease in toxicity and virulence,U36
Decomposition, 96
Delhi boil, 554
Demonstration of ammonia, 91
Dengue, 487
Denitrincation, 97
Deodorant, 118
Deuterotoxins, 89
Diarrhoea, relation of bacteria in milk
to, 50 i
Dilution methods, 71, 175
Diphtheria, agglutinin development, 165
antitoxin, 211
deleterious effects, 218
globulin preparation, 221
nature of, 211, 220
neutraUzing characteristics of,
211
persistence of, in blood, 223
production of, for therapeutic
purposes, 212
refining by separation of anti-
toxin, 220
result of, treatment of, 217,
218
results of use of refined, 221,
222
standardizing of testing, 216
testing of, 214, 215, 216
unit of, 217
testing of, 212
use of, in treatment and im-
munization, 217
bacillus, 195-231
agglutination of, 223
animal inoculation as test for
virulence of, 229
biology of, 199
characteristic appearances of,
197. 19S
658
GENERAL INDEX,
Diphtheria bacillus in diagnosis, value
of detection, 225
examination of exudate, 229
exudate due to, contrasted
with that due to other
bacteria, 226
growth on agar, 199
in ascitic bouillon, 201
on blood serum, 199
in bouillon, 200
on gelatin, 201
in milk. 201
in healthy tnroats, 204
human inoculation of, 202
isolation by means of serum
bouillon, 201
of, from plate cultures,
200
morphology, 196, 198
non- virulent forms of, 206
pathogenesis, 201
persistence of, in throats, 205
of characteristics in types
of, 207
pseudodiphtheria, 206
resistance to heat, drying, and
chemicals, 210
staining of. 196, 197. 198
varieties of, 208, 209
virulence in cases of diphtheria,
204, 208
virulent, in healthy throats,
204
causes of death in, 202
characteristic appearances, 225
comparative virulence of different
cultures, 203
direct microscopic examination of
exudate of, 229
examination of cultures for diag-
nosis, 226
historical, 195
-like bacilli, virulent for guinea-
pigs, but not producing diph-
theria toxin, 206
mixed infection in, 221
relation of bacteriology to diag-
nosis, 225
susceptibility to and immunity
against, 211
tecfiiique of bacteriological diag-
nosis, 227
toxiiij^ 202%
Ehrlich's partial saturation
method of study, 214-2i6
injections in horses, 213
neutralizing value of a fatal
dose, 214
production of, in culture
media, 202
relation between toxicity and
neutralizing value of, 214,
215, 216
union with antitoxin, 212
toxoid, 216
toxon, 216
Diphtheria, transmission of, 210, 212
by milk, 518
value of cultures in diagnosis of.
226
Diphtheritic inflammations, location of.
226
tissue changes in, 202
Diplococcus intracellularis meningitidis,
392-401
biology, 393
morphology, 392
staining, 393
of pneumonia, 38 1 - 39 1 . See Pneu-
mococcus.
biology, 382
growth on media, 383
morphology, 381
staining, 382
Discharges, disinfection of, 115
Disease, liability of bacteria to cause,
131, 132
in beer and wines, 102
Disinfectants, 113
gaseous, 107
organic, 10.)
strengtn^of, 105
Disinfection *1 13- 130
agents for, 113
of books, 124
in contagious diseases, 115
definition, 103
by heat, 114
by moisture, 119
practical, of house, person, instru-
ments, and food, 103, 113, 113,
116, 123, 127, 128
preventive, 117
rooms, etc., 117
Dobell's solution, 128
Dri^lski and Conradi medium, 301
Drying, effects of, on bacteria, 53
Ducrey, bacillus of soft chancre, 410
Dunham's peptone solution, 63
Dysentery, amebic, 535. See abo
Amebfip.
bacillary, 275
bacillus, 274 281
agglutination characteristics,
279, 280
biology of, 274
differential diagnosis of, 546
Flexner Philippine type, 278
mannite fermenting varieties,
278, 281
morphology of, 274
Moimt Desert type, 277
pathogenesis of, 275
relation to paradysentery ba-
cilli, 276
types of, 279-281
historical notes of, 274
pathology of, 276
£
Ectoparasites, 531
Ehrlich's side-chain theory, 150
GENERAL INDEX,
659
Eimeria schubergi, 590
Elective staining properties, 30, 31
Electricity, influence on bacteria, 56
on protozoa, 52S
Elimination of bacteria through milk,
143
through skin and mucous mem-
brane, 143
through urine, 286
of foreign bacteria from prepara-
tions, 27
Endocarditis, 406
Endospores, 20
Endotoxins, 87
Ensilage, 102
Entamoeba^ 529. See also Amebae.
buccahs, 533
coli, 533
histolytica, 533
Entameba tetragena, 533
Enteriditis sporogenes, bacillus of, 442
Enterococcus, 253
Enzymes, 83
Erlenmeyer's flask, 68
Erysipelas, streptococci in, 373
Esmarch's method of growing colonies,
73
Essential oils as disinfectants, 112
Estivo-autuminal parasite of malaria,
598
Ethyl alcohol, 94
Eucalyptol, 112
Examination, of air, 489
for tubercle bacilli, 498
of fseces and urine for typhoid
bapilli, 303
of hanging drop, 41
of soil, 489
of sputum, 341-346
of unstained bacteria, 41
of water, 489
Extracellular toxins, 87
Eye-piece, 37, 39
Facultative aerobic and anaerobic
bacteria, 51
Fsecalis alcaligenes, bacillus, 272
Faeces, disinfection of. 115
examination of, tor amebse, 537
for tubercle bacilli, 344
for typhoid bacilli, 303
Farcy, 418
Fats, decomposition of, 91
Favus, 476
Fermentation, 82
alcoholic, 102
by bacteria, 82
tube, 93
of urea, 85
Ferments, characteristics of, 83-88
diastatic, 84
inverting, 84
proteolytic, 84
rennin-like, 84
Film preparations, 27
Filter beds, 100
Filtration of water, 495, 496
Finkler and Prior, spirillum of, 455
Fish, tuberculosis in, 341
Fixation of smears, 28
Flagella^ 14, 523
staming of, 35
Flagellata, 530, 550
classification, 530, 551
general characteristics of, 550
life cycle of, 551
materials and methods, 552
natural habitat, 551
Flies, relation to trypanosomiasis, 56 1
Focusing, 39
Food,. 48
Foot and mouth disease, 486
Foraminifera, 530
Formaldehyde as disinfectant, 109, 115
lime method of generating, 122
poisonous effects, 110
m room disinfection, 118-125
permanganate of potash method,
122
Wilson's rapid generator, 122
Formalin, 114, 120, 121
Formic acid^ 94
Formulae of stain combinations, 32
Fractional sterilization, 54
Framboesia tropica, 580
Friedlander, bacillus' of, 267, 381
Fungi pathogenic varieties, 474-479
Fungus of favus, 476
of pityriasis, 477
ray (actinomyces), 458
of ringworm, 474
of thrush (soor), 478
G
Gabbett's solution, 343
Gall-sickness, 559
Gametocytes, 602.
from carbohydrates, 93
Gas production by bacteria, 93
test for, 94
Gauze, sterilization of, 127
Gelatin media, 62
Gemmule formation, 614
Gentian violet, 29
Germicidal action, method of determin-
ation, 103
Germination of spore, 20
Giemsa stain, 624
Glanders bacillus, 417-422
biology of, 417
cultivation of, 418
isolation of, 418
morphology, 417
pathogenesis, 418
diagnosis of, 419
immunity against, 419, 420
staining, 417
test for (mallein), 420
Globulin, relation of serum globulin to
diphtheria antitoxin, 211, 212
660
GENERAL INDEX,
Glossina palpalis in relation to trypano-
somiasis, 565
morsitans, in nagana, 559
Glucose bouillon, 61
Glycerin agar, 63
Goldhorn stains, 570, 598
Gonococcus, 402 -4 OS
bacteria resembling, 400, 408
culture media for, 404
diseases excited by, 405
in endocarditis, 406
occurrence of, 405
staining reactions, 403
toxins of, 405
Gonorrhcea, bacteriological diagnosis
of, 406
Gram-negative and Gram-positive bac-
teria, list of, 191
stain, 33, 398
Granules, metachromatic, 14
Grass bacillus, 348-352
Green methyl, 29
pus, bacillus of, 412-416
Gregarinida, 531
Group agglutinins, 166
reaction, 167
Growth of bacteria, 48
Gruber-Widal reaction, 292-293
persist- nee of reaction,
297
relation of, to typhoid,
297
use of dead cultures for,
297
of dried blood for,
292
of serum for, 294
Guarnidri vaccine bodies, 615
Gymnamoebida (see Amebae), 529-532
HiBMOLYsiNs, Ehrlich's studies on, 161
Haemolysis, 161
Haemolytic sera, 161, 163
Hsemosporidia, 531, 596
Haemoproteus, 606
Haffkine's preventive inoculations for
cholera, 452
for plague, 426
Halteridium, 606
Hand brushes, disinfection of, 115, 128
Hanging drop for study of bacteria, 41
mass, 42
Haptophore group, 152, 153
Head spore, 20
Heat, effect of dry on bacteria, 53
disinfection, 54
of moist on bacteria, 53
Hermann's fluid, 522
Herpes tonsurans, 478
Herpetomonas, 553
donovani, 553 554
infantile, 554
tropica, 554
Heterommastigida, 530
Heterotrichida, 530
Hiss's capsule stain, 34
media for t^hoid bacillus, 299
serum media, 66, 384
Histoplasma capsulatum, 554
effect on human host, 556
morphology, 556.
Historical sketch of bacteriology, 1
of protozoa, 520
Hollow slide, 40
Holotrichida, 530
Horse, injections of diphtheria toxin in,
213
Hydration, 82
Hydrogen, 92
peroxide, 108
Hydrophobia (see Rabies), 484, 622
Hypersusceptib.lity, 162
Hjrpochlorites, 108
Hyphomycetes, 472, 480
Ice, bacteria in, 305^ 306
typhoid bacilli m, 305
Icteroides, bacillus, 2i6, 637
Immune body, 151
multiplicity of, 158
ori^n of, 158
Immunity^ active, 147-149, 221
duration of, 223
passive, 147
production of, 146
specific, 146
tneories of, 163
Impetigo contagiosa, 484
Increase of toxicity and \irulence of
bacteria, 136
Incubators, 77
low temperatures in, 77
India ink method of examining bac-
teria, 47
Indol, 92, 446
test for. 91, 260
Infection, oi blood, 142
influence of quantity in, 135
mixed, 137
modes of entrance, 140
protection afforded by skin and
mucous membranes, 138-9
spread of, 140
Inflammation due to bacteria, 134
Influence of one species upon growth of
another, 49
reaction of media upon growth, 49
Influenza bacillus, 353-358
agglutination of, 358
bacteriological diagnosis, 357
biology, 354
cultivation, 354
detection of, in sputum, 354
distribution in the body, 357
effect on animals, 355
epidemology, 356
examination of sputum for,
* 358
immunity to, 355
morphology of, 353
GENERAL INDEX.
661
Influenza bacillus, pathogenesis, 355
presence in blood, 356
resistance, 354
serum therapy, 358
staining characteristics, 353
in tuberculosis, 356
vaccines, 358
Infusion, meat, 60
Infusoria, 530
Inoculation, anterior eye chamber, 186
body cavities, 186
cutaneous, 185
inhalation, 186
intestinal tract, 186
intravenous, 186
subcutaneous, 185
trachea^ 186
trephinmg, 186
Inorganic compounds in disinfection,
106
Instruments, dressings, etc., disinfec-
tion of, 127
Interbody, 157
Intestinal flora, changes in, 247
Intestines, anaerobic conditions in, 246
development of bacteria in, 139,
245
methods used in examination of
ffipces, 247
regional distribution, 246
significance of bacteria in, 245
Intolerance to tuberculin, 332
Intracellular toxins, 88
Inulin in serum media, 66
Invisible microorganisms, 485
Involution forms of bacteria. 17
lodin-alcohol, 522
Iodine as disinfectant, 108
Iodoform, 111
Iron sulphate as a disinfectant, 106
Ixodes redivius, 609
Japanese worm, 482
Kala-azar, 553-556
diagnosis of, 556
Leishman bodies in, 553
Karyo-'ome, 524 #
Kine^^ic nucleus, 524
Koch-Ehrlich aniline water solution, 32
phenomenon of, 160
Koch- Weeks bacillus of conjunctivitis,
359
biological characters, 359
differential, diagnosis of,
360
immunity, 360
pathogenesis, 360
resistance, 360
staining, 359
transmission, 360
Koch's original tuberculin, 328
Labarraque's solution, lOS
Lactic acid, 94
milks, 248
producing bacilli, 248, 256
Lactose bouillon, 61
Lamblia, 530, 551
intestinalis, 586
Levarania malarise, 598, 599
animalcula, 520
Leishman method, 174
bodies, 553
Leishmania (Herpetomonas) donovani,
553
bed bugs as carriers, 556
morphology, 556
Leprosy bacillus, 349-352
biological characters of. 350
differential diagnosis of, 351
morphology of, 349
pathogenesis of, 350
Leptothnx, 458, 459
Leukocytes, extract of, 172
part played by, in immunity, 158
production of exudates rich in, 187
for testing phagocytosis, 187
Leukocytic extract, 183
Life of bacteria in absence of moisture,
57
Ligatures, disiinfecton of, 127
Light, production of, by bacteria, 81
by bacteria, 81
influence of, on bacteria, 55
Lime, milk of, 114
Listerine, 128
Litmus, as indicator, 66
media, 63
Locomotor nucleus, 524
LoeflSer's alkaline solution of methylene
blue for staining diphtheria, 32,
36
blood serum, 65
Lungs, growth of bacteria in, 139
Lustgarten's bacillus, 348. See Smeg-
ma.
Leydenia gemmipara, 547
Lysol as disinfectant, 112, 114
M
Maorogametocyte, 602
Madura foot, 465
Mai de caderas, 559
Malaria, 596-61 1 . See also Plasmodium
malarise.
diagnosis, 605
historical note, 596
immunity, 604
infection, how acquired, 596^ 602
-like organisms in other ammals,
606
materials and methods for study,597
mosquitoes in relation to, 596, 598,
602, 604
parasites, 598
662
GENERAL INDEX.
Malaria, technique of blood examina-
tion in, 597, 598
Malic acid, 94
Malignant oedema, bacillus of, 439
pustule, 434
Mallei, bacillus, 417
Mallein test for glanders, 420
Malta fever, 408
spread by goat's milk, 409
Manmte fermenting dysentery bacilli,
278, 281
in media, 62
Marble broth, 384
Margaropus annulatus, 609
Marsh gas, 92
Massol, bacillus of, 249
Masti^ophora, 530
Matenal for bacteriological examina-
tion, procuring of, from
those suffering from disease,
188
routine technique of examina-
tion, 190
media to be used, 193
Measles, 484, 620
Meat infusion, 60
poisoning, 271, 272
Media, preparation and sterilization of,
60
reaction of, 49, 66
special, 384
storage of, 68
various kinds of, 60 66
Melitensis, micrococcus, 408-410
Meningitis, bacteriological diagnosis of,
398
various organisms exciting, 400
Meningococcus, 392-401
agglutination, 395
bacteriological diagnosis, 398
biological characteristics, 393
morphology, 392
pathogenesis, 394
presence in nares of botli sick and
healthy persons, 394
in blood, 395
resistance, 394
serum treatment, 395
staining, 393
Mercaptans, 92
Mercurjr bichloride, as a disinfectant, 106
biniodide, as a disinfectant, 106
Merozoites, 600
Mesophilic bacteria, 52
Metachromatic granules, 14
Metchnikoff, spirillum of, 172, 456
Methyl green, 29
violet, 29
Methylene blue, 29
Meyerstein's enriching method, 304
Microchemical reactions, 22
Micrococcus catarrhahs, 400, 401
biology, 400
culture media, 400
gonorrhoea, 402, 405. See Gono-
coccus.
serum and vaccine, 406
Micrococcus gonoerhoea, staining, 403
intracellularis, 392-401. See Men-
ingococcus,
lanceolatus, 381-391. See Pncu-
mococcus.
melitensis, 408-410
pharyngis siccus, 401
tetragenus, 367, 368
biologv, 367
growth on media, 368
morphology, 367
pathogenesis, 368
stainijg, 367
zymogens, 410
Microphotography, 47
Microscope, different parts of, 37
Microscopic methods, 27
examination of unstained bactem,
41
Miescher's tubes, 594
Milk, bacterial contamination of, 50S
bacteriology of, in relation to dis-
ease, 500
as culture medium, 63
ehmination of bacteria through,
143
examination of, 500
heated vs. raw, in feeding, 503-50^
identification of bacteria in, 501
influence of cleanliness on, 514
of temperature on growth of
bacteria in, 510
number of bacteria in, 500, 508
pasteurization of, 129, 511
pathogenic properties of, 502
smear method of estimating num-
ber of bacteria, 500
sterilization of, 129
streptococci in, 503
time required for multiplication of
bacteria in, 502
transmission of disease through,
516
Milzbrand, 429. See Anthrax.
Mixed infection, 137
Moeller's method of staining spores, M
Moisture, 119
Monadida, 530, 551
Monotricha, 15
Mosquitoes as agents of infection in
malaria, 596, 59s, t;02,
604. 605
in yellow fever. 637, 63s
trypanosomes in, 562
Mordants, 32
Morphology of bacteria, 7
permanence of, 9
of protozoa, 522
Mosaic diseases cf tobacco, 487
Motility of bacteria, 41
organs of, 14
Moulds, pathogenic, 472 -483
Mucor, 473
corymbyfer, 473
Mucous membranes, ability of battens
to penetrate, 139
disinfection of, 128
GENERAL INDEX.
663
Multipartial sera, 159
Mumps, 485
Mycelium, 474, 475
Mycetoma, 465
Mycetozoa, 530, 547
Myxidium, 531
Myxobolus, 531
Myxosporidia, 593
N
Nagana, 559
Negri bodies in rabies, 623
morphology of, 626
smear metnod of demonstrating,
624
Neisser stain for diphtheria bacilli, 197
and Wechsberg phenomenon, 159
Neosporidia, 531, 594
Neucleophaga, 530
Nessler's reagent, 91
Neuroryctes nydrophobife, 623.
Neutral red, 64
Nitrate bouillon, 64
of silver, 106
Nitric acid, 92
Nitrification, 92, 97
Nitrifying bacteria, 92
Nitrites and nitrates produced by bac-
teria, 92
Nitrogen combination, 96
fixing bacteria^ 97
Nitroso-indol reaction, 91
Nocardia, 458, 465
Noguchi method, 579
Noma, 485
Nosema bombycis, 531, 593
lophii, 593
Novy method of making anaerobic cul-
tures, 79
Nucleus of a bacterium, 13
of a protozoon, 523
Nutrient bouillon, 61
(Edema, malignant (anthrax), 434
Oil-immersion lens, 37
Ookinet (zygote), 602
Ophthalmo-tuberculin test, 337
Opsonic index, 174
accuracy of, 176, ISO
diagnostic value, 180
Simon's method, technique,
174
test, 178
variation in healthy persons,
178
therapy, 181
Opsonins, 151, 172, 180
diagnostic value of, 183
Osmic acid as a fixative, 521
Oxvgen, behavior of bacteria toward,
50
absorption of, 80
Paracolon bacilli, 269
Paradysentery bacilli, 276
Paratyphoid bacilli, 269
infection, 269
serum reaction in, 270
Pasteur filter, 497
flask, 68
treatment of rabies, 634
by mail, 636
Pasteurization, 54, 129
P^brine, 5, 593
PeUagra, 485
Pemphigus neonatorum, 484
Penicillium minimum, 474
Peppermint, oil of, 112
Peptone, 92
solution, Dunham's, 63
Peritricha. 15
Peritrichida, 531
Permanganate of potash and lime, 122
Peroxide of hydrogen as a disinfectant,
108
Pest (bubonic plague), 423
Petri dish, 70
Petrusky's litmus-whey, 63
Pfeiflfer's phenomenon, 150
Phagocvtosis, 187
Phenol,* 92
Phenolphthalein as indicator, 67
Physiologic salt solution, 522
Phytoflagellida, 530
Pigment olue, 85
production by bacteria, 85
red and yellow, 85
violet, 85
Piroplasma (Babesia), 607-611
bigeminum, 607
blood organisms, 611
morphology, 608
pathogenesis, 609
prognosis, 610
prophylaxis, 610
symptoms, 610
ticks as carriers of, 609
treatment, 610
canis, 611
staining, 608
V. Pirquet cutaneous tuberculin test,
336
and Shick's theory, 219
of serum sickness, 218
Pityriasis versicolor, 477
Plague, bubonic, 42 i
Plants, bacterial disease of, 102
Plasmodiophora brassicae, 530, 547
Plasmodium, 531
falciparum, 598
of malaria, 5 6. See also Malaria,
aestivo-autumnal parasite, 598
classification, 59i>
cycle in mosquito, 601
examination of blood for, 598
pathogenesis, 604
quartan parasite, 598
staining methods for, 597, 598
tertian parasite, 598
malaria;. 596, 598
664
GENERAL INDEX.
Plasmodium, prophylaxis, 604
toxin production, 604
vivax, 598
Plate cultures, streaked surface of, 72
study of colonies in, 73, 75
technique of making, 70
Plectridium^ 20
Pleuritic fluid in culture media, 64
Pleuropneumonia, contagious, of cattle,
488
Pneumobacillus of Friedlander, 267
Pneumococcus, 381
agglutination reaction, 390
attenuation of virulence, 385
biological characteristics, 382
elTects of drving and sunlight on,
384
immuity to infection by, 390
morphology, 381
mucosus, 389, 390
occurrence in man in health, 385
special media for cultivation, 383,
384
staining, 382
pathogenesis, 385
presence in diseases other than
pneumonia, 387
in lobar and bronchqpneu-
monia, 386
restoration of virulence, 385
therapeutic experiments, 390
toxin production, 385
vaccines, 391
varieties of, 389
Poisons, similar vegetable and animal,
88
Polymastigida, 530, 551
Polymerization, 82
Polyvalent serum, 159
Potatoes as culture medium, 64
Precipitation of extracellular toxins, 87
Precipitins, 171
Pressure, influence of, on bacteria, 56
Prior, spirillum of Finkler and, 455
Proprionic acid, 94
Protective defen es of body, 150
Proteins, bacterial, 84
Proteosoma, 606
Proteus, bacillus, 414
Protista, 7, 519
Prototoxins, 89
Protozoa, 519
blepharoplast, 524
centrosome, 524
chemical composition, 52S
chromidia, 524
classification, 529
cultivation, 527
cyst-formation, 526
cytoplasm, 522
definition of, 519
ectoplasm, 523
effect of physic and chemic agents
on, 527
entoplasm, 523
general characteristics of, 522
Protozoa, habitat, 527
history of, 520
irritability, 525
karyosome, 524
material and methods for study,
521 .
morphology, 522
nucleus, 523
locomotor or kinetic, 524
nutrition, 525
origjn, 521
pathogenesis, 528
relationship to other microorgan-
isms, 7, 519
reproduction, 525
respiration, 525
sexual phenomena, 526
structure, 522
vital phenomena, 525
Protozoan-like bodies in smallpox and
allied diseases, 613
Pseudodiphtheria bacilli, 206
Pseudoinnuenza bacillus, 357
Pseudomembranous inflammations due
to bacteria other than diphtheria ba-
cilli, 224
Pseudomeningococcus, 401
Pseudotuberculosis, strep tothrix in, 466
Pseudoworm, 482
Psittacosis bacillus, 269
Psychrophilic bacteria, 52
Ptomaines, 86
Public conveyances, disinfection of, 118
Pure cultures, 75, 527
in tubed media, 76
Pure-mixed cultures, 52/, 538
Pustule, malignant, 434
Putrefaction, 91
Pyelonephritis, 416
Pyocyanase, 413
Pyocyaneus, bacillus, 412
Pyocyanin, 413, 86
Pyogenic cocci, 361-3^0
Pyrosoma bigeminum, 607
Quartan parasite of malaria, 598
Quarter evil, 436
Rabbit vaccine, 619
Rabic virus, effect of chemic and
physic agents on, 629
Rabies, 484, 622-637
cauterization of wounds in, 636
complement, binding test, 629
diagnosis of, 628
experimental infection, 630
fixed virus, 629, 633
M a 1 1 o r y ' s eosi n-methylene-blue
method, 625
material and methods for study,
624
methods of immunization, 636
natural infection, 629
GENERAL INDEX,
665
Rabies, Negri bodies in, 623
Pasteur's treatment, 633
pathogenesis, 629.
preventive inoculation against, 633
smear method, 624
symptoms, 631
Radiolana, 530
Radium, influence on bacteria, 56
Rainey's tubes, 594
Rauschbrand, 436. See Symptomatic
anthrax.
Ray fungus, 458. See Actinomyces.
Reaction of media, correction of, 49
adopted by American Public
Health Association, 66
Vosges' and Proskauer's test, 499
Receptors, 153
Recovery of poison production, 25
Red, basic fuchsin, safranin, 29
Reduction processes, effect by bacteria,
90
Refrigerators, disinfection of, 118
Relapsing fever, spirillum of, 583
Relation between agglutinating and
bactericidal power, 171
Reproduction among bacteria, 16
higher bacteria, 21
in protozoa. 525
Rhinoscleroma, bacillus of, 268
Rhinosporidium kinealyi, 592
Ricin, 88
Rinderpest, 487
Ri2opodia, 529
Rocky Mountain spotted fever, 426
Rosen-Runge's method, 304
Ross, method of examining malarial
blood; 598
Von Ruck's watery extract, 330. See
Tuberculin.
Saccharomyoes, 481
Busse, 481
cerevisije, Hansen, 483
neoformans, 483
subcutaneus tumefaciens, 481
Safranin, 29
Saprophytes, facultative, 48
strict, 4S
Sarcinae, 10
Sarcocystin, 595
Sarcocystis, 531
muris, 594
Sarcodina^ 529
Sarcosporidia, 594
Sarcosporidiosis, 594
Sauerkrautj 101
Sausage poisoning, 271
Scarlet fever, 484, 619
conveyance of, by milk, 517
etiolo^, 620
historic note, 619
Mallory's protozo6n-like
bodies in, 620
streptococci in, 379
Schizogony, 526
Schizomycetes, 480
Schizonts, 600
Schizosaccharomyces octosporus, 481
Schottmiiller's method, 304
Scurvy, 485
Sea-water, bactericidal properties of,
498
Sections, preparation of, 36
Sensitizer, 151
Septic tank, 99
Septicaemia, various organisms con-
cerned in, 423
Sera, antitoxic, 148
bactericidal, 148
bacteriolytic, 155
cytolytic, 155
demonstration of nature of,
156
therapeutic value, 148
multipartial, 159
poljrvalent, 159
Serum, alkaline blood, 65
antimeningococcus, 395
antipneumococcus, 390
antistreptococcus, 376, 377
antityphoid, 290
bactericidal, 148
bacteriolytic, 155
bouillon media, 65
collection of, for diagnostic pur-
poses, 188
cytolytic, 155
diagnosis, 290. See Gruber-Widal
reaction.
haemolytic, 155, 156
limit of curative power, 148
Loeffler's blood, 65
media, 64
production of protective, 146
sickness, 218, 219
water media, 66
Sewage, bacteria in, 99
disposal of, 497
farming, 100
Sexual cycle, 526
Shiga, dysentery bacillus, 274-281.
Silkworm disease, 593
Silver nitrate as a disinfectant, 106
Sinks, disinfection of, 116, 117
Skatol, 92
Skin, ability of bacteria to penetrate,
138
disinfection of, Fiirbinger's method,
128
Sleeping sickness, 555, 550
Smallpox, 484, 612-620
pathogenesis, 615
protozoan bodies in smaUpox and
allied diseases, 613
relation to vaccinia, 612
Smear method in diagnosis of rabies,
623. 624
for direct examination of milk,
500
Smears, staining of, 27
GENERAL INDEX.
Smegma baciilua, 348
biological and pathogenic
Sroperties, 34S
srential diaKDOsis, 34»
Soapsuds as cleansing solution, 113
Koda solution as disinfectant, 113
Sodium compounds as disinfectants, 107
Soft chancre, 410
Soil, bacteria in, 98. 489
examination of, 499
Soor, fungus of, 478
South African horse sickness, 487
Species, influence of one upon growth of
another, 49, 55
permanence of, 24
Specific agglutinins, 166
Specificity of agglutinins, 164 -
Specimens, preservation of, 37
Spirilla, general characteristics of, II
allien] to cholera, 443
morphologic charae-
Spirillum of Asiatic cholera, {tee Chol-
era), 443 45t
of Finkler and Prior, 455
of Metchnikoff, 456
of relapsing fever, 580
undulans, 15
Spirocheta, 551, 560
bala nitidis, 571
balbiani, 571
buccal is, 572
carteri, 583
dentium, 572
duttoni, 582
obermeieri, 580
bioloeical characteristics, 581
morpnology, 581
pathozenesis, 581
pallida, 573. See Treponema pal-
refringens, 572
vincenti, 572
Spirochete, cultures of, 571
in frambcesia tropica, 580
in mouth, 572
methods for study, 569
miscellaneous, 572
relation to protozoa and to bac-
teria, 580
in tumors, 580
Spirochetes from relapsing fever in
America, 5S3
Spittoons, disinfection of, 118
Sporoioa, 531, 551, 588
life cycle, 590
SporoEoites, ^26.
Spotted fever, 426. See also Menin-
gitis.
Sputum, disinfection of, 115
methods of examination for
tubercle bacilli in, 341-346
of other bacteria in, 346
washing, 345
Staining bacteria, 30
principles underlying, 30
Stains, blue, 29
brown, 20
Gabbett, 343
Giemsa, 624
Goldhom, 570, 598
Gram
33
in,' 29
LoefHer's, 32, 35, 36
Mallory, 625
Moeller, 34
Neiseer, 197
Nocht-Romanowsky, 597
red, 29
m, 624
if, 30
heat, 53
i6, 527
.,„...^29
Van Gie
Welch, a
Wright, 597
Ziehl-Neelsen, 33
Standardizing of antitoxin, 216
Staphylococcus, 10
epidermidis albus, 366
pyogenes, 361
albus, 366
citreus and other staphylo-
cocci, 367
biology, 361
morpnologv, 361
cultivalioni 361
immunity, 366
occurrence in man, 365
pathogenesis, 363
products of growth, 363
resistance, 362
staining, 361
thera{)eutic use, 366
varieties, 367
Steam disinfection chambers, 126
Stegomyia calopus (vellow-fever mo*-
quito), 637, 638
Sterilization, 103
fractional, 54
of milk, 129
Sterilizer, dry heat, 60
Stitch abscess, 367
Stock vaccines, 183
Stomach, as protection against bacterial
Storage of media, 68
Streaked surface plate cultures. 72
Streptococci, bacteriological diagnosis.
GENERAL INDEX.
667
Streptococci, definition of, 10
general characteristics, 368
in relation to disease, 503
Streptococcus mucosus capsulatus, 389.
See Pneumococcus mucosus.
pyogenes, 369, 620
biology, 370
cultivation, 370
duration of life, 371
hsemol^rtic substance, 371
immunization against, 375
influence of serum on, 376
injected in sarcoma, 374, 375
morphology, 369
occurrence in man, 373
pathogenesis, 372
m scarlet fever, 620, 379
staining. 370
susceptibility to, 375
toxic substances produced by,
375
Streptothrix, 458
biology, 469
infection by, 465
in pseudotuberculosis, 466, 471
spore formation by, 470
Structure of protozoa, 522
Subcutaneous connective tissue % 139
Sublimate alcohol as a fixative, 521
Substage, 39
Suctoria, 531
Sulphate of copper as disinfectant, 106
Sulphur dioxide gas in house disinfec-
tion, 107, 125, 126
Sulphuretted hydrogen in reduction
processes, 90
tests for, 90
Sunlight, influence on bacteria, 55
Surgical instruments, disinfection of,
127
Symbioses, 49, 50
Symptomatic anthrax, 436
Synthesis, 82
Syn toxoids, 216
Synura, 530
Syphilis, 575
immunity, 577
Lustgarten's bacillus in, 573
in man, 576
in monkey, 575
in rabbit, 576
treponema pallidum in, 573
Syringes, disinfection of, 128
Taba-RDIllo, 427
Telesporidia, 531
Temperature, effect of, on antitoxin,
214
on bacteria, 51, 52, 53
on protozoa, 528
Test for indol, 91
nitrites, 90
Tetanolysin, 236
Tetanospasmin, 236
Tetanus^ 232, 234, 235
antitoxin, 237
method of administration of,
242
persistence in blood, 238
unit, 238
bacillus, animal experiments, 239
biology of, 233
duration of life, 236
^owth in media, 233
in intestines, 232
isolation of pure cultures, 234
morphology, 232
non-virulent type, 244
occurrence in soil, 232
pathogenesis, 234
rapidity of absorption, 240
spores of, 233
. staining of, 233
diagnostic procedures in, 243, 244
dififerential diagnosis, 243
immunization against, 242
in man, 235
natural infection, 235
toxiuj 236
absorption, 240
action of, in body, 236
neutralization of, 241
in body, 242
presence in blood, 237
union with antitoxin in body,.
239
treatment with antitoxin, 241
Tetrads, 9
Tetrogenus, micrococcus, 367, 368
Texas fever, parasite of, 607-611
prophylaxis, 610
Thermic effects of bacteria, 81
Thermophilic bacteria, 52
Thermo-regulator, 78
Thionine blue, 29
Thrush fungus, 478
Thymol as disinfectant, 112
Ticks, Boophilus bovis, 609
Ixoides redivius, 609
in relation to disease, 609
Timothy grass bacillus, 352
Tinea barbse, 474
circinata, 474
sycosis,474
tonsurans, 474
Tissue, characteristics, 138
examination of bacteria in, 36
Titration of culture media, 67
Toxins, 92
Ehrlich's theory as to the nature
of, 89
extracellular, 87
ferment characteristics of, 88
intracellular, 88
variation in amount, 136, 137
Toxoids, 153, 216
Toxon. 216
Toxophore group, 153, 216
Trachoma, 621
Traps, disinfection of, 118
668
GENERAL INDEX.
Treponema pallidum, 573-580. (Syph-
attenuated virus, 577
cultivation, 574
morphology of, 573
passive immunization, 577
pathogenesis, 575
staining, 574
Wassermann reaction, 577
pertenuis, 580
Trichomonas, 551
hominis, 530, 586
vaginalis, 586
Trichomycetes, 458
Trichonympha, 530
Trichophyton megalosporon, 474
nucrosporon, 474
tonsurans-M., 475
Tricresol, 112, 114
Trioxy methylene, 121
Tritotoxins, 89
Tropical malaria, 598. See iEstivo-
autumnal malaria.
Trypanosoma, 557
brucei, 558, 561
comparative characteristics of
different species, 560
cruzi, 558, 559
cultivation of, 563
equinum, 558
equiperdum, 557, 558
evensi, 558, 560
examination for, 566
cytoplasm, 561
gamoiensi, 558, 559
lewisi, 557, 55S
life cycle of, 562
in man, 564
morphology, 560
motility, 561
nucleus, 561
noctusB, 561
pathogenesis, 563
pathogenic forms, 557
reproduction, 561
table of pathogenic forms, 558
theileri, 558, 561
transvaalense, 559
Trypanosomata, 551
Trypanosomiasis, 564, 565
diagnosis, 565
duration, 565
methods of examination for, 566
pathological changes, 565
prophylaxis, 566
serum therapy in, 567
symptoms, 564
treatment, 566
Tsetse flies, 559
Tubed cultures, 176
Tube-length, 40
Tubes, preparation and Ailing of, 69
Tubercle bacillus, 310-351
agglutination, 338
attenuation, 325
avian, 340
biology, 311
Tubercle bacillus, bovine, 339
calf virulence, 340
of cattle, pigs and sheep, 339
chemical constituents of, 32S
cultivation, 313, 314, 315
diagnosis by animal inocula-
tion, 347
differences between human
and bovine types, 339
discovery, 310
distribution, 310
examination of material for.
341
growth, 314
human, 339
immunization against, 328,
330
media for isolation, 316
method of examining milk for,
322
making pure cultures,
tures, 315-317
methods of examination for,
341-342
microscopic examination, 341
in mixed infection, 325, 344
morphologjr, 310
pathogenesis, 316
point of entrance, 318
poisons, 317
action on tissues, 318
rabbit virulence, 339
resistance, 311, 325
stability, 341
staining peculiarities, 311
in tissues, 347
toxins, 317
transmissibility, 325
in urine and faeces, 344
viability, 319
Tuberculin, bacillus emulsion of new,
" B. E.," 329
bouillon filtrate of, 329
diagnostic use of, 334, 335
intolerance to, 332
Moro's test, 337
precipitation of "T. P.," 329
therapeutic use of, 330, 334
Von Pirquet's test, 336
Tuberculins. 328, 329, 330
original, 328
Tuberculosis, agglutination, 338
in animals, 313
bovine infection of man, 322
of different regions, 318-320
in fish, 341
immunization against, 326. 327
individual susceptibility, 325
mixed infection, 325
mode of infection, 319
prophylaxis, 338
serum treatment of, 338
U. S. Govt, directions for inspect-
ing herds for, 334
Tumors, injection of streptococcus and
prodigiosus toxins in, 374
Turpentine, oil of, as disinfectant, 112
GENERAL INDEX.
669
Typhoid bacillus, 282
agglutination of, 290, 291
biological characteristics, 282
carriers, 287
relation to milk infection,
517
cultures, 283, 284
distribution in human subject,
285
duration of life outside the
body, 288
elimination of, through urine
endo medium, 302
and faeces, 286, 304
in faeces, 303
in healthy persons, 287
identification, 307
in ice, 305
importance of, in mixed in-
fection, 286
isolation of, 298
in blood, 304
Capaldi method, 300
Hiss' method, 299
Drigalski and C o n r a d i
Endo medium method,
301
from water, 494
morphology, 282
occurrence in water, oysters,
and milk, 289, 304
staining, 282
unusual localization, 286
in urine, 304
-colon intermediates, 257
communicability, 28S
diagnosis by means of serum test,
296
differential diagnosis, 307
due to infected milk, 288
Gruber-Widal reaction in, 292, 293
technique of test, 293
use of dead cultures, 297
immunization against, 290
Typhus fever, 427
individual susceptibility, 289
vaccination against, 290
Tyrotoxicon, 86
U
Ultramicroscopic organisms, 485
examinations, 46
Urea, fermentation of, 85
Urine, bacteria eliminated through, 140,
143
tubercle bacilli, examination for,
341
typhoid bacilli in, 286, 287, 298, 304
Urinals, cleansing and disinfection of,
118
Vaccination, immunity conferred by,
616. (Smallpox.)
Vaccinia, 612
Vaccine, bacterial autogenous, 183
as immunizing agents, 184
bacterial, 172
preparation of, 182,
sensitized, 182
stock, 183
therapy, 181
dosage in, 182
for variola, 616
bacteria in, 619
bodies, 615
durability of, 618
preparation of, 616
Valerianic acid, 94
Van Ermengen's method, 35
Van Gieson's staining method for rabies,
624
Variola, etiology of, 613. See Smallpox.
Venins, 88
Vibrio i erolinenais, 455
of cholera, 443
danubicus, 455
metchnikovi, 456
water, 457
Vincent's angina, 230
bacilli in, 231
Vinegar, production of, 101
Violet crystal, 29
gentian, 29
methyl, 29
Virulence, relation between, and build-
ing of immune bodies, 158
variation in degree of, possessed
by bacteria, 136
Vital phenomena in protozoa, 525
Vosges reaction, 499
W
Waldeyer, reaction, 577
test, 578
Water, bacilli of colon group in, 492
bacteriological examination of, 489
interpretation of results,
492
collection of samples of, 490
contamination and purification of,
489, 495
proteus bacilli in, 494
purification of, for domestic pur-
poses, 495, 496
on large scale, 495
quantitative analysis of, 489
sea, 498
streptococci in, 494
typhoid bacilli in, 494
Weeks' bacillus, 359. See Koch- Weeks.
Weigert's law of overproduction, l53
Weigert-Ehrlich, hypotnesis of over-
production of antitoxin, 153
Welch's capsule stain, 33, 382
Whooping-cough, 484
agglutinins of, A>A
bacilli found in, 4S4.
Widal reaction. See Gruber-Widal,
292- 298
Williams' smear method, 624
670
GENERAL INDEX.
Wilson, apparatus for Tormaldehyde
disinfection, 121, 122
method for anaerobic cultures, 78,
7i), 80
Wolf -Eisner, ophthaiimo- tuberculin
test, 337
WolfFhttgel's apparatus, 72
Woodwork, disinfection of, 118
Wool-sorters' disease, 435
Worm, Japsnese, or pseudo-, 482
Wright's method, accuracy of, 175
Yeasts, 472, 479
culture, 480
pathogenic, 472
relationship to bacteria, 22
wild, 480
Yellow fever, 487, 637, 63S
bacillus icteroides, 637, &
mosquitoes in, 637, 638
trypant
Zenker's fluid, 625
Ziebl-Neelsen carbol-fuchsin solution for
tubercle bacilli, 33
Zinc chloride, 107
Zymophore group, 153
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11